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The results presented in Table 1 demonstrate that materials with all three enrichments evaluated in this work can be identified in a single laser-driven neutron pulse. Note that the uncertainties listed for the integral DN counts include the uncertainty on the bubble chamber readings used for normalization and also the uncertainty in the background corrected measured DN counts determined as the square root of the number of counts observed within the DN interval. A clear increasing trend of integral delayed neutron counts with 235 U enrichment is observed for fast as well as thermal modes, as expected. In fast mode, the detection response is nearly linear, which makes particularly straightforward the determination of the amount of fissile material present in the canisters. The sensitivity of the active interrogation is significantly enhanced in the thermal mode, however the interrogation in thermal mode exhibits non-linear behavior for higher 235 U enrichment of the items 47 . At low enrichment, the 235 U mass is more dilute in the sample, and as the 235 U enrichment increases, the self-shielding effect reduces the thermal neutron penetration through the sample, so fast mode neutron interrogation is more representative.
|
39753631_p33
|
39753631
|
Detection of nuclear material by active interrogation
| 4.160518 |
biomedical
|
Study
|
[
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0.00032199182896874845,
0.001257284777238965
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[
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0.000037389407225418836
] |
en
| 0.999997 |
As one approach, one could utilize a thermal mode high-sensitivity detector arrangement for high-throughput active interrogation for screening purposes. For the cases where fissile material is detected, one could set aside the item to repeat the assay offline safely with a fast mode detection arrangement (with increased neutron dose if necessary) in order to determine more accurately the amount of special nuclear material, and possibly to do additional tests as warranted (e.g., passive muon imaging, nuclear spectroscopy, etc.) before attempting internal inspection of the item.
|
39753631_p34
|
39753631
|
Detection of nuclear material by active interrogation
| 3.673505 |
biomedical
|
Other
|
[
0.9900394082069397,
0.0013091453583911061,
0.00865152757614851
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[
0.11542336642742157,
0.8827021718025208,
0.0014406610280275345,
0.00043380711576901376
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en
| 0.999995 |
A series of experiments were conducted at Los Alamos National Laboratory to explore the potential and technical advantages of active interrogation using a high-intensity, laser-driven neutron source. The results presented in this paper provide the first direct evidence of the feasibility of single-pulse laser-driven neutron interrogation of special nuclear materials through the detection of delayed neutrons. The reported data demonstrate the ability to detect the presence of fissile material unequivocally and with a level of consistency, which is necessary for practical quantitative applications. With the first successful integrated demonstration of active interrogation with LDNS to detect fissile material, this research advances the technology from a conceptual stage to a phase where validation has occurred in a laboratory environment. From this platform optimization and engineering studies can be initiated. Below, we suggest further work to further advance the technology out of the laboratory and towards the deployment of a production system.
|
39753631_p35
|
39753631
|
Discussion
| 4.042341 |
biomedical
|
Study
|
[
0.9818691611289978,
0.00039317485061474144,
0.017737673595547676
] |
[
0.9988289475440979,
0.0008585118921473622,
0.00026910624001175165,
0.00004346995774540119
] |
en
| 0.999996 |
In considering the future development and deployment of the LDNS active interrogation concept demonstrated in this study, it is pertinent to discuss the progression of its Technology Readiness Level (TRL). Specifically, the focus is on advancing to TRL 5, which involves “pre-prototype testing in a simulated environment in the laboratory.” To construct a pre-prototype, one must first define the specific mission to derive the necessary user requirements. A particular or representative application must be selected, contingent upon funding for research and development by a committed sponsor. This holds true not only for an LDNS but also for neutron sources driven by conventional accelerators, especially within the context of fissile material detection. For instance, the design of the detector system would vary significantly depending on whether the object under interrogation is the size of a suitcase, a radioactive-waste drum, or a semitrailer. Correspondingly, the required D-beam properties would be different as well.
|
39753631_p36
|
39753631
|
For further work towards a production system
| 3.572018 |
biomedical
|
Study
|
[
0.7078653573989868,
0.0007209375035017729,
0.29141369462013245
] |
[
0.9029946327209473,
0.0952250063419342,
0.0015390169573947787,
0.00024130864767357707
] |
en
| 0.999996 |
Advancing to TRL 5 for most applications involving LDNS that we envision will necessitate the development of high-repetition-rate lasers and compatible target systems. This requirement is particularly critical for active interrogation applications that can demand high throughput and substantial neutron doses. The criteria to determine what constitutes ‘suitable’ equipment may differ among researchers and sponsors; nonetheless, we provide our brief evaluations here to inform and guide the broader enabling research in this field.
|
39753631_p37
|
39753631
|
For further work towards a production system
| 2.703726 |
biomedical
|
Other
|
[
0.915312647819519,
0.000639484089333564,
0.084047831594944
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[
0.43074461817741394,
0.5541017651557922,
0.01453057024627924,
0.0006230351864360273
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en
| 0.999999 |
Our baseline concept for an LDNS is based on a laser-based D accelerator using the BOA mechanism based on the demonstrated level of performance and sufficient maturity. That requires sub-micron thick D-rich targets that can be replenished and repositioned accurately at the laser focal plane at a rate ranging from ~ 0.1–10 Hz, depending on specific application requirements. The present labor-intensive methodology for laboratory-based experiments involving mounting spin-coated plastic foils on a tiny holder that is manually mounted on the target holder in the chamber is clearly unsuitable. Promising techniques already demonstrated with high-power high-energy lasers are liquid crystals 48 , 49 and liquid cryogenic D 2 jets 50 . In the meantime, it is possible that alternative ion acceleration mechanisms such as collisionless-shock acceleration 14 in thin deuterated foam targets ~ 10 μm thickness may demonstrate equivalent performance for this application. In that case, dedicated research and development may be warranted to develop a foam-target system compatible with high repetition-rate operations.
|
39753631_p38
|
39753631
|
Laser targets
| 4.077703 |
biomedical
|
Study
|
[
0.9954997897148132,
0.0002849431475624442,
0.004215251188725233
] |
[
0.9985539317131042,
0.0010026542004197836,
0.0004007195238955319,
0.000042758536437759176
] |
en
| 0.999996 |
Although the CO 2 -based laser at the Brookhaven ATF merits continued attention, present state of the art performance in high-energy high-power short-pulse lasers and laser-driven ion acceleration is achieved with solid-state lasers, so we restrict our discussion to the latter. There is a clear need for transitioning away from general-purpose research laser facilities such as Trident, typically large in size and optimized for flexibility and access, towards more compact laser systems engineered and optimized with a view towards deployment in the field, with a much cheaper cost per shot and per J of laser energy. Improving the low repetition rate of lasers from the Trident generation is also important. While the technology of the low-energy high-repetition-rate front end on Trident was improved over time or even pioneered there (in the case of pulse cleaning), the low shot-repetition rate ~ 10 − 4 Hz was dictated by the cooling time of the glass laser amplifiers, which were based on 1970’s laser technology. Much newer technology for relevant PW-class sub kJ laser pulses (per beamline) has already been demonstrated at repetition-rates up to 10 Hz, which is sufficient for most envisioned applications. While the exact laser system suitable for a TRL 5 demonstration is not quite available for purchase off-the-shelf, we believe that it could be delivered as a custom system under contract with a national laboratory or one of the large laser companies worldwide. We justify our assessment below and outline what such a laser might look like.
|
39753631_p39
|
39753631
|
High repetition lasers
| 3.760895 |
biomedical
|
Study
|
[
0.8596137762069702,
0.0006300137611106038,
0.13975627720355988
] |
[
0.8085290193557739,
0.1798177808523178,
0.011296180076897144,
0.00035696601844392717
] |
en
| 0.999996 |
Two main relevant technology branches exist in high repetition-rate solid-state laser amplifiers: Ti: S crystals pumped by glass lasers and actively cooled glass disks. Ti: S amplifiers are the path to the highest powers and shortest pulse lengths (≈ 10–50 fs), limited to ~ 10 J per beamline at 800 nm wavelength by factors such as the availability of suitable crystals. They can operate at longer pulse lengths if required, which is a valuable feature for parametric studies in a multi-purpose facility. Intrinsically, they offer the tradeoff of higher intensity in shorter pulses in exchange for lower pulse energy and lower wall-plug efficiency, the latter in part because of the pumping step going from glass to Ti: S. On the other hand, state-of-the-art high repetition-rate glass disk amplifiers operate at 1 μm wavelength pumped by either flash lamps or diode arrays. They feature higher energy short-pulses per beam (> 100 J demonstrated at high repetition-rates) and higher energy efficiency (~ 10% if the diode is pumped). They are also intrinsically cheaper per J of delivered laser energy. However, they usually cannot support the bandwidth to go below sub-ps pulse duration, providing lower intensity for a given pulse energy. It is easy to see that both technologies have positive attributes, e.g., higher intensity is desirable (it may lead to a hotter D spectrum that exploits a higher nuclear-stripping cross-section in the neutron converter), but so is higher energy (it leads to more D ions). Therefore, the laser-pulse length is a key optimization parameter in LDNS-relevant D acceleration.
|
39753631_p40
|
39753631
|
High repetition lasers
| 4.286178 |
biomedical
|
Study
|
[
0.946410596370697,
0.0007779713487252593,
0.05281134322285652
] |
[
0.9764263033866882,
0.008446517400443554,
0.014982923865318298,
0.0001443654764443636
] |
en
| 0.999997 |
So far, the best ion acceleration results we have obtained have been based on BOA with sub-ps laser pulses. The ion dynamics of target-plasma disassembly that limit ion acceleration are ~ sub-ps. For a given laser-pulse energy, optimal target thickness decreases with shorter pulses 51 , which makes them more susceptible to destruction by pre-pulse, therefore increasing the pulse cleaning requirements possibly beyond the state of the art. A simple optimization for this regime (including laser cost) yields sub-ps optimum laser-pulse duration 21 . Consistent with this assessment, ion acceleration results from the highest energy Ti: S laser facilities have not matched those in glass-laser facilities. Therefore, while we strongly encourage continued research on ion acceleration with ~ fs laser pulses, at present glass lasers would be the right choice for reaching TRL 5 quickly with least risk. Within that branch, the application-specific requirements would determine whether ~ 1/minute repetition-rates (i.e., flash-lamp-pumped amplifiers) are sufficient or 10 Hz (i.e., diode-pumped amplifiers) are necessary.
|
39753631_p41
|
39753631
|
High repetition lasers
| 4.152997 |
biomedical
|
Study
|
[
0.9986856579780579,
0.00024014154041651636,
0.0010742120211943984
] |
[
0.9990666508674622,
0.0003639620263129473,
0.0005262926570139825,
0.00004306720074964687
] |
en
| 0.999996 |
We highlight two existing modern LDNS-relevant laser facilities that demonstrate the existence of sufficiently mature laser technology. Such lasers include the Penelope PW laser 52 with diode-pumped glass amplifiers at the Helmholtz-Zentrum Dresden-Rossendorf (150 J in 0.15 ps at 1 Hz repetition-rate), and the L4 ATON4 laser beamline at ELI Beamlines (Prague) 53 featuring flash-lamp-pumped glass amplifiers, which operates in 1 PW (150 J in 0.15 ps) and 10 PW (1.5 kJ in 0.15 ps) configurations at 1 shot/minute. As we explain below, a modern glass laser in the 1 PW class can be engineered to have a compact footprint suitable for deployment in the field to drive a LDNS. With similar pulse cleaning technology in the front end as the 200 TW Trident, it should be capable of driving a higher neutron dose/J by virtue of a higher possible intensity. But, even with the same efficiency, 1 PW should be adequate for most active interrogation applications we envision, likely with a single shot. Therefore, experiments at either L4 1 PW or Penelope should be strongly considered for further development of LDNS-based active interrogation, provided the demonstrated laser contrast is high enough and that regulatory hurdles relating to neutron generation and the interrogated sample can be overcome. Such a successful test at those facilities, if otherwise the rest of the hardware is a reasonably close prototype of a fieldable system, might be judged to be a TRL 5 demonstration.
|
39753631_p42
|
39753631
|
High repetition lasers
| 4.164416 |
biomedical
|
Study
|
[
0.9911651015281677,
0.0003160974883940071,
0.008518788032233715
] |
[
0.9904764890670776,
0.004129221197217703,
0.005310696549713612,
0.00008354507008334622
] |
en
| 0.999996 |
A simplified version of the HAPLS laser 54 (10 Hz repetition rate) built by LLNL for the L3 beamline at ELI Beamlines (Prague) is an attractive design basis for an LDNS prototype in a fieldable system. The footprint of the whole HAPLS laser is 4.6-by-17 m, plus 4 square meters for the laser pulse compressor, which represents an upper bound of what would be needed. HAPLS consists of two interconnected Livermore-designed laser systems: a diode-pumped, solid-state Nd: glass laser that pumps the second system, a Ti: S 30 fs, 30 J short-pulse laser. The pump laser is frequency doubled to deliver 200 J of energy at a repetition rate of 10 Hz. HAPLS has surpassed a much higher bar than would be needed for TRL 5. The design simplification to be considered is feeding a cleaned sub-ps pulse to a stretcher, then directly to the HAPLS glass pump amplifier, then finally to the pulse compressor back to sub-ps. Such a simplified design dispenses with all the unneeded fs-pulse and frequency-doubling hardware, the latter increasing the net amplifier energy output beyond 200 J, thus tripling the Trident pulse energy. So, in principle, a 1-second, 10-shot burst from such a modified-HAPLS laser driving a LDNS would have allowed us to perform the same measurement reported in this paper with at least 30x the neutron yield, probably more if the higher intensity capability (from higher pulse energy) were exploited.
|
39753631_p43
|
39753631
|
High repetition lasers
| 4.199391 |
biomedical
|
Study
|
[
0.9693608283996582,
0.0005034743226133287,
0.030135665088891983
] |
[
0.9937414526939392,
0.005844690836966038,
0.00033696411992423236,
0.00007692055078223348
] |
en
| 0.999996 |
From the neutron detection perspective, potential solutions include traditional 3 He-based systems but also alternatives such as scintillation detectors 32 , 34 , 36 , 37 , 40 . Scintillation detectors offer improved response times (both recovery and counting rate), a potential advantage with an LDNS pulse system. Excellent gamma-ray rejection will always be a prominent consideration. Recent developments in position-sensitive detectors based on 6 Li scintillation composite technology provide spatial resolution for locating special nuclear material accurately 55 . Continued research and development will be crucial to realizing the full potential of LDNS-based active interrogation systems with detector selection depending on application specific goals, commercial and other considerations (reliability, stability, supply change, disposal costs etc.).
|
39753631_p44
|
39753631
|
Detections solutions
| 3.947217 |
biomedical
|
Study
|
[
0.9491510987281799,
0.0004453688161447644,
0.05040356516838074
] |
[
0.8187392354011536,
0.10186279565095901,
0.0790567621588707,
0.0003411757934372872
] |
en
| 0.999997 |
The Los Alamos National Laboratory experiments reported here illustrate the potential of a high-intensity, laser-driven neutron source for active interrogation. The data obtained mark a significant milestone, providing the first empirical validation of single-pulse laser-driven neutron interrogation of special nuclear materials through delayed neutron detection. The ability to unequivocally detect the presence of fissile material and the consistency of the results demonstrate this technology’s practical feasibility.
|
39753631_p45
|
39753631
|
Conclusions
| 3.433041 |
biomedical
|
Study
|
[
0.8660658597946167,
0.0006338112871162593,
0.133300319314003
] |
[
0.970030665397644,
0.029092935845255852,
0.0006918933358974755,
0.00018449599156156182
] |
en
| 0.999998 |
This successful demonstration of active interrogation with LDNS signifies a progression of the technology from a theoretical concept to a validated laboratory proof-of-concept. It sets the stage for future advancements in this field, potentially revolutionizing the way we detect and handle fissile materials.
|
39753631_p46
|
39753631
|
Conclusions
| 2.192163 |
biomedical
|
Other
|
[
0.9019888043403625,
0.001270510139875114,
0.09674067050218582
] |
[
0.13135842978954315,
0.863143801689148,
0.004561614245176315,
0.0009361021220684052
] |
en
| 0.999997 |
During the experimental campaign, as a result of several refinements, we achieved the highest LDNS yield yet reported, predominant forward-directed yield per solid angle, to drive the D-ion beam that, in turn, drives the neutron source. It is essential to emphasize the significance of these results for both the non-destructive assay and related experimental physics communities and theoretical plasma physics. The data presented in the paper offer an opportunity for theoretical physicists to validate and refine their plasma models and simulations, as well as for the designers of next-generation laser-driven neutron sources to develop platforms for practical applications.
|
39753631_p47
|
39753631
|
Conclusions
| 3.905885 |
biomedical
|
Study
|
[
0.9935927987098694,
0.0002124841557815671,
0.006194747518748045
] |
[
0.9898695349693298,
0.009451347403228283,
0.0005620462470687926,
0.00011699295282596722
] |
en
| 0.999997 |
This novel capability shows considerable promise for a wide range of applications in the realm of active interrogation, including high-throughput interrogation of transport containers at ports, treaty verification applications (nuclear warhead and nuclear material signatures), assay of radioactive debris, stockpile stewardship and certification, and spent nuclear fuel assay at storage facilities and in casks. The potential of LDNS for further applications and extensions to other areas of use is vast, including their use as alternatives with lower regulatory burden and cost than nuclear reactors or conventional accelerators for neutron production at universities. Areas such as basic material science research, industrial testing and in situ maintenance (e.g., jet turbine blades, bridge components) would benefit significantly from LDNS. Additionally, the high intensity and prevalent directionality of the laser-driven neutron pulse make it suitable for pulsed radiography of static and dynamic experiments 14 , 23 , 56 .
|
39753631_p48
|
39753631
|
Conclusions
| 3.818797 |
biomedical
|
Study
|
[
0.9379593729972839,
0.0004897415055893362,
0.06155091151595116
] |
[
0.5397202372550964,
0.33943578600883484,
0.12014881521463394,
0.00069515808718279
] |
en
| 0.999997 |
The technology and science of LDNS are currently progressing rapidly, and we anticipate the availability of mobile sources with automated target changers, optimized repetition rates, and tailored neutron energy spectra and directionality to meet the increasing safety, security, and safeguards needs.
|
39753631_p49
|
39753631
|
Conclusions
| 1.78849 |
biomedical
|
Other
|
[
0.8001866340637207,
0.0017785798991099,
0.19803480803966522
] |
[
0.010600542649626732,
0.9883003234863281,
0.0007923672092147171,
0.0003068318183068186
] |
en
| 0.999998 |
Both of the AWCCs used in the experiment were thoroughly characterized prior to commencement of the laser-driven interrogation measurements to assure comparable performance. Count rates of cps and cps were obtained in thermal mode for the same reference 252 Cf spontaneous fission sealed neutron source in the master (assay) and reference (background) counters, respectively and confirmed better than 1% agreement in efficiency between the two detectors.
|
39753631_p50
|
39753631
|
Neutron counters
| 4.045728 |
biomedical
|
Study
|
[
0.9985645413398743,
0.00021523272152990103,
0.0012202447978779674
] |
[
0.9992824196815491,
0.00047191299381665885,
0.00020275675342418253,
0.00004298433486837894
] |
en
| 0.999996 |
To obtain integral delayed neutron counts directly from the interrogated nuclear material a background neutron contribution had to be removed from the detected signal. This was performed using the data from the reference AWCC to account for the contribution of 9 Li delayed neutron component. Note that in the very short times following the interrogating pulse (up to ~ 1s), the ambient neutron background is affected by a contribution of 9 Li delayed neutrons produced through the interactions in the Be converter, and this contribution needs to be considered to ensure accurate background subtraction. The neutron background from the reference AWCC does not necessarily need to be equivalent to the background measured in the master AWCC with the nuclear material (slightly different location of each detector, differences in signal processing electronics). Therefore, the neutron background from the reference AWCC was scaled to match the AWCC used for nuclear material measurements. This was accomplished by extracting ambient background from the AWCC with nuclear material during the period immediately before the interrogating pulse. This period provides the best estimate of the ambient neutron background for this AWCC. The ambient neutron background from the reference AWCC was measured in the similar time period and the ratio of these two measurements provided the necessary scaling factor to correct for any residual differences between the reference AWCC and the AWCC with nuclear material. The signal from the reference AWCC for each shot is scaled by the appropriate scaling factor. The scaling factor was determined on a shot-by-shot basis.
|
39753631_p51
|
39753631
|
Neutron counters
| 4.200698 |
biomedical
|
Study
|
[
0.9983103275299072,
0.0002891404728870839,
0.0014005214907228947
] |
[
0.9994224309921265,
0.0003620038914959878,
0.00017842200759332627,
0.000037085424992255867
] |
en
| 0.999997 |
Bubble detectors produced by Bubble Technology Industries (BTI) 57 were used to measure the neutron dose. Multiple bubble detectors with three different bubble-to-dose sensitivities (in the range 5–19 bubble/mrem) were placed at 0° with respect to the laser propagation direction at the chamber’s exit flange. The neutron doses reported in the paper are the weighted average values of the detectors, and the uncertainty is the associated standard deviation of the values.
|
39753631_p52
|
39753631
|
Neutron bubble detectors
| 3.908068 |
biomedical
|
Study
|
[
0.9956303834915161,
0.00024743389803916216,
0.004122219979763031
] |
[
0.998931348323822,
0.0008436336065642536,
0.00017301482148468494,
0.00005203037289902568
] |
en
| 0.999998 |
The neutron yield in neutron/sr is obtained as follows. The neutron dose in mrem was obtained from bubble detectors, and the neutron energy distribution was obtained from the nTOF measurements . The neutron yield in neutron/cm 2 at the bubble detector location is calculated by multiplying neutron dose for the integral, in the neutron energy range, of the ratio between the normalized energy spectrum and the neutron sensitivity 11 , 57 , 58 , we then convert the yield to neutron/sr by knowing the distance of the bubble detector to the neutron source (104 cm for the exit flange on axis) 12 . Bubble detector neutron sensitivity data from the cited literature were linearly interpolated to be used in the calculation.
|
39753631_p53
|
39753631
|
Neutron yield determination
| 4.16243 |
biomedical
|
Study
|
[
0.9980916380882263,
0.0001889127161120996,
0.001719363033771515
] |
[
0.9985315799713135,
0.0012168791145086288,
0.00020494768978096545,
0.00004657062527257949
] |
en
| 0.999999 |
A neutron time-of-flight (nTOF) detector system, based on a NE102 plastic scintillator (76.2 mm diameter, 18.2 mm thick) coupled to XP4362B with 6 dynodes Photonis PMT was positioned 6.2 m away from the Be converter along the central beam axis (the laser-propagation direction and the symmetry axis of the converter), outside the Trident building. Positioning the detector outside the building reduced the neutron scattering contributions into the scintillation detector significantly. The nTOF was operated in current mode and was connected to a fast oscilloscope. The nTOF spectrum consists of a digitized oscilloscope trace of the photomultiplier output. 102 mm of lead was placed in front of the detector to attenuate the X-ray flash associated with the pulse. A pure X-ray shot was produced in Trident, using a 238 U thin target instead of the CD target, to acquire the X-ray response function of the neutron detector system for subtraction to obtain a neutron–only spectrum. The neutron energy spectrum was then unfolded by fitting the data with response vectors calculated using the MCNP-Polimi radiation transport code (using ENDF VII cross-section libraries) 59 . The response vectors were matched to the 20-ns nTOF bins in the experimental spectrum. The nTOF spectrum was described as a linear combination of overlapping response functions. All the recoil and reaction contributions in the scintillation material (proton, alpha, carbon) and the corresponding light output contributions to the signal were calculated as needed for the neutron response function of the detector.
|
39753631_p54
|
39753631
|
Neutron time-of-flight (nTOF)
| 4.275718 |
biomedical
|
Study
|
[
0.99859219789505,
0.0003217877238057554,
0.0010860082693397999
] |
[
0.9991699457168579,
0.0004964786930941045,
0.0002771726285573095,
0.000056425593356834725
] |
en
| 0.999997 |
Below is the link to the electronic supplementary material.
|
39753631_p55
|
39753631
|
Electronic supplementary material
| 0.976989 |
other
|
Other
|
[
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] |
[
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0.0016435992438346148,
0.0009437996195629239
] |
en
| 0.999998 |
Supplementary Material 1
|
39753631_p56
|
39753631
|
Electronic supplementary material
| 1.299746 |
biomedical
|
Other
|
[
0.8858742117881775,
0.0030190381221473217,
0.11110673099756241
] |
[
0.1275506466627121,
0.8626377582550049,
0.0076365359127521515,
0.0021750046871602535
] |
en
| 0.999998 |
Alzheimer’s disease (AD) can be divided into at least two entities—the sporadic and the familial. While it is quite clear that the genetic variant results from mutations or duplications in a limited number of genes (the amyloid precursor protein gene and the gamma-secretase complex genes PS1 and 2 1 ), the underlying mechanisms of the sporadic variant are still not fully understood. However, there is a consensus in the field that a failure or overshooting of immune reactivity is an important contributor to pathogenesis 2 . Brain immune cells respond to oligomeric and deposited Aβ peptides, hallmarks of the disease, in a time-dependent manner: early contact induces phagocytic activity and activation 3 , 4 , while prolonged exposure may lead to senescence or overshooting activity that further damages the surrounding cells 5 – 7 . Aβ can bind directly to several receptors on the microglial surface. Triggering receptor expressed on myeloid cells 2 (TREM2), toll-like receptors (TLRs), and formyl peptide receptors (FPRs) can recognize extracellular Aβ as a ligand and other microglial proteins such as CD36 further contribute to the Aβ-evoked cellular response cascades (summarized in ref. 8 ). However, in addition to exposure time, there is also a spatial component, as few reports have described that cells close to deleterious peptide deposits respond differently from cells further away. For example, Olmedillas and colleagues 9 used three virally transduced fluorophores to show that in the APPPS1 AD mouse model, the vicinity of plaques ( < 50 µm) contained a higher degree of migration-, death- and division-events of microglia as compared to behavior of all microglia in the AD brain or in the brains of wild type mice. They therefore identified the vicinity of plaques as hot spots of microglial turnover. By dividing the area around plaques into five concentric rings and performing spatial transcriptomics in AppNL-G-F AD model mice, it was shown that Apoe and CtsI are expressed exclusively in microglia within the inner ring (within 10 µm), which the authors defined as the amyloid plaque niche 10 . Other methodological approaches, such as laser capture microdissection, used for spatial studies, cannot achieve single cell resolution in most cases 11 . In addition, the use of a high-energy laser beam can damage the region of interest and introduce artefacts into the analytical processes. This may be even more relevant when attempting to sample subcellular structures with dimensions of only a few µm. Alternatively, organelles can be harvested by differential centrifugation or immunocapture after cell or tissue lysis 12 , 13 . These methods are time-consuming, which can lead to degradation of organelle function, and do not allow selection of samples from living cells representing a particular state (e.g. activated phenotype).
|
39753747_p0
|
39753747
|
Introduction
| 4.729478 |
biomedical
|
Study
|
[
0.9986743927001953,
0.0007565952255390584,
0.000569046416785568
] |
[
0.9519643783569336,
0.0009792428463697433,
0.04654358699917793,
0.0005128123448230326
] |
en
| 0.999996 |
Single cell nanobiopsy methods have been established to collect small amounts of cellular material from individual cells, for example, based on scanning ion conductance microscopy 14 . The used syringes or nanopipettes comprise a diameter in the range of 150 to a few hundred nanometers and by this, prevent disruption of the cells (e.g 15 .). With such methods, pico- or femtoliters can be aspirated and longitudinal measures are feasible. Nevertheless, these custom-built devices need a high degree of technical expertize and sometimes lack supplies that can sustain cellular viability (e.g. extraction procedure performed at room temperature 15 ). Moreover, some functional assays may require larger sampling volumes.
|
39753747_p1
|
39753747
|
Introduction
| 4.029511 |
biomedical
|
Study
|
[
0.9994016885757446,
0.00012057590356562287,
0.0004777142603415996
] |
[
0.9724408984184265,
0.012268321588635445,
0.015131738968193531,
0.00015900666767265648
] |
en
| 0.999995 |
To overcome these limitations, we tested the Single Cellome™ System SS2000 (Yokogawa), which promises to collect subcellular material by robotic aspiration through glass capillaries combined with confocal microscopy allowing the visualization of amyloid deposits and mitochondria or nuclei in each cell before collecting organelles. We were able to demonstrate the applicability of the system by obtaining samples with the respective characteristics such as Lumitracker Mito Red-related fluorescence only in mitochondrial samples or specific detection of subcellular mRNA markers. Moreover, mitochondrial samples gained from close vicinity of Aβ deposits showed reduced oxygen consumption and a differential expression pattern compared to those collected in greater distance. Fig. 1 Subcellular compartment sampling of SIM-A9 cells. Cells were seeded at a density of 20,000 cells per well and incubated for 24 h. After staining with Lumitracker Mito Red and DAPI, cells were used for sampling of mitochondrial or nucleic material via tips with a diameter of 3 µm using the Single Cellome™ System SS2000. a A schematic of the instrument is shown. b Example images of cells before and after sampling are shown. The position of the nucleus is indicated by a dashed line, as is the area of aspirated material after sampling, which is also indicated by an arrowhead. Scale bar: 10 µm. c An example image of a tip with aspirated Lumitracker Mito Red-stained mitochondrial material is shown. A magnification of the part of the tip with extracted material (white arrowheads) is shown in the black box. d Both mitochondrial and nuclear material was extracted. Material from three cells was pooled to obtain one sample and fluorescence measured using 540 nm Exc/ 580 nm Em (red fl.) or 485 nm Exc/ 520 nm Em (blue fl.). Means of corrected relative fluorescence intensity (RFU) adjusted for background measurements (PBS) and SD are shown. e After fluorescence measurement, the subcellular compartment material was lysed and subjected to one-step RT-qPCR using primer pairs for Atp8 (mitochondrial gene product) or Gapdh (nucleic gene product). The values obtained for the mean of the nucleic samples were set to 100%. f Oxygen consumption was assessed immediately after sampling, with sample buffer as a negative control. Consumption was calculated as slope per minute (RFU/min) after correction for the slope of the negative control. Two independent experiments ( n = 4 each) were performed. g Samples from the mitochondrial compartment with or without complex III inhibitor (antimycin A) were analyzed for oxygen consumption rate. Two independent experiments ( n = 3 each) were performed. Unpaired two-tailed t-tests were used to compare the measurements obtained with Welch correction if needed (*** p < 0.001; ** p < 0.01; * p < 0.05).
|
39753747_p2
|
39753747
|
Introduction
| 4.215569 |
biomedical
|
Study
|
[
0.9993569254875183,
0.0004315910045988858,
0.00021142669720575213
] |
[
0.9992725253105164,
0.00024563458282500505,
0.0003970483667217195,
0.00008479558164253831
] |
en
| 0.999996 |
SIM-A9 cells were plated in a 96-well format and mitochondrial and nuclear material was collected using organelle-specific stains (Lumitracker Mito Red and DAPI). To assess the accuracy of mitochondrial and nuclear sampling, we visually followed the extraction process and measured organelle-specific dye fluorescence as well as marker gene expression in the collected material . For example, mitochondrial material showed a significantly lower blue fluorescence signal compared to nucleic material , indicating the comparatively low but still DAPI-stainable DNA content in these organelles 16 . Expression of the nucleic marker gene Glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) was found exclusively in the nucleus with no measurable signals in mitochondrial material , whereas the mitochondrially encoded ATP synthase membrane subunit 8 ( mt-Atp8 ) was almost exclusively expressed in mitochondria-derived samples . To assess the functionality of the isolated mitochondrial material, we analyzed oxygen consumption and obtained a tenfold higher consumption rate in mitochondrial material compared to nucleic material . This oxygen consumption was significantly inhibited by the administration of a complex III inhibitor .
|
39753747_p3
|
39753747
|
Accuracy of organelle sampling
| 4.218853 |
biomedical
|
Study
|
[
0.999460756778717,
0.00031060699257068336,
0.00022859210730530322
] |
[
0.9993196725845337,
0.00016749727365095168,
0.0004413135757204145,
0.00007149992597987875
] |
en
| 0.999997 |
Having confirmed selective organelle sampling, we exposed SIM-A9 cells to Aβ-deposits to explore the applicability of subcellular sampling in a pathological environment. Cells were categorized according to their distance from a deposit . A scrambled peptide (Sc, lacking biological activity) was used as a control in addition to mere solvent administration control. Measurement of oxygen consumption in extracted mitochondria showed that only mitochondria from cells close to toxic Aβ-deposits and not to scrambled peptide aggregates showed a reduced respiration rate . Fig. 2 Spatial collection of organelle material and analysis of mitochondrial respiration from Aβ-deposit-exposed SIM-A9 cells. a Cells were exposed to pre-aggregated peptides or solvent control (PBS). Sample images were taken after 24 h. Scale bar: 25 µm. Peptide deposits have been artificially stained black for better visualization . b After 24 h exposure, mitochondrial and nucleic material was collected from cells in close proximity (close, C) to peptide deposits or further away (far, F) after staining of mitochondria as indicated in the scheme. Far and near distances were defined using the half-maximum distance between two neighboring peptide deposits, selecting the 10% distance near the deposit and the 90-100% of the radius away from the deposit. Distances to the edge of the deposits are expressed as mean + SD ( n = 36 per group, values derived from all samples aspirated). Brown–Forsythe ANOVA test with Dunnett’s T3 multiple comparisons test was performed for statistical analysis (*** p < 0.001). The box plot indicates min and max. c Oxygen consumption rate was measured in freshly sampled mitochondrial material (ScAβ: scrambled peptide; Aβ: Aβ peptide). Four independent experiments were performed ( n = 4). Each sample consisted of material from three cells ejected in 6 µL buffer. Random samples were taken for the PBS control. Statistical analysis was performed by one-way ANOVA followed by Sidak’s multiple comparison post-test (* p < 0.05; ns, p > 0.05).
|
39753747_p4
|
39753747
|
Assessment of gene expression pattern in mitochondria and nuclei in dependency from distance to peptide deposits
| 4.159174 |
biomedical
|
Study
|
[
0.9993999004364014,
0.0003758574603125453,
0.0002242672344436869
] |
[
0.9993869066238403,
0.00016784659237600863,
0.0003673417668323964,
0.00007798449223628268
] |
en
| 0.999996 |
Nuclear and mitochondrial material was subjected to organelle-specific RT-qPCR , taking into account genes whose expression has previously been shown in the literature to be affected by amyloid plaque exposure. Gapdh and mt-Atp8 showed no changes upon exposure to either scrambled peptide deposits or Aβ aggregates, irrespective of the distance to the deposited material. Fig. 3 Gene expression patterning in nuclei and mitochondria from Aβ-deposit-exposed SIM-A9 cells in relation to deposit proximity. a To assess the effect of proximity of microglial cells to peptide deposits, eight genes were selected and their gene products analyzed in either nucleic or mitochondrial material sampled as described in Fig. 2 . Three are nucleic encoded and affect mitochondrial homeostasis (green), two are mitochondrially encoded (red) and three genes (yellow) are not fundamentally related to mitochondrial function but have been described to respond to proximity to Aβ deposits. Gapdh and mt-Atp8 served as housekeeping controls. b Quantification of mitochondrial and nucleic mRNAs and mt-DNA. Mean of uncorrected threshold cycles are shown as a heat map. c Respective mt-RNAs were quantified from mitochondrial samples and related to mt-Atp8. The amount of mt-DNA was normalized to Gapdh mRNA values. d , e Nucleic mRNAs were quantified from nucleic samples and related to Gapdh in two independent experiments ( n = 3 each; single values were excluded if outlier analysis indicated an outlier (ROUT, Q = 1%)). Values are expressed as mean + SD. Statistical analysis was performed by one-way ANOVA followed by Sidak’s multiple comparison post-test (*** p < 0.001; ** p < 0.01; * p < 0.05). f Summary of findings regarding mitochondrially and nucleic encoded genes in relation to proximity of amyloid deposits.
|
39753747_p5
|
39753747
|
Assessment of gene expression pattern in mitochondria and nuclei in dependency from distance to peptide deposits
| 4.180176 |
biomedical
|
Study
|
[
0.9994543194770813,
0.00033239260665141046,
0.00021322259271983057
] |
[
0.999273955821991,
0.00017653133545536548,
0.00047253206139430404,
0.00007695654494455084
] |
en
| 0.999999 |
Most of the investigated gene products were only differentially regulated in cells in close proximity to Aβ aggregates and were not affected by the scrambled peptide administration. For example, mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (mt-Nd1) and cytochrome c oxidase subunit 1 (mt-Co1) were both reduced in mitochondria derived from cells close to active peptide deposits . Additionally, mt-DNA amount was increased in samples derived from mitochondria in close proximity to Aβ peptide deposits . The peroxisome proliferator-activated receptor gamma coactivator 1α (Pgc1α) mRNA was strongly reduced, regardless of whether the nucleic material was obtained far (F) or close (C) from the Aβ-deposits .
|
39753747_p6
|
39753747
|
Assessment of gene expression pattern in mitochondria and nuclei in dependency from distance to peptide deposits
| 4.147295 |
biomedical
|
Study
|
[
0.9995142221450806,
0.0002448850718792528,
0.00024087197380140424
] |
[
0.9993860721588135,
0.00019940985657740384,
0.0003533813578542322,
0.00006109234527684748
] |
en
| 0.999994 |
Next, we assessed expression of nucleic encoded genes that are not fundamentally involved in mitochondrial maintenance and homeostasis but have been found to be responsive to Aβ and are pivotal for microglial function: Itgax (integrin subunit alpha X; codes for CD11c), inositol polyphosphate-5-phosphatase (Inpp5d), and cyclooxygenase 2 (Cox2, Ptgs2). Itgax mRNA increased selectively in close proximity to deposits , while the other two gene products were increased independently from distance to deposit.
|
39753747_p7
|
39753747
|
Assessment of gene expression pattern in mitochondria and nuclei in dependency from distance to peptide deposits
| 4.107961 |
biomedical
|
Study
|
[
0.9995181560516357,
0.00023004190006759018,
0.00025175471091642976
] |
[
0.9994733929634094,
0.00023627374321222305,
0.00023725911159999669,
0.00005309129483066499
] |
en
| 0.999996 |
In the area of single cell-based technology, it is mandatory to be able to also collect intact material from single cells and probably even more – subcellular regions. We here explored the Single Cellome™ System SS2000 to investigate local effects of Aβ deposits (hallmarks of AD) on microglial mitochondria. While the system offers carbon dioxide supply to be integrated, we chose the option to work with CO 2 -independent culture medium. This ascertained a stable viability of the cells during samplings conducted within one hour (allowing about nine to twelve samplings). The system offers a range of fluorescent labels to be used for identification of subcellular regions. In the here presented experiments, DAPI and Lumitracker Mito Red were applied. By titrating the concentration of the mitochondrial stain, a threshold was experienced that—while cells were still clearly visibly stained – did not allow the system to identify the fluorescence for sampling. However, this was in a range of 12.5–2.5% of the stain concentration suggested by the manufacturers of comparable mitochondrial stains (minimal usage of 12.5 nM instead of 500–100 nM). Using only 25 nM of the Lumitracker Mito Red stain was sufficient to identify and extract mitochondrial material. This is of importance because of the potential photosensitizing effects such stains have been reported to show when administered at higher or comparable concentrations 17 , 18 .
|
39753747_p8
|
39753747
|
Technical applicability of the subcellular sampling system
| 4.149022 |
biomedical
|
Study
|
[
0.9995236396789551,
0.000271900586085394,
0.0002044748980551958
] |
[
0.9994035959243774,
0.00020896845671813935,
0.0003283487167209387,
0.00005909546962357126
] |
en
| 0.999997 |
Mitochondria can make up approximately 35% of the cell volume 19 ; however, due to physiological conditions or pharmacological treatment, this volume can even increase up to 40-50% 20 . When calculating the amount of mitochondrial material aspirated from SIM-A9 cells, approximately 25% of the cellular content was sampled, taking into account the measured cell diameter and assuming a perfect spherical shape of the cell. The finding that red fluorescence was exclusively found in mitochondrial samples and not in nucleic material, underlines purity of the sample. However, a certain signal derived from excitation for DAPI staining was observed in mitochondrial material. This might hint at cross-contamination with nucleic material but it might also derive from staining of mitochondrial nucleoids as has been described before 16 , 21 . This is supported by the fact that only minor signals could be observed for the nucleus-encoded gene Gapdh in mitochondrial lysates while the signal for mt-Atp8 was rather strong. This finding for Gapdh could be also confirmed on the protein level by Western blotting . However, contamination of the collected nuclear or mitochondrial material with other cellular material cannot be fully excluded. We tried to detect Calnexin as an endoplasmic reticulum (ER) marker in both fractions and did observe a respective band of about 90 kDa . This indicates that co-collection of ER material cannot be avoided, probably due to the wide and branched distribution of this organelle throughout the cell. Nevertheless, oxygen consumption could also only be detected in mitochondrial samples and be blocked to a high extent by antimycin A. Oxygen consumption in addition points at preserved functionality of the aspirated mitochondria; however, it is difficult to estimate the exact grade of functionality. More traditional methods for isolation of mitochondria via centrifugation take much longer than the direct sampling (e.g., 90 min 22 ) and thus would not allow a side-by-side comparison of both methods with material derived from the same cell samples. Nevertheless, in sum our data revealed that sampling of mitochondria was feasible and delivered material with both, purity (in regard to nucleic contamination) and functionality, and comparable content of biological material. A limitation of our study is that we did not assess cellular viability after the extraction procedure. However, as the capillary diameter is much larger with 3 µm as compared to nanoscale extraction tools (openings in the range of 50–400 nm 23 ,), we assume that the cells were damaged due to severe membrane disruption. This would not allow longitudinal sampling from one cell as has been demonstrated for nanoscale tools, which only lead to minimal disruptions (e.g 24 : puncturing of nucleus: >60% living cells). Nevertheless, using the microscale extraction method that we present here might also bear advantages: (1) mitochondria (for example, rodent smooth muscle cell mitochondria: 0.90 ± 0.20 µm in length and 0.63 ± 0.12 µm in width 25 ) might be affected by flow stress due to too small pipette diameter. (2) Sampling of multiple organelles might better allow conducting functional assays such as the oxygen consumption assay, while for high sensitivity assays with amplification steps, such as RT-qPCR, material in the pL range from nanoscale isolation is sufficient. (3) The extraction chamber as well as the sample collection chamber of the commercial instrument are temperate (37 and 4°C). This allows keeping the cells in an optimal state during the process and immediately protects the samples after extraction. This might not always be the case in custom-built devices. For example, Chen et al. 15 . described that while cells were kept at 37°C before and after the extraction process, the extraction via Live-seq itself was performed at room temperature.
|
39753747_p9
|
39753747
|
Technical applicability of the subcellular sampling system
| 4.364582 |
biomedical
|
Study
|
[
0.9992425441741943,
0.0004745328624267131,
0.0002829546865541488
] |
[
0.9991212487220764,
0.00020436763588804752,
0.0005698073073290288,
0.00010455033770995215
] |
en
| 0.999995 |
After confirming that characteristic subcellular material could be collected, SIM-A9 cells were exposed to Aβ-based plaque-like deposits 26 . The scrambled peptides which are biologically inactive, surprisingly also tended to built deposits after seven days of incubation. It was reported that while a prediction algorithm did not allocate amyloidogenic regions to either scrambled or reverse variants of Aβ, the peptides nevertheless formed fibrils 27 . However, ThT fluorescent signal was lower at 48 h of incubation for both as compared to wild type Aβ and – even more importantly – toxic effects on rat primary rat neurons were only minimal when compared to the wild type peptide after seven days of incubation. This is confirmed by the here presented ThT assay data and toxicity measures. One limitation of our study might be that we used aggregates of pure Aβ peptides. Plaques occurring in the brain of patients, however, comprise a wide variety of other proteins that also lead to distinguishable plaque subtypes. Complement and inflammatory response proteins as well as proteins from the lipid transport and metabolism section, proteins involved in blood coagulation and hemostasis and many others can be found – depending on the methods used for analysis (reviewed in ref. 28 ). For example, attached fibrinogen seems to be directly involved in microglia activation via fibrinogen-Cd11b binding as shown in 5xFAD AD model mice 29 . Thus, the here used peptide deposits only provide a simplified model of plaques to demonstrate feasibility of combined subcellular sampling and spatial resolution in response to Aβ-deposits. In close vicinity to the deposits, however, we found that oxygen consumption was reduced in mitochondria. In 5xFAD mice, for example, already at the age of two months progressive alterations of mitochondrial morphology and function such as oxygen consumption was observed in hippocampus 30 . This is coincident with timing of plaque deposition in the rather aggressive disease model and underlines the early impact Aβ deposits have on energy homeostasis in the brain. Microglia per se have a high energy-demand to maintain immune functions. In the resting state, they rely on OXPHOS to gain ATP; however, they can switch to anaerobic glucose usage in case of being stressed or activated 31 , 32 .This would coincide with reduced oxygen consumption as seen in the SIM-A9 cell-derived mitochondria in contact to Aβ deposits.
|
39753747_p10
|
39753747
|
Spatial resolution of Aβ-deposit evoked damage in microglia
| 4.418814 |
biomedical
|
Study
|
[
0.9993343949317932,
0.000400625605834648,
0.0002649585367180407
] |
[
0.998785674571991,
0.00024925492471084,
0.0008462710538879037,
0.00011881397222168744
] |
en
| 0.999994 |
Expression of mitochondrially encoded genes was affected only in cells in close proximity to the Aβ deposits. Such downregulation of mt-Nd1 has been previously described in BV-2 cells exposed to Aβ oligomers 33 with an effect size of 40%. We observed a much stronger reduction (70%), which could be explained at least in part by local extraction. The mt-Co1 , which was reduced by more than 80%, was also shown to be reduced in cells after Aβ administration, as were nucleic encoded mitochondrial transcription factor A ( Tfam ) mRNA levels 34 . The Pgc1α -mRNA was strongly reduced, regardless of whether the nucleic material was obtained far or close from the Aβ-deposits. This is consistent with the finding that a reduction in the expression of this gene is an event that occurs, for example, at an early age in AD mouse models 35 , indicating a rapid response that might not need the highly concentrated localized deposits and preceding mitochondrial damage. Finally, mt-DNA amount was increased in samples derived from mitochondria in close proximity to Aβ peptide deposits. Most reports indicate a reduction of mt-DNA in material derived from patients with AD or other neurodegenerative disorders 36 . However, defects in the fission machinery might also lead to a disturbed distribution of newly replicated mt-DNA nucleoids and subsequently to accumulation 37 . Moreover, the mt-DNA amount of a cell is determined by its energy demand and thus, the observed increase might be interpreted as an early attempt of the mitochondrial machinery to compensate for decreased energy provision resulting from altered gene expression.
|
39753747_p11
|
39753747
|
Locally affected gene expression pattern
| 4.444667 |
biomedical
|
Study
|
[
0.9993951320648193,
0.0003831730573438108,
0.0002216958673670888
] |
[
0.9987249970436096,
0.0002999464049935341,
0.0008443675469607115,
0.00013070525892544538
] |
en
| 0.999998 |
Concerning nucleic encoded genes that are not fundamentally involved in mitochondrial maintenance and homeostasis but have been found to be responsive to Aβ, CD11c has been identified as a key marker for disease-associated microglia (DAM) and to be expressed in proximity to plaque foci 38 . Inpp5d was found to correlate with plaque density in human brain and expressed in plaque-associated microglia in a previous study 39 ; however, distance to plaque was not taken into account. Cox-2 deficiency has been found recently to influence microglial density and morphology in mice 40 and treatment of BV-2 microglia with Aβ led to increase of Cox-2 expression via NFκB signaling 41 . Thus, a global upregulation of the latter two genes coincides with these reports.
|
39753747_p12
|
39753747
|
Locally affected gene expression pattern
| 4.252106 |
biomedical
|
Study
|
[
0.9995867609977722,
0.00018666191317606717,
0.00022660136164631695
] |
[
0.9992243051528931,
0.0002522400754969567,
0.0004606067668646574,
0.00006283502443693578
] |
en
| 0.999997 |
Our research demonstrates the feasibility of extracting intact, functional mitochondrial material using the Single Cellome™ System SS2000. This commercial system allowed to first visualize mitochondria in single cells using confocal microscopy and second to decipher mitochondrial properties and function. This is a huge improvement regarding other methods, where mitochondria need to be sampled from thousands to millions of cells or from bulk tissue.
|
39753747_p13
|
39753747
|
Conclusion
| 4.020912 |
biomedical
|
Study
|
[
0.9997001886367798,
0.00011956926755374297,
0.00018031620129477233
] |
[
0.9980295300483704,
0.0012359947431832552,
0.0006566056399606168,
0.00007780588930472732
] |
en
| 0.999995 |
By applying the system to an investigation of Aβ-deposit-derived changes in nucleic and mitochondrial gene expression in microglia, a clear distinction due to the relative positioning of the cells was found . In future, this system might be used for sampling other particle-shaped organelles and it might be considered that mitochondria that can be collected in such rapid, function-sparing, and defined manner might also serve as a valuable source for mitochondrial transfer experiments 42 . Keeping in mind that a recent exciting paper from the Picard group 43 shows distinct mitochondrial profiles in different brain areas, this method might allow digging even deeper in elucidating cell-specific mitochondrial function between neuronal populations, glia cells, and astrocytes. In addition, the system also could allow sampling selectively from cells containing pre-dominantly fragmented versus from such with fused mitochondria and exploring their differential gene expression.
|
39753747_p14
|
39753747
|
Conclusion
| 4.11575 |
biomedical
|
Study
|
[
0.9996676445007324,
0.0001383782655466348,
0.00019396643619984388
] |
[
0.9974754452705383,
0.0005248739616945386,
0.0019318602280691266,
0.00006780545663787052
] |
en
| 0.999997 |
Spontaneously immortalized mouse microglia (SIM-A9, BIOZOL Diagnostica Vertrieb GmbH, KER-END001) 44 were cultivated in 10 cm culture dishes (Sarstedt AG & Co. KG) in a CO 2 incubator (Forma™ STERICULT CO 2 Incubator, Thermo Fisher Scientific Inc.) at 37 °C, 95% humidity, and 5% CO 2 . The culture medium consisted of phenol red-free Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM:F-12), supplemented with 10% v/v heat-inactivated fetal bovine serum (Gibco®, Life Technologies), 5% v/v heat-inactivated donor horse serum (Gibco®, Life Technologies), 1% v/v Penicillin–Streptomycin (Sigma-Aldrich), and 1% v/v L-Glutamine (Sigma-Aldrich). Upon reaching 80% confluence, the cells were passaged. Therefore, the conditioned medium was aspirated, and the cells were washed with 5 ml of pre-heated phosphate-buffered saline (PBS, 37 °C). Subsequently, 2.5 ml of Trypsin/EDTA-solution (Sigma-Aldrich) were added for 5 min at 37 °C, 95% humidity, and 5% CO 2 . The trypsin reaction was halted by adding 7.5 ml of culture medium. Following resuspension, the cells were transferred to a 50 ml CELLSTAR®, BLUE SCREW CAP Tube (Greiner Bio-One International GmbH) and centrifuged in a Megafuge 1.0 R (Heraeus) for 6 min at 600xg. The cell supernatant was removed, and the cell pellet was suspended in 20 ml of culture medium. Finally, 10 ml of suspended cells were distributed evenly between two culture dishes. Work with the SIM-A9 cell culture was conducted within a sterile biosafety cabinet (MSC-Advantage™, Thermo Fisher Scientific Inc.). For experiments, aliquots from cell suspension following the termination of the trypsin reaction were diluted with PBS and cells counted using the Cell Scepter TM 3.0 (Merck KGaA). Twenty-thousand cells per well were seeded in black, glass bottom 96-well plates (Greiner Bio-One GmbH).
|
39753747_p15
|
39753747
|
SIM-A9 Cell Culture
| 4.255779 |
biomedical
|
Study
|
[
0.9992693066596985,
0.00047456618631258607,
0.00025612072204239666
] |
[
0.9974101185798645,
0.0019320532446727157,
0.0004996893112547696,
0.00015804429131094366
] |
en
| 0.999997 |
The 4’,6-diamidino-2-phenylindole stock solution (1 mg/mL) was diluted 1:2000 with 1×PBS and 50 μL were added per well. The plate was incubated in darkness for 10 min at 37 °C using the Thermomixer comfort (Eppendorf AG). Following incubation, the DAPI solution was aspirated, and the cells underwent two washing steps with 200 μL of 1×PBS each. Subsequently, staining with LumiTracker Mito Red CMXRos was carried out.
|
39753747_p16
|
39753747
|
Staining of Nuclei with DAPI
| 4.031339 |
biomedical
|
Study
|
[
0.9978219270706177,
0.001352806342765689,
0.00082523183664307
] |
[
0.6564010381698608,
0.341235488653183,
0.0013214168138802052,
0.0010421181796118617
] |
en
| 0.999997 |
The LumiTracker Mito Red CMXRos stock solution was diluted 1:40,000 in pre-warmed CO 2 -independent culture medium (Gibco®, Life Technologies). 50 μL of the prepared solution were added per well, followed by a 15 min incubation at 37 °C in the Single Cellome™ System 2000 (Yokogawa Electric Corporation; Tokyo, Japan). After incubation, organelle material extraction was performed.
|
39753747_p17
|
39753747
|
Staining of Mitochondria with LumiTracker Mito Red CMXRos
| 3.997692 |
biomedical
|
Study
|
[
0.9992430210113525,
0.0003712274774443358,
0.00038587491144426167
] |
[
0.9388065934181213,
0.060061320662498474,
0.000682795129250735,
0.00044927801354788244
] |
en
| 0.999997 |
For peptide aggregation, aliquots containing 40 µL of 350 µM of Aβ1-42 or scrambled Aβ1-42 in sterile 1xPBS were incubated at 37°C as described previously 26 . To induce aggregation, these aliquots were subjected to 50 pipette strokes using 20 µL filter-pipette tips. The pipetting regimen commenced immediately on day 1, followed by repetitions after 48 hours on day 3, and on day 6. After 168 hours (on day 7 from the start of incubation), the resulting deposits of Aβ1-42 or scrambled Aβ1-42 were utilized for cell culture experiments. Aggregate formation within the Aβ1-42 solution was confirmed by ThT assay and by assessing toxicity using Cell Titer Glo assay . Toxicity was probably not elicited by the larger fibrillary aggregates that could be visualized during extraction but by oligomeric forms 45 . Ten µL of aggregated peptide solution or of 1xPBS were added to the cells (in 90 µL culture medium volume). The added peptide preparation or solvent were carefully mixed with the cultivation medium by ten pipette strokes with 50 µL volume. The cells where subsequently cultivated for 24 h in an incubator (Forma™ STERICULT CO 2 Incubator, Thermo Fisher Scientific Inc.) at 37 °C, 95% humidity, and 5% CO 2 .
|
39753747_p18
|
39753747
|
Peptide Aggregation
| 4.188917 |
biomedical
|
Study
|
[
0.9993682503700256,
0.00045518495608121157,
0.000176538378582336
] |
[
0.9989191293716431,
0.0005584760801866651,
0.00041483205859549344,
0.00010753374954219908
] |
en
| 0.999997 |
ThT-solution was freshly diluted to 10 µM with PBS . Thirty µl of peptide-containing solution were mixed with 55 µl of ThT solution in black 96 well plates and fluorescence measured at 37°C with Ex/Em=440 nm/484 nm (Fluostar Omega, BMG Labtech).
|
39753747_p19
|
39753747
|
ThT assay
| 4.002603 |
biomedical
|
Study
|
[
0.999526858329773,
0.00025517118046991527,
0.00021798427042085677
] |
[
0.9919769167900085,
0.007451784331351519,
0.0003955608990509063,
0.00017573777586221695
] |
en
| 0.999995 |
Using the built-in custom ruler tool integrated with the pixel-to-metric unit translator of the Single Cellome™ System 2000, the distance between two deposits was measured by delineating a line from the edge of one deposit to the edge of the other. The midpoint of this line was identified as the farthest point, while the starting points of the line were defined as the nearest point. Subsequently, cells were categorized into three groups: those far from the deposits, situated more than 10 µm away from the midpoint between two deposits; those near the plaques, within 10 µm of the edge of the deposit; and those in between. Only cells categorized as far or near to a deposit were selected for organelle harvesting.
|
39753747_p20
|
39753747
|
Measuring Cell-Peptide-Deposit Distance
| 4.079803 |
biomedical
|
Study
|
[
0.9994996786117554,
0.00024242633662652224,
0.0002578209387138486
] |
[
0.9991735816001892,
0.0005660228198394179,
0.00020341368508525193,
0.00005691853948519565
] |
en
| 0.999998 |
Mitochondrial and nuclear materials were extracted employing the Single Cellome™ System 2000. The Single Cellome™ System 2000 was programmed to manual entry of the cell and automatic discharge of collected material into a collection plate. The collection loader was cooled to 4 °C, while the cell incubation loader was heated to 37 °C. The microscope channels were configured for fluorescence visualization, with 561 nm Exc/ 617 nm Em (red fl.) or 405 nm Exc/ 447 nm Em (blue fl.). The objective lens was adjusted to a magnification of “40x Dry.” Upon selection of the respective cell, the tip was inserted into the cell with controlled velocity (10 μm/s) and pressure (1 Pa). The material was extracted by changing the pressure to −10 Pa for 5 s and automatically discharged into the collection plate (MicroAmp™ optical 96-well reaction plate, Applied Biosystems™), pre-filled with 6 μL of sterile 1×PBS for use in RT-qPCR and qPCR or 6 μL of sterile measurement buffer (250 mM sucrose (Sigma-Aldrich), 15 mM KCl (Carl Roth GmbH + Co. KG), 5 mM MgCl 2 (Carl Roth GmbH + Co. KG), 30 mM K 2 HPO 4 (Carl Roth GmbH + Co. KG), 50 mM succinate (Sigma-Aldrich), pH 7.4) for the Oxygen Consumption Assay. For each sample, material from three cells from the same well was extracted and pooled. The total extraction process took approximately 4 minutes, with several distinct steps. The identification of a suitable cell required around 30 seconds, followed by 2 to 3 min for internal machine positioning and loading of the extraction tips. The subsequent alignment of the tip relative to the cell took an additional 20–30 s. Finally, the actual extraction of the cellular material was completed in approximately 10 s.
|
39753747_p21
|
39753747
|
Organelle Material Extraction Using Single Cellome™ System 2000
| 4.298485 |
biomedical
|
Study
|
[
0.9990938901901245,
0.0006452101515606046,
0.00026094214990735054
] |
[
0.9917725920677185,
0.007102964911609888,
0.0008579952991567552,
0.00026643028832040727
] |
en
| 0.999998 |
To ascertain that the extracted material did not vary in general amount of to be measured molecules (DNA, RNA, and protein), we assessed the respective data by using the absorption at 260 nm for nucleic acids (Nanodrop) and the ProteOrange kit for measuring small amounts of protein. All sample means did not differ statistical from each other or from the control .
|
39753747_p22
|
39753747
|
Organelle Material Extraction Using Single Cellome™ System 2000
| 3.635123 |
biomedical
|
Study
|
[
0.9991565942764282,
0.00023260412854142487,
0.0006107473745942116
] |
[
0.998553454875946,
0.00108378566801548,
0.00028778924024663866,
0.00007496320904465392
] |
en
| 0.999997 |
The extracted material was transferred to a black 384-well plate with transparent bottom (Greiner Bio-One GmbH), and supplemented with 4 μL of nuclease-free water. Fluorescence signals were measured using the FLUOstar® Optima microplate reader (BMG Labtech GmbH), with 485 nm Exc/ 520 nm Em (DAPI blue fl.) or with 540 nm Exc/ 580 nm Em (LumiTracker Mito Red CMXRos, red fl.).
|
39753747_p23
|
39753747
|
Fluorescence Analysis of Extracted Organelle Samples
| 4.070223 |
biomedical
|
Study
|
[
0.9995489716529846,
0.00023594641243107617,
0.0002151211374439299
] |
[
0.9934050440788269,
0.006039176136255264,
0.00040220239316113293,
0.00015366432489827275
] |
en
| 0.999997 |
Six μL of samples were combined with the Cell lysis mix , excluding DNAseI, resulting in a total volume of 40 μL. 32 μL were transferred into a new vial and the lysis protocol proceeded according to the manufacturer’s instructions. For qPCR reactions, the remaining 8 μL of sample lysis mix underwent the manufacturer’s protocol, substituting DNAseI with 10 μM RNase A (Carl Roth GmbH + Co. KG) and incubation for 10 min at 37 °C. RT-qPCR was carried out with Luna ® Cell Ready One-Step RT-qPCR Kit (New England Biolabs) and qPCR with primaQUANT SYBRGreen qPCR Blue with ROX (Steinbrenner Laborsysteme GmbH) adhering to the manufacturer’s guidelines. The sample volume was adjusted to 20 μL per sample and a primer concentration of 0.4 μM (Qiagen or produced by EurofinsMWG). The reactions were carried out on a StepOnePlus™Real-Time PCR System (Applied Biosystems™). For primer sequences see Table 1 . Table1 Primer sequences Primer Sequence 5ʹ- > 3ʹ Reference mt-Atp8_for GGCACCTTCACCAAAATCAC 46 mt-Atp8_rev TTGTTGGGGTAATGAATGAGG mt-Co1_for CCTAGATGACACATGAGCAAAAG (forward primer modified) 47 mt-Co1_rev AGCGTCGTGGTATTCCTGAAA mt-Nd1_for ACGCTTCCGTTACGATCAAC 46 mt-Nd1_rev ACTCCCGCTGTAAAAATTGG mt-DNA_for CTAGAAACCCCGAAACCAAA 48 mt-DNA_rev CCAGCTATCACCAAGCTCGT mGapdh QuantiTect Primer Assay (Qiagen) Tfam_for CCAAAAAGACCTCGTTCAGC 49 Tfam_rev ATGTCTCCGGATCGTTTCAC Pnpt1_for AATCGGGCACTCAGCTATTTG PrimerBank ID 12835817a1 50 Pnpt1_rev CAGGTCTACAGTCACCGCTC PGC1α_for CGGAAATCATATCCAACCAG 51 PGC1α_rev TGAGGACCGCTAGCAAGTTTG Cox2_for GGGTGTGAAGGGAAATAAGG 52 Cox2_rev TGTGATTTAAGTCCACTCCATG Itgax_for CCATGCTGGCTGTAGATGACC 53 Itgax_rev GTCATCCTGGCAGATGTGGTC Inpp5d_for GAGCTACTTTCCAGAGCCG 54 Inpp5d_rev CACAATTCCGGAACAGCACG
|
39753747_p24
|
39753747
|
Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) and qPCR
| 4.287495 |
biomedical
|
Study
|
[
0.9993137121200562,
0.00038427067920565605,
0.0003020101285073906
] |
[
0.9938086867332458,
0.00539743946865201,
0.0006300804670900106,
0.00016381009481847286
] |
en
| 0.999997 |
The Oxygen Consumption Rate Assay Kit was performed in accordance with the manufacturer’s guidelines with the reagent volumes adjusted to 40 μL in a black 384-well plate with transparent bottom (Greiner Bio-One GmbH), using 6 μL of organelle material in measurement buffer. Antimycin A (1 μM; Cayman Chemicals) was employed for inhibition, while glucose oxidase was utilized as a positive control for oxygen consumption. Fluorescence signal was measured by the FLUOstar® Optima microplate reader (BMG LABTECH) with an excitation wavelength of 380 ± 12 nm, and an emission wavelength of 630 ± 12 nm. The linear phase from the kinetic measurement was used for calculation of oxygen consumption.
|
39753747_p25
|
39753747
|
Oxygen Consumption Assay
| 4.144167 |
biomedical
|
Study
|
[
0.9995542168617249,
0.00026206692564301193,
0.00018371416081208736
] |
[
0.9971691966056824,
0.002267979783937335,
0.00045060590491630137,
0.00011221485328860581
] |
en
| 0.999996 |
Proteins were subjected to 10% SDS PAA gels. As control, 40 µg (lysate high, Lh) and 14 µg (lysate low, Ll) of protein derived from lysed SIM-A9 cells was used and 14 µg of protein from extracted material (nuclear or mitochondrial). Proteins were transferred to nitrocellulose membrane and the membrane was blocked with I-block solution (0.2% in PBS) (Thermo Fisher Scientific) including 0.05% Tween 20 (AppliChem). Primary antibody incubation took place overnight at 4 °C with anti-Calnexin or anti-Gapdh in combination with HRP-labeled secondary antibodies . Signals were detected after incubation with SuperSignal West Femto chemiluminescent substrate using a CCD-camera imaging system (Stella Camera, Raytest, Straubenhardt).
|
39753747_p26
|
39753747
|
Western blotting
| 4.110462 |
biomedical
|
Study
|
[
0.999488115310669,
0.00023456172493752092,
0.0002772661973722279
] |
[
0.9971069693565369,
0.002480599330738187,
0.00031792177469469607,
0.00009449903154745698
] |
en
| 0.999998 |
Data were obtained from at least two independent experiments as indicated in the figure legends. All statistical analyses were conducted using GraphPad Prism 6 or 8 software (GraphPad Software). All data are presented as mean ± standard deviation. Statistical significance of differences between two groups was determined using two-tailed Student’s t-tests. One-way analysis of variance (ANOVA) was used for three or more groups and a post-hoc pairwise comparison as indicated. Outlier analysis was performed with GraphPadPrism (ROUT 1%). A p -value < 0.05 was considered statistically significant.
|
39753747_p27
|
39753747
|
Statistics and Reproducibility
| 3.543136 |
biomedical
|
Study
|
[
0.999442994594574,
0.000205004500458017,
0.0003519404854159802
] |
[
0.9737608432769775,
0.023954160511493683,
0.0019903688225895166,
0.00029465131228789687
] |
en
| 0.999997 |
Schematics were created by using Biorender.
|
39753747_p28
|
39753747
|
Images
| 1.40632 |
biomedical
|
Other
|
[
0.9415069818496704,
0.0035091976169496775,
0.054983776062726974
] |
[
0.06406097859144211,
0.926696240901947,
0.0064081428572535515,
0.002834669314324856
] |
en
| 0.999999 |
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
|
39753747_p29
|
39753747
|
Reporting summary
| 0.843296 |
biomedical
|
Other
|
[
0.5440846681594849,
0.0033791586756706238,
0.4525361955165863
] |
[
0.011882489547133446,
0.9854373931884766,
0.0019243218703195453,
0.0007558096549473703
] |
en
| 0.999998 |
Supplementary Information Description of Additional Supplementary File Supplementary data 1 Reporting Summary
|
39753747_p30
|
39753747
|
Supplementary information
| 1.603749 |
biomedical
|
Other
|
[
0.9694399237632751,
0.001160050043836236,
0.029400015249848366
] |
[
0.14051735401153564,
0.8525478839874268,
0.00561775965616107,
0.001317030400969088
] |
en
| 0.999998 |
As of 2024, endometrial cancer (EC) ranks as the sixth most common malignancy in women globally, contributing significantly to cancer-related morbidity and mortality . Despite recent advancements in the early detection of EC and its treatment modalities, a significant number of patients with EC continue to suffer from poor prognoses, particularly those with advanced-stage and high-grade disease . This persistent challenge underscores the limitations of current prognostic tools and therapies, thereby necessitating a more nuanced understanding of the molecular and immunological aspects of EC.
|
39751650_p0
|
39751650
|
Introduction
| 3.967898 |
biomedical
|
Review
|
[
0.9979860782623291,
0.0010628938907757401,
0.0009510900708846748
] |
[
0.0803922563791275,
0.004396265372633934,
0.9146897792816162,
0.0005217301659286022
] |
en
| 0.999998 |
The Cancer Genome Atlas (TCGA) has been instrumental in identifying four key genomic subgroups in EC: POLE (ultramutated), microsatellite instability (MSI) (hypermutated), copy-number low (CN low) (endometrioid), and copy-number high (CN high) (serous-like) . These findings have significantly advanced our approach to personalized EC treatments and prognoses. Notably, tumors with POLE mutations have shown better outcomes, even in high-grade cases, whereas the CN high subtype, particularly with TP53 mutations, often indicates a poorer prognosis . This diversity in outcomes among the EC subgroups underscores the necessity for additional markers to refine the risk stratification and personalize treatments . The proactive molecular risk classifier for endometrial cancer (ProMisE), based on TCGA's genomic subgroups, represents a significant step forward in this context . By integrating POLE exonuclease domain mutations and immunohistochemistry for mismatch repair proteins and TP53, the ProMisE categorizes EC into four distinct groups: POLE-EDM, MMR-D, p53wt, and p53abn . It extends TCGA's foundational work by providing comprehensive prognostic information, enhancing patient management within the International Federation of Gynecology and Obstetrics 2023 staging system .
|
39751650_p1
|
39751650
|
Introduction
| 4.499127 |
biomedical
|
Study
|
[
0.9984373450279236,
0.0008535799570381641,
0.0007089930004440248
] |
[
0.5644580125808716,
0.0028213495388627052,
0.43195390701293945,
0.0007667781319469213
] |
en
| 0.999996 |
The distribution of tumor-infiltrating lymphocytes (TILs) was reported to be associated with the prognosis of patients with colorectal cancer in 2006 . The concept of immunophenotypes (i.e., inflamed, excluded, and desert) based on the spatial distribution of CD8 + TILs, which recognized as vital players in modulating tumor progression and influencing responses to immunotherapies, was introduced in 2016 . These immunophenotypes are known to affect the effectiveness of treatments, particularly treatment with immune checkpoint inhibitors (ICIs) in some solid tumors . In EC, the amount of CD8 + TILs has been reported to be associated with patients’ prognosis ; however, the association between the distribution of CD8 + TILs and clinical outcomes has not been investigated. Although ICIs are increasingly being applied clinically in the treatment of EC, research on the tumor immune microenvironment of EC is lacking.
|
39751650_p2
|
39751650
|
Introduction
| 4.081398 |
biomedical
|
Study
|
[
0.9996044039726257,
0.0001963321556104347,
0.00019923763466067612
] |
[
0.9955867528915405,
0.00033768449793569744,
0.003991348203271627,
0.00008423181861871853
] |
en
| 0.999998 |
We sought to bridge this knowledge gap by exploring the distribution and prognostic relevance of CD8 + TIL-based immunophenotypes in EC, stratified by ProMisE molecular classification. We hypothesized that each molecular subtype may be associated with a distinct immunophenotype, which could have significant implications for patient prognoses and treatment responses. Our goal is to integrate genomic and immunological data to provide a more holistic view of the EC landscape, potentially paving the way for novel, targeted immunotherapeutic strategies for EC.
|
39751650_p3
|
39751650
|
Introduction
| 4.102594 |
biomedical
|
Study
|
[
0.9995381832122803,
0.00032492782338522375,
0.00013688878971152008
] |
[
0.9976060390472412,
0.00042508961632847786,
0.0018657143227756023,
0.00010313771053915843
] |
en
| 0.999998 |
A prospective analysis was conducted to examine the relationship between immunophenotype and molecular classification, as well as survival outcomes. The cases of 60 patients with EC treated during the period from January 2019 to December 2022 at Nagoya University Hospital (Nagoya, Japan) were prospectively enrolled. Following a definitive pathological diagnosis, these patients underwent surgical intervention. Ethical approval of this study was granted by the Institutional Review Board of Nagoya University . Both fresh frozen and formalin-fixed paraffin-embedded (FFPE) tumor tissues were collected for analysis.
|
39751650_p4
|
39751650
|
Prospective cohort
| 4.049722 |
biomedical
|
Study
|
[
0.9979051351547241,
0.0018359562382102013,
0.00025899242609739304
] |
[
0.9990512728691101,
0.0006028121570125222,
0.00017956169904209673,
0.0001662789873080328
] |
en
| 0.999996 |
We retrospectively analyzed the cases of a separate cohort of patients with high-grade EC ( n = 85, including endometrioid grade 3, serous, and clear cell histology) treated between January 2002 and December 2017 at Nagoya University Hospital. FFPE tumor tissues were collected for analysis.
|
39751650_p5
|
39751650
|
Retrospective cohort
| 3.806277 |
biomedical
|
Study
|
[
0.9987239241600037,
0.0009688363061286509,
0.00030719381175003946
] |
[
0.9992363452911377,
0.00044926241389475763,
0.00020167999900877476,
0.00011274792632320896
] |
en
| 0.999998 |
All 145 of the patients underwent a simple or semiradical hysterectomy with a bilateral salpingo-oophorectomy, pelvic, and/or para-aortic lymphadenectomy. The surgical specimens were staged according to the FIGO 2008 staging system. Postoperative treatment followed the Japan Society of Gynecologic Oncology guidelines . Risk-based adjuvant therapy was administered; low-risk patients received no additional treatment, and the intermediate- to high-risk patients received radiotherapy or platinum-based chemotherapy.
|
39751650_p6
|
39751650
|
Diagnosis and treatment
| 3.956275 |
biomedical
|
Study
|
[
0.8908725380897522,
0.10838084667921066,
0.0007465503294952214
] |
[
0.9453375935554504,
0.04200030863285065,
0.0027433340437710285,
0.009918774478137493
] |
en
| 0.999997 |
Hotspot mutations in exons 9, 13, and 14 of the POLE gene were identified using Sanger sequencing. Genomic DNA extraction was conducted in accord with the manufacturers' protocols for both fresh frozen (NucleoSpin® DNA Rapidlyse, Macherey–Nagel, Düren, Germany) and FFPE tissues (QIAamp DNA FFPE Advanced UNG Kit, Qiagen, Hilden, Germany). The samples were enriched using the Blend Taq Plus (Toyobo, Osaka, Japan). The polymerase chain reaction (PCR) amplification and conditions were as described . A portion of the PCR products was run on a 2% agarose gel in 1× TAE buffer to verify the presence of a single band approx. 200–300 base pairs in size. The rest of the PCR products was then purified using the QIAquick Gel Extraction Kit (Qiagen), following the manufacturer's instructions.
|
39751650_p7
|
39751650
|
Sanger sequencing for POLE exonuclease domain
| 4.138988 |
biomedical
|
Study
|
[
0.9995056390762329,
0.0003453933459240943,
0.00014888541772961617
] |
[
0.998766303062439,
0.0007842041668482125,
0.00034011510433629155,
0.0001093155806302093
] |
en
| 0.999998 |
The DNA concentrations were measured with a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, USA). The subsequent DNA sequencing was outsourced to Eurofins Genomics (Tokyo). For the sequence analysis, we used SnapGene Viewer ver. 6.1.2 (GSL Biotech, San Diego, CA) to examine the waveform patterns. Mutation identification was conducted using the Nucleotide BLAST tool (Basic Local Alignment Search Tool) from the U.S. National Center for Biotechnology Information (NCBI). In this study, we defined ' POLE pathogenic variants' as the nine single-nucleotide substitutions on exons 9, 13, and 14: c.857 C > G (P286R) (exon9), c.884 T > G (M295R) (exon9), c.890 C > T (S297F) (exon9), c.1231 G > T/C (V411L) (exon13), c.1270 C > A (L424I) (exon13), c.1307 C > G (P436R) (exon13), c.1331 T > A (M444K) (exon13), c.1366 G > C (A456P) (exon14), and c.1376 C > T (S459F) (exon14) .
|
39751650_p8
|
39751650
|
Sanger sequencing for POLE exonuclease domain
| 4.126885 |
biomedical
|
Study
|
[
0.9994733929634094,
0.0003486259374767542,
0.00017797209147829562
] |
[
0.9991540908813477,
0.0005654809065163136,
0.00017889575974550098,
0.00010148641013074666
] |
en
| 0.999998 |
Immunohistochemistry (IHC) was conducted on 4-µm sections of FFPE tumor tissues, which included sections from both the central tumor (CT) and the invasive margin (IM). The primary antibodies used for the IHC included anti-human CD8 (clone C8/144b, Dako, Glostrup, Denmark; 1:100), PMS2 (clone A16-4, Biocare Medical, Walnut Creek, CA; 1:100), MSH6 (clone BC/44, Biocare Medical, 1:100), and p53 (clone DO-7, Dako; 1:100). The sections were deparaffinized and rehydrated, subjected to antigen retrieval in 10 mM sodium citrate (pH 6.0) or 1× Immunoactive (pH 9.0, Matsunami, Osaka, Japan) for 20 min at 95 °C in a microwave, and treated with 0.3% hydrogen peroxide in methanol for 20 min. Blocking was performed using the Histofine SAB-PO kit (Nichirei, Tokyo), followed by overnight incubation at 4 °C with the diluted primary antibodies. After the primary antibody incubation, the sections were incubated with biotin-labeled secondary antibody, peroxidase-labeled streptavidin, and developed using 3,3'-diaminobenzidine (DAB) substrate-chromogen for specific time durations. Then, the sections were counterstained with hematoxylin, dehydrated, and mounted.
|
39751650_p9
|
39751650
|
Immunohistochemistry analysis
| 4.186735 |
biomedical
|
Study
|
[
0.9993128776550293,
0.0005014144699089229,
0.00018570144311524928
] |
[
0.9971089959144592,
0.0021073927637189627,
0.0006273849285207689,
0.00015631067799404263
] |
en
| 0.999996 |
Our application of immunohistochemistry for PMS2 and MSH6 was based on reports suggesting their effectiveness in screening for mismatch repair deficiency (MMRd) . MMRd was identified by the complete absence of nuclear staining for either protein with internal positive controls including unaltered nuclear staining in adjacent normal endometrium, stromal cells, and inflammatory cells. Representative examples of loss of MSH6 or PMS2 expression are shown in Supplementary Fig. S1 A–D. Abnormal p53 staining (p53abn) was characterized as either a strong, diffuse nuclear staining pattern in > 80% of carcinoma cells, or a complete lack of staining ("null pattern"), using adjacent non-tumor cells as an internal control. Wild-type tumor cells exhibited weak and heterogeneous staining patterns . Representative examples of normal and abnormal p53 immunohistochemical staining patterns are shown in Supplementary Fig. S1 E-G.
|
39751650_p10
|
39751650
|
Immunohistochemistry analysis
| 4.143143 |
biomedical
|
Study
|
[
0.9994834661483765,
0.000323768996167928,
0.00019275287922937423
] |
[
0.9993621706962585,
0.00024039750860538334,
0.0003251005837228149,
0.00007235258090076968
] |
en
| 0.999998 |
Multiplex immunofluorescent (IF) staining was performed by the TSA method using Opal IHC kit (PerkinElmer, Waltham, MA) according to the manufacture’s instructions. Anti-pan cytokeratin (clone AE1/AE3, Abcam, Cambridge, UK; 1:600) and anti-human CD8 (clone C8/144b, Dako, Glostrup, Denmark; 1:100) were used as primary antibodies. The antigen retrieval process was carried out as described above, and blocking was performed using the opal kit reagent, followed by incubation at room temperature for 30 min for anti-pan cytokeratin and overnight incubation at 4 °C for anti-CD8. After the primary antibody incubation, the sections were incubated with peroxidase-labeled secondary antibody, followed by incubation with opal 520 or opal 620 reagent. Then, the sections were stained with 4’, 6-diamidino-s-phenylindole (DAPI) (Dojindo, Kumamoto, Japan) and mounted. Multiplexed fluorescent labeled images were captured with a BZ-X800 (Keyence, Osaka, Japan).
|
39751650_p11
|
39751650
|
Immunohistochemistry analysis
| 4.165201 |
biomedical
|
Study
|
[
0.9994592070579529,
0.0003426572075113654,
0.0001981433160835877
] |
[
0.993770182132721,
0.00540211470797658,
0.0006346910377033055,
0.00019295699894428253
] |
en
| 0.999995 |
The assessment of TILs in this study followed the guidelines established by the International Immuno-Oncology Biomarker Working Group . We did not differentiate between intratumoral TILs and stromal TILs during this evaluation. After capturing stained slide images with a VS120-S5 (Evident, Tokyo, Japan), CD8 + TILs were quantified automatically in both the CT and IM using QuPath ver. 0.3.0 . This quantification was performed over five distinct areas, each being a square with 0.25 mm on each side . The average number of CD8 + TILs per square millimeter was calculated for these regions.
|
39751650_p12
|
39751650
|
Immunophenotyping
| 4.072474 |
biomedical
|
Study
|
[
0.9995438456535339,
0.00028488095267675817,
0.00017118656251113862
] |
[
0.9993178844451904,
0.0003515901626087725,
0.0002654635172802955,
0.00006516242865473032
] |
en
| 0.999997 |
The distinction between the three immunophenotypes (inflamed, excluded, and desert) was based on previous research , but as there is no established definition for the density of CD8 + TILs in EC, we adopted 1000 cells/mm 2 in this study. Tumors with a CD8 + TIL density ≥ 1000 cells/mm 2 in both the CT and IM regions were classified as 'inflamed' phenotype. The tumors with a CD8 + TIL density < 1000 cells/mm 2 in the CT but > 1000 cells/mm 2 at the IM were designated as the 'excluded' phenotype. Conversely, tumors were classified as the 'desert' phenotype when the density of CD8 + TILs was < 1000 cells/mm 2 in both the CT and IM areas.
|
39751650_p13
|
39751650
|
Immunophenotyping
| 4.076801 |
biomedical
|
Study
|
[
0.9994933605194092,
0.0002540592395234853,
0.0002525841409806162
] |
[
0.9993784427642822,
0.0003230453876312822,
0.00025022815680131316,
0.00004831719343201257
] |
en
| 0.999997 |
We conducted the molecular classification of tumors using an adapted approach from the ProMisE methodology, aligned with the steps outlined in the World Health Organization (WHO) classification . This approach involved a sequential assessment of specific molecular markers. Initially, all tumor samples underwent Sanger sequencing to identify mutations in the POLE exonuclease domain, specifically targeting exons 9, 13, and 14 as noted above. Tumors harboring pathogenic variants in these regions were classified as 'POLEmut.' Next, the tumors exhibiting a complete absence of nuclear staining for PMS2 or MSH6 protein by IHC were categorized as 'MMRd.' Then, the tumors demonstrating abnormal p53 expression patterns by IHC were classified as 'p53abn.' Finally, tumors that did not exhibit any of the aforementioned molecular characteristics were classified as 'NSMP,' indicating a no specific molecular profile.
|
39751650_p14
|
39751650
|
The ProMisE molecular classification
| 4.12149 |
biomedical
|
Study
|
[
0.9994450211524963,
0.00036587018985301256,
0.00018913066014647484
] |
[
0.998828113079071,
0.00039316152106039226,
0.0006967636290937662,
0.00008196639828383923
] |
en
| 0.999997 |
We defined progression-free survival (PFS) as the duration from the initiation of a patient's treatment to the point of observed disease progression. Overall survival (OS) was determined as the period from the commencement of treatment to either the death of a patient due to any reason or the patient's last confirmed survival status, with data collected up until March 2023 in retrospective cohort or until November 2024 in prospective cohort. We used the Kaplan–Meier method to estimate the 145 patients' PFS and OS rates. To assess the impact of immunophenotypes on the prognosis of EC patients within each molecular classification, survival curves were compared across the three immunophenotypes (inflamed, excluded, and desert) within each ProMisE subtype.
|
39751650_p15
|
39751650
|
Survival analysis
| 4.15277 |
biomedical
|
Study
|
[
0.9974029660224915,
0.002387515502050519,
0.00020949541067238897
] |
[
0.9981060028076172,
0.0011311962734907866,
0.0005429196171462536,
0.0002198413567384705
] |
en
| 0.999998 |
Given the limited sample size of our cohort, the abundance of CD8 + TILs from the Cancer Genome Atlas (TCGA) database was estimated using the CIBERSORT algorithm in R . Patients in the TCGA database were stratified into high and low CD8 + T-cell groups within the four genomic subgroups of EC (POLE, MSI, CN low, and CN high) using a receiver operating characteristic (ROC) curve. An optimal cutoff value of 0.0536085 was adopted for predicting 1-year OS.
|
39751650_p16
|
39751650
|
Survival analysis
| 4.096845 |
biomedical
|
Study
|
[
0.9994767308235168,
0.00028470699908211827,
0.00023847856209613383
] |
[
0.9995156526565552,
0.00022209039889276028,
0.00020560504344757646,
0.000056611424952279776
] |
en
| 0.999996 |
The RNA-sequencing analysis was carried out on samples from the 40 of the 60 patients in prospective cohort from whom RNA samples were available. The extraction of RNA from fresh frozen tumor tissue was done using the NucleoSpin RNA Plus kit (Macherey–Nagel), following the manufacturer's guidelines. We measured the total RNA concentration with the NanoDrop One spectrophotometer. RNA-sequencing was then performed by Novogene Japan (Tokyo). The obtained raw FASTQ data were uploaded to Galaxy, an open-source web-based platform tailored for data-intensive biomedical research. Quality control of the data was executed using FastQC and Trimmomatic. The clean, paired-end data were then processed for gene expression quantification using the Kallisto quant tool, referencing the GENCODE GRC38.p13 transcript (genecode. v41.transcript).
|
39751650_p17
|
39751650
|
RNA-sequencing analysis
| 4.11084 |
biomedical
|
Study
|
[
0.9995148181915283,
0.00029734038980677724,
0.00018796029326040298
] |
[
0.9993603825569153,
0.0003217098710592836,
0.00024273166491184384,
0.00007529588037868962
] |
en
| 0.999996 |
Post-processing, the data were aggregated using the tximport package (ver. 1.18.0) in R software (ver. 4.0.3) and RStudio. For the subsequent analyses, scaled transcripts per million (TPM) counts were used. The TPM counts were processed with the use of the web portal for integrated differential expression and pathway analysis (iDEP) . We also used iDEP 2.01 for a principal component analysis (PCA). A gene set enrichment analysis (GSEA) was conducted employing the GSEA software (ver. 4.3.2), allowing for the identification of significantly altered pathways and gene sets in the dataset.
|
39751650_p18
|
39751650
|
RNA-sequencing analysis
| 4.07827 |
biomedical
|
Study
|
[
0.9994956254959106,
0.0001608775492059067,
0.0003434491518419236
] |
[
0.9989172220230103,
0.0007220542174763978,
0.00030816736398264766,
0.000052615039749071
] |
en
| 0.999997 |
We used GraphPad Prism software, ver. 9.2.0 (GraphPad Software, San Diego, CA) for the statistical analyses. To compare the relationships between different groups, we applied two distinct statistical tests depending on the data structure and distribution: the Wilcoxon matched-pairs signed-rank test was used for paired data comparisons, and the nonparametric Mann–Whitney U-test was applied for unpaired data sets. To compare distributions between the observed and expected data, we used the χ 2 -test.
|
39751650_p19
|
39751650
|
Statistical analysis
| 3.644018 |
biomedical
|
Study
|
[
0.9992092251777649,
0.0001844880753196776,
0.0006062726606614888
] |
[
0.9805481433868408,
0.018478624522686005,
0.0008070127223618329,
0.0001662077265791595
] |
en
| 0.999999 |
The Kaplan–Meier method was used for the survival analyses, i.e., the PFS and OS rates. This allowed us to plot survival curves and estimate survival probabilities over time. The differences in survival rates between groups were evaluated by the log-rank test. Throughout the analyses, a p value threshold < 0.05 was set for determining statistical significance.
|
39751650_p20
|
39751650
|
Statistical analysis
| 3.908514 |
biomedical
|
Study
|
[
0.9995005130767822,
0.0002795895270537585,
0.00021986923820804805
] |
[
0.9976478219032288,
0.0017413586610928178,
0.0005177836283110082,
0.00009301928366767243
] |
en
| 0.999997 |
We conducted a prospective study over a 4-year period, enrolling consecutive 60 EC patients representing a full spectrum of histology. The clinicopathological characteristics of these patients are provided in Table 1 . The cohort had a median follow-up of 38.4 months (range 0.6–66.0 months) and a median age of 58 years (range 32–84 years). Most of the patients (61.7%) presented with endometrioid G1/2 histology, and 66.7% were diagnosed at early stages . The ProMisE classification revealed that 11.7% of these patients fell into the POLEmut subtype, 25.0% into MMRd, 48.3% into NSMP, and 15.0% into p53abn (Table 2 ). The detailed POLE pathogenic variants are presented in Supplementary Table S1 . The distribution of these categories aligns with another investigation of Japanese patients with EC . Notably, higher ages were observed in the p53abn subtype, and the NSMP subtype predominantly consisted of endometrioid G1 and G2 histology. The serous histology tumors were exclusively categorized as p53abn. Table 1 Prospective cohort; EC patients in 2019–2022 Total patients, n 60 Follow–up period, mos.; median (range) 38.4 (0.6–66.0) Age, yrs; median (range) 58.0 (32–84) Histology Endometrioid G1/2 37 (61.7) Endometrioid G3 20 (33.3) Serous 3 (5.0) Stage I 35 (58.3) II 5 (8.3) III 18 (30.0) IV 2 (3.3) Risk of recurrence Low 13 (21.7) Intermediate 18 (30.0) High 29 (48.3) The data are numbers and percentages EC endometrial cancer; FIGO international federation of gynecology and obstetrics Table 2 ProMisE molecular classification in prospective cohort POLEmut ( n = 7) MMRd ( n = 15) NSMP ( n = 29) p53abn ( n = 9) Proportion, % 11.7 25.0 48.3 15.0 Age, yrs; median 57.0 58.0 58.0 66.0 (range) (48–75) (50–65) (32–84) (52–75) Histology Endometrioid G1/2 5 6 23 3 Endometrioid G3 2 9 6 3 Serous 0 0 0 3 Stage I 6 7 19 3 II 0 1 4 0 III 1 6 6 5 IV 0 1 0 1 Risk of recurrence Low 2 2 8 1 Intermediate 4 4 10 0 High 1 9 11 8 ProMisE proactive molecular risk classifier for endometrial cancer; POLEmut polymerase-epsilon mutation; MMRd mismatch repair deficiency; NSMP no specific molecular profile; p53abn p53 abnormality; FIGO international federation of gynecology and obstetrics
|
39751650_p21
|
39751650
|
The clinicopathological characteristics and ProMisE classification of prospective cohort
| 4.209706 |
biomedical
|
Study
|
[
0.998971700668335,
0.0008013054030016065,
0.00022699905093759298
] |
[
0.9992750287055969,
0.00027298330678604543,
0.0003370408376213163,
0.0001149654999608174
] |
en
| 0.999996 |
Figure 1 provides representative histological images of the three immunophenotypes; inflamed , excluded , and desert . The classification was based on the distribution patterns of CD8 + TILs. The inflamed phenotype showed abundant CD8 + TILs in both the CT and the IM , whereas the excluded phenotype had a higher concentration in the IM than the CT . The desert phenotype was characterized by sparse CD8 + TILs in both areas . To further evaluate the spatial relationship between tumor cells and CD8 + TILs, we performed multiplex IF staining for pan cytokeratin (tumor cell marker) and CD8. In the inflamed phenotype, CD8 + TILs were abundant in both the CT and the IM , and CD8 + TILs well infiltrated into the tumor cells in both regions. In the excluded phenotype, there were many CD8 + TILs in the IM , but few in the CT . In the desert phenotype, there were few CD8 + TILs in both the CT and the IM . Fig. 1 Representative images of three immunophenotypes based on the distribution patterns of CD8 + tumor-infiltrating lymphocytes (TILs) in endometrial cancer. The immunohistochemistry images of CD8 + TILs in the inflamed phenotype ( A , D , G ), excluded phenotype ( B , E , H ), and desert phenotype ( C , F , I ) are shown. The middle rows ( D , E , F ) show CD8 + TILs in the CT, and the bottom rows ( G , H , I ) show those in the IM. Abbreviations: CT, central tumor; IM, invasive margin
|
39751650_p22
|
39751650
|
Three distinct immunophenotypes based on the distribution of CD8+ TILs
| 4.126618 |
biomedical
|
Study
|
[
0.9994951486587524,
0.00032526697032153606,
0.0001795618300093338
] |
[
0.9991843104362488,
0.000254719314398244,
0.00048170232912525535,
0.00007929361163405702
] |
en
| 0.999996 |
The CT and IM regions on the hematoxylin and eosin stain are shown in Fig. 2 A. CD8 + TILs were automatically quantified five distinct areas in both the CT and IM, as illustrated in Fig. 2 B. The average density of CD8 + TILs (positive cells per square millimeter) in the CT (blue bar) and IM (red bar) for 60 EC patients is shown in Fig. 2 C. Among all 60 patients, 17 (28.3%) were classified as the inflamed phenotype, 22 (36.7%) as the excluded phenotype, and 21 (35%) as the desert phenotype. The median density of CD8 + TILs in the CT was significantly lower than that in the IM in the inflamed phenotype , in the excluded phenotype , and in the desert phenotype . Fig. 2 The relationship between the ProMisE classification and the immunophenotypes in prospective cohort. The approximate areas of the CT and IM in the hematoxylin and eosin stain are shown ( A ). CD8 + tumor-infiltrating lymphocytes (TILs) were automatically counted at five areas in the CT ( surrounded by blue lines ) and IM ( surrounded by red lines ) ( B ). The average density of CD8 + TILs (positive cells per square millimeter) in the CT ( blue bar ) and IM ( red bar ) for 60 EC patients is shown ( C ). Comparison of the density of the CD8 + TILs in the CT and IM in the inflamed phenotype ( D ), excluded phenotype ( E ), and desert phenotype ( F ). Comparison of the density of the CD8 + TILs by the four ProMisE subtypes in the CT ( G ) and IM ( H ). Abbreviations: CT, central tumor; IM, invasive margin; TILs, tumor-infiltrating lymphocytes; POLEmut, polymerase-epsilon mutation; MMRd, mismatch repair deficiency; NSMP, no specific molecular profile; p53abn, p53 abnormality
|
39751650_p23
|
39751650
|
The relationship between the ProMisE classification and the immunophenotypes in prospective EC cohort
| 4.124865 |
biomedical
|
Study
|
[
0.9992486834526062,
0.0005005151615478098,
0.0002507969329599291
] |
[
0.9994946718215942,
0.00022912415442988276,
0.00020762236090376973,
0.00006850773934274912
] |
en
| 0.999998 |
Figure 2 G depicts the density of CD8 + TILs in the CT in each ProMisE category. The median density of CD8 + TILs in the CT was not significantly different between the POLEmut and MMRd subtypes or between the NSMP and p53abn subtypes . This value was significantly higher in the POLEmut subtype compared to the NSMP and p53abn subtypes and was significantly higher in the MMRd subtype versus the NSMP and p53abn subtypes .
|
39751650_p24
|
39751650
|
The relationship between the ProMisE classification and the immunophenotypes in prospective EC cohort
| 4.033228 |
biomedical
|
Study
|
[
0.9993988275527954,
0.0003528881352394819,
0.00024829135509207845
] |
[
0.999464213848114,
0.0002556093968451023,
0.00021796934015583247,
0.00006228005076991394
] |
en
| 0.999998 |
Figure 2 H illustrates the density of CD8 + TILs in the IM in each of the ProMisE categories. The median density of CD8 + TILs in the IM was not significantly different between the POLEmut and MMRd subtypes but was significantly higher in p53abn subtype than in NSMP subtype unlike the densities in the CT. In terms of the median density of CD8 + TILs in the CT and IM, the POLEmut and MMRd subtypes showed the inflamed phenotype with more CD8 + TILs in both areas; the NSMP subtype showed the desert phenotype with fewer CD8 + TILs in both areas; and the p53abn subtype showed the excluded phenotype with fewer CD8 + TILs in the CT and more in the IM. However, looking at individual cases, some of the POLEmut and MMRd subtypes showed the non-inflamed phenotypes, and some of the NSMP subtype showed the inflamed phenotype.
|
39751650_p25
|
39751650
|
The relationship between the ProMisE classification and the immunophenotypes in prospective EC cohort
| 4.095935 |
biomedical
|
Study
|
[
0.9993681311607361,
0.00034024828346446157,
0.0002915870863944292
] |
[
0.999497652053833,
0.0001994389749597758,
0.000248976779403165,
0.000053895044402452186
] |
en
| 0.999995 |
To further investigate the relationship between the ProMisE classification and immunophenotype, we added that a retrospective cohort consists of 85 EC patients treated prior to 2017 to our analysis. These patients' clinicopathological characteristics are summarized in Supplementary Table S2 . The median follow-up period was 75.8 months (range 0.7–173.3 months). Most of the retrospective cohort (70.6%) presented with endometrioid G3 histology; the other patients presented serous, clear, and mixed histology. The ProMisE classification revealed that 7.1% of the retrospective cohort were the POLEmut subtype, 22.3% had the MMRd subtype, 42.4% showed NSMP subtype, and 28.2% were classified as p53abn subtype (Supplementary Table S3 ). The detailed POLE pathogenic variants are presented in Supplementary Table S4 .
|
39751650_p26
|
39751650
|
The analysis of the relationship between the ProMisE classification and the immunophenotypes in prospective and retrospective EC cohort
| 4.119604 |
biomedical
|
Study
|
[
0.9992393255233765,
0.0005242613842710853,
0.00023649699869565666
] |
[
0.9995205402374268,
0.0001596342772245407,
0.00024261687940452248,
0.00007726362673565745
] |
en
| 0.999998 |
Table 3 summarizes the distribution of the three immunophenotypes in each ProMisE category in prospective and retrospective cohort. Notably, the inflamed phenotype was most frequently observed in the POLEmut and MMRd subtypes, while the desert phenotype was predominant in the NSMP subtype; however, other immunophenotypes were also observed. No cases of the inflamed phenotype were observed in the p53abn subtype. The distribution of the three immunophenotypes in each cohort is shown in Supplementary Table S5 (prospective cohort) and S6 (retrospective cohort). Table 3 Relationship between the ProMisE classifications and immunophenotypes in prospective and retrospective cohorts POLEmut ( n = 13) MMRd ( n = 34) NSMP ( n = 65) p53abn ( n = 33) p value Inflamed 6 22 9 0 p < 0.0001 Excluded 6 9 13 23 Desert 1 3 43 10 ProMisE proactive molecular risk classifier for endometrial cancer; POLEmut polymerase-epsilon mutation; MMRd mismatch repair deficiency; NSMP no specific molecular profile; p53abn p53 abnormality; FIGO international federation of gynecology and obstetrics
|
39751650_p27
|
39751650
|
The analysis of the relationship between the ProMisE classification and the immunophenotypes in prospective and retrospective EC cohort
| 4.100888 |
biomedical
|
Study
|
[
0.9994199275970459,
0.00035063986433669925,
0.00022950720449443907
] |
[
0.9994656443595886,
0.00020089096506126225,
0.0002762535586953163,
0.00005722581045120023
] |
en
| 0.999996 |
The survival analysis revealed distinct prognostic differences across the ProMisE classifications and immunophenotypes. The POLEmut and MMRd subtypes exhibited favorable PFS and OS rates, whereas the p53abn and NSMP subtypes were associated with poorer outcomes . Moreover, the patients with the excluded or desert phenotypes demonstrated significantly worse survival rates compared to those with the inflamed phenotype , highlighting the prognostic relevance of this immunophenotypic categorization. Within each ProMisE subtype, the prognostic trends of the immunophenotypes were generally maintained, with the inflamed phenotype associated with better outcomes compared to the excluded and desert phenotypes. However, due to the small number of patients in each subgroup, these trends did not reach statistical significance. Survival analysis by immunophenotype in each ProMisE subtype is depicted in Supplementary Figure S3 . Fig. 3 Survival analysis of 145 patients stratified by ProMisE classification, and the immunophenotypes. The progression-free survival and overall survival rates according to four ProMisE categories comprising POLEmut ( light blue line ), MMRd ( yellow-green line) , NSMP ( orange line ), and p53abn ( red line ) subtypes are shown ( A , B ). The progression-free survival and overall survival rates according to three immunophenotypes comprising the inflamed ( red line ), excluded ( violet line ), and desert (blue line ) phenotypes are shown ( C , D ). Abbreviations: ProMisE, proactive molecular risk cassifier for endometrial cancer; POLEmut, polymerase-epsilon mutation; MMRd, mismatch repair deficiency; NSMP, no specific molecular profile; p53abn, p53 abnormality
|
39751650_p28
|
39751650
|
Survival analysis of the 145 EC patients stratified by the ProMisE classification and their immunophenotypes
| 4.121298 |
biomedical
|
Study
|
[
0.9991262555122375,
0.0006168709951452911,
0.00025691380142234266
] |
[
0.9991664886474609,
0.00024973266408778727,
0.0004992548492737114,
0.00008452178008155897
] |
en
| 0.999998 |
We utilized the TCGA database to verify whether the CD8 + T-cell fraction, estimated using the CIBERSORT algorithm, serves as a prognostic factor across the four molecular subtypes of EC in a larger cohort. Since the TCGA database does not provide direct immunophenotype data, we categorized cases based on the estimated CD8 + T-cell fraction. Cases with high CD8 + T-cell fractions were classified as CD8-high (analogous to the inflamed type), while those with low CD8 + T-cell fractions were grouped as CD8-low (representing non-inflamed types, including excluded and desert phenotypes). There were few cases in the CD8-low group in POLE subtype, and no differences in prognosis were observed between the two groups . Although it was not significant, there was a trend for the prognosis of the CD8-low group to be worse than that of the CD8-high group in MSI and CN low subtypes. In the CN high subtype, the prognosis was significantly worse in the CD8-low group than in the CD8-high group .
|
39751650_p29
|
39751650
|
Survival analysis of the differences in the abundance of CD8+ T-cells in the four genomic subgroups of EC in the TCGA database
| 4.096725 |
biomedical
|
Study
|
[
0.999406099319458,
0.0003480463637970388,
0.00024594893329776824
] |
[
0.9994926452636719,
0.00018439862469676882,
0.00026394418091513216,
0.00005904473800910637
] |
en
| 0.999997 |
Finally, to investigate factors that produce the different distribution patterns of CD8 + TILs between the non-inflamed (excluded and desert) and inflamed phenotypes, we performed an RNA-sequencing (RNA-seq) analysis. The principal component analysis (PCA) was performed with RNA-seq data of 40 EC samples from prospective cohort. The first two principal components (PCs) are plotted and colored according to the ProMisE classification or immunophenotype . The GSEA of the MMRd subtype with the non-inflamed phenotypes compared to that with the inflamed phenotype revealed that the expressions of MYC target gene sets were more enriched in the non-inflamed phenotypes versus the inflamed phenotype . Figure 4 D shows the enrichment plots for the top two datasets that were enriched in the GSEA hallmark analysis. The heat map of the top 50 marker genes for each phenotype in the MMRd subtype with the non-inflamed phenotypes and that with the inflamed phenotype is provided as Fig. 4 E. The top two genes that were upregulated in the non-inflamed phenotype were CD99 and NLGN1 . The top gene that was upregulated in the inflamed phenotype was BRINP1. Fig. 4 The results of the RNA-sequencing analysis in Cohort 2. Principal component analysis plots for the data of 40 samples in Cohort 2 are shown. The first two principal components are plotted and colored according to the ProMisE classification ( A ) or immunophenotype ( B ). The results of a gene set enrichment analysis (GSEA) of the MMRd subtype with the non-inflamed (excluded and desert) phenotypes versus that with the inflamed phenotype comparison are illustrated, as are the results of the GSEA hallmark analysis showing significantly enriched gene sets (FDR < 25% and a nominal p value < 5%). A positive normalized enrichment score indicates enrichment in the MMRd subtype with the non-inflamed phenotypes ( C ). Enrichment plots for the top two datasets enriched in the GSEA hallmark analysis, showing the profile of the running enrichment score and the positions of gene set members on the rank-ordered list ( D ). The heat map of the top 50 marker genes for each phenotype with the non-inflamed phenotypes ( left column ) versus that with the inflamed phenotype ( right column ). Expression values are represented as colors and range from red (high expression), pink (moderate), and light blue (low) to dark blue (lowest expression) ( E ). Abbreviations: PC, principal component; POLEmut, polymerase-epsilon mutation; MMRd, mismatch repair deficiency; NSMP, no specific molecular profile; p53abn, p53 abnormality
|
39751650_p30
|
39751650
|
Comparison of gene expression between the non-inflamed (excluded and desert) and inflamed phenotypes
| 4.200213 |
biomedical
|
Study
|
[
0.9994043111801147,
0.00034038297599181533,
0.0002553451049607247
] |
[
0.9994358420372009,
0.00019763009913731366,
0.0002987385669257492,
0.00006781948468415067
] |
en
| 0.999999 |
Our GSEA of the NSMP subtype with the non-inflamed phenotypes compared to that with the inflamed phenotype revealed that the expressions of type-1 interferon response gene sets were more enriched in the non-inflamed phenotypes versus the inflamed phenotype . The enrichment plots for the top two datasets enriched in the GSEA hallmark analysis are given in Fig. 5 B. The heat map of the top 50 marker genes for each phenotype in the NSMP subtype with the non-inflamed phenotypes and that with the inflamed phenotype is shown in Fig. 5 C. The top gene that was upregulated in the non-inflamed phenotype was OVOL2 . The top gene that was upregulated in the inflamed phenotype was HDC. Fig. 5 Gene set enrichment analysis (GSEA) results of the NSMP subtype with the non-inflamed (excluded or desert) phenotypes versus that with the inflamed phenotype in Cohort 2. The results of the GSEA hallmark analysis showing significantly enriched gene sets (FDR < 25% and a nominal p value < 5%). A positive normalized enrichment score indicates enrichment in the NSMP subtype with the non-inflamed phenotypes, and a negative score indicates enrichment in that with the inflamed phenotype ( A ). Enrichment plots for the top two datasets enriched in GSEA hallmark analysis, showing the profile of the running enrichment score and the positions of gene set members on the rank-ordered list ( B ). Heat map of the top 50 marker genes for each phenotype with the non-inflamed phenotypes ( left column ) versus that with the inflamed phenotype ( right column ). Expression values are represented as colors and range from red (high expression), pink (moderate), and light blue (low) to dark blue (lowest expression) ( C ). Abbreviation: NSMP, no specific molecular profile
|
39751650_p31
|
39751650
|
Comparison of gene expression between the non-inflamed (excluded and desert) and inflamed phenotypes
| 4.176056 |
biomedical
|
Study
|
[
0.9993776679039001,
0.00035522188409231603,
0.0002671488036867231
] |
[
0.9994921684265137,
0.00020160048734396696,
0.00024506915360689163,
0.00006118028977653012
] |
en
| 0.999999 |
Our investigation into the distribution and prognostic significance of CD8 + TILs in EC according to a molecular classification revealed crucial findings. Most notably, we identified three distinct immunophenotypes—inflamed, excluded, and desert—based on CD8 + TILs in EC patients. These immunophenotypes provide a more granular understanding of the immune landscape in EC, reflecting the diverse immunological responses triggered by tumor development. Our results demonstrated that inflamed phenotypes were associated with better prognosis, while excluded and desert phenotypes correlated with poorer outcomes. Importantly, the integration of immunophenotypes with the ProMisE molecular classification underscores the complexity of EC and highlights the need for personalized therapeutic strategies that consider both molecular and immunological characteristics.
|
39751650_p32
|
39751650
|
Discussion
| 4.191726 |
biomedical
|
Study
|
[
0.9994639754295349,
0.00034727973979897797,
0.0001886919344542548
] |
[
0.9979532957077026,
0.00025169638684019446,
0.0016959793865680695,
0.00009907047206070274
] |
en
| 0.999998 |
Interestingly, we observed that the EC patients with the excluded or desert phenotypes had poorer prognoses than those exhibiting inflamed phenotypes. This observation aligns with the increasing body of evidence suggesting that a robust anti-tumor immune response, represented by a high level of CD8 + TILs, is associated with better outcomes in various cancers, including EC . Additionally, TCGA-based analysis using the CIBERSORT algorithm supported this observation, revealing that cases with high CD8 + T-cell fractions (analogous to inflamed phenotypes) demonstrated better prognoses. These results emphasize the value of incorporating immunophenotypic evaluation, alongside molecular classification, into prognostic assessments for EC.
|
39751650_p33
|
39751650
|
Discussion
| 4.135058 |
biomedical
|
Study
|
[
0.9995218515396118,
0.0002909668837673962,
0.00018723240646068007
] |
[
0.9990623593330383,
0.00021336817007977515,
0.0006480502197518945,
0.00007624901627423242
] |
en
| 0.999996 |
When evaluated by the median density of CD8 + TILs in the CT and IM, the POLEmut and MMRd subtypes showed the inflamed phenotype; the NSMP subtype showed the desert phenotype; and the p53abn subtype showed the excluded phenotype. This insight suggests a potential correlation between the genomic background and CD8 + T-cell anti-tumor response in EC. It has been reported that POLEmut and MMRd subtypes have high tumor mutation burdens which contribute to the expression of neoantigens and they cause a strong anti-tumor response by CD8 + T-cell . The limited CD8 + T-cell response in the NSMP subtype suggests that this subtype is less likely to elicit a CD8 + T-cell response due to its low immunogenicity and may contribute to its resistance to immunotherapy strategies. Our observation that the p53abn subtype exhibited the excluded phenotype suggests that this subtype, despite eliciting an immune response, is resistant to immunotherapy because it has mechanisms that exclude CD8 + TILs from the central tumor. However, looking at individual cases, some of the POLEmut and MMRd subtypes showed the non-inflamed phenotypes, and some of the NSMP subtype showed the inflamed phenotype. It is thought that the formation of different immunophenotypes is due to some factors other than the number of genetic mutations or differences in immunogenicity.
|
39751650_p34
|
39751650
|
Discussion
| 4.254195 |
biomedical
|
Study
|
[
0.9994481205940247,
0.0003304209094494581,
0.00022152249584905803
] |
[
0.9990831613540649,
0.0002508223697077483,
0.0005831972812302411,
0.00008277082815766335
] |
en
| 0.999999 |
Our RNA-seq analysis provided valuable insights into the molecular mechanisms underlying the formation of inflamed and non-inflamed phenotypes within the ProMisE subtypes. In the MMRd subtype, non-inflamed phenotypes exhibited upregulation of CD99 and NLGN1, both of which have been implicated in immune suppression and tumor progression. There have been reports that high expression of CD99 in tumors is involved in the infiltration of immunosuppressive macrophages, in addition to the malignant transformation of tumor cells themselves , and some study reported that high expression of NLGN1 was a poor prognostic factor in colorectal cancer, prostate cancer, and pancreatic cancer . In contrast, inflamed phenotypes showed upregulation of BRINP1, a gene associated with immune cell differentiation and PD-L1 regulation in tumor cells, suggesting its role in fostering robust anti-tumor immune responses . In the NSMP subtype, the non-inflamed phenotypes were characterized by elevated expression of OVOL2, a potential tumor suppressor gene , However, the role of OVOL2 in tumor immunity remains unclear. In contrast, inflamed phenotypes showed increased expression of HDC, an enzyme involved in histamine production, which has recently been associated with immune modulation in the tumor microenvironment . There is scope for further exploration of the impact of these genes on tumor immunity.
|
39751650_p35
|
39751650
|
Discussion
| 4.367057 |
biomedical
|
Study
|
[
0.9994245767593384,
0.00035709995427168906,
0.00021835890947841108
] |
[
0.9981860518455505,
0.0003030765801668167,
0.0013970118016004562,
0.0001138294319389388
] |
en
| 0.999997 |
Our RNA-seq analysis also provided another layer of insight, suggesting that MYC target genes or type-1 interferon response genes might play a role in determining these immunophenotypes. The MYC oncogene is a well-documented driver of tumorigenesis in many cancers, and the type-1 interferon pathway is an important pathway in the antiviral response; however, their roles in the formation of the tumor immunosuppressive microenvironment remain unclear. Our findings suggest that the MYC signaling pathway or type-1 interferon pathway may, directly or indirectly, influence the distribution of CD8 + TILs and form a different immunophenotype, representing a novel avenue for future research.
|
39751650_p36
|
39751650
|
Discussion
| 4.188513 |
biomedical
|
Study
|
[
0.9995742440223694,
0.00021953867690172046,
0.00020623925956897438
] |
[
0.9993038177490234,
0.0002284653455717489,
0.00040334570803679526,
0.00006434023089241236
] |
en
| 0.999998 |
There are some limitations to our research. First, we only assessed CD8 + TILs in the evaluation of the immune microenvironment of endometrial cancer, To clarify the immune microenvironment, it is necessary to assess the distribution patterns of immune cells other than CD8 + T-cells and the interaction of each immune cell and also necessary to assess the expression of immune evasion molecules such as PD-L1 expression on tumor cells, which is a future task. Next, there is a lack of functional analysis of the MYC target genes and type-1 IFN genes in terms of their effects on immune phenotypes and EC patients’ clinical outcomes, which is another topic for future research. Furthermore, because the number of tumors in which RNA-seq was performed was limited, it was not possible to find the factors that cause the differences between the excluded and desert phenotypes. As it is predicted that it will be difficult to find the differences in these two non-inflamed phenotypes using RNA-seq of only one region in the CT, we believe that it will be necessary to compare the gene expression in the CT and the IM in future research.
|
39751650_p37
|
39751650
|
Discussion
| 4.048442 |
biomedical
|
Study
|
[
0.9995443224906921,
0.00021513162937480956,
0.00024058193957898766
] |
[
0.999208390712738,
0.00022032081324141473,
0.0005148059572093189,
0.00005648122532875277
] |
en
| 0.999995 |
In conclusion, our results suggest that evaluating not only the molecular classification but also the immunophenotype may lead to more accurate patients’ prognosis prediction in EC. Future studies exploring the role of the MYC signaling pathway or type-1 interferon pathway in shaping the immune landscape will undoubtedly provide further insights into the complex biology of EC. Elucidating the mechanisms that underlie the formation of the three immunophenotypes could lead to the discovery of novel immunotherapy targets.
|
39751650_p38
|
39751650
|
Discussion
| 4.035459 |
biomedical
|
Study
|
[
0.9995903372764587,
0.00024894715170376003,
0.00016080641944427043
] |
[
0.9967760443687439,
0.0003536942240316421,
0.002783561358228326,
0.00008670848910696805
] |
en
| 0.999995 |
Below is the link to the electronic supplementary material. Supplementary file 1 Supplementary file 2 (PDF 41 KB)
|
39751650_p39
|
39751650
|
Supplementary Information
| 1.044788 |
other
|
Other
|
[
0.2562398314476013,
0.0028378127608448267,
0.7409223318099976
] |
[
0.009614438749849796,
0.9888092875480652,
0.0010562385432422161,
0.0005200589657761157
] |
en
| 0.999996 |
Malignant neoplasm of the breast was the fifth leading cause of death among women in Germany in 2020, with 18,500 deaths, according to the last available statistics of the German Federal Statistical Office . Socioeconomic inequalities in mortality due to breast cancer (BC) have been documented in Germany both at the individual and district level , where low-income women or women living in areas with higher levels of deprivation entail a higher mortality risk.
|
PMC11699213_p0
|
PMC11699213
|
Introduction
| 2.541626 |
biomedical
|
Other
|
[
0.981641411781311,
0.0009609742555767298,
0.017397593706846237
] |
[
0.3543992340564728,
0.6337065696716309,
0.010915271006524563,
0.0009789153700694442
] |
en
| 0.999998 |
The European Commission encouraged Member States to implement organised screening programmes (OSP) in 2003, with invitations being sent out on a biannual basis to women aged between 50 and 69 . Since then, numerous European studies have reported a decrease in breast cancer mortality rates and a reduction in inequalities in access to screening services .
|
PMC11699213_p1
|
PMC11699213
|
Introduction
| 2.110677 |
biomedical
|
Other
|
[
0.9540270566940308,
0.003922629170119762,
0.042050402611494064
] |
[
0.02295364998281002,
0.956952691078186,
0.019348541274666786,
0.000745164230465889
] |
en
| 0.999996 |
Germany initiated the implementation of the OSP in 2005 and achieved full implementation by 2009 . The participation rate following an invitation has fluctuated between 43% and 55% over the past two decades, failing to reach the 70% benchmark recommended by the European Commission . Additionally, in 2020, 10.38% of the targeted women reported that they had never attended BCS in their lifetime .
|
PMC11699213_p2
|
PMC11699213
|
Introduction
| 1.866198 |
biomedical
|
Study
|
[
0.8613728880882263,
0.008029746823012829,
0.13059736788272858
] |
[
0.6009232401847839,
0.3960455358028412,
0.0018203724175691605,
0.001210808171890676
] |
en
| 0.999998 |
Several studies have investigated the sociodemographic characteristics of women who are at higher risk of not participating in breast cancer screening (BCS) programmes. In the most recent international systematic review, Mottram et al. observed that migrant women, women with lower socioeconomic status, without home ownership, and those who experienced false positives had the lowest attendance rates . In a scoping review of the German context, Pedrós Barnils et al. identified native women, women with lower incomes, women living in rural areas, and those not cohabiting with their partners as those with the lowest lifetime BCS attendance rates. However, the author also highlighted considerable heterogeneity in methods and, therefore, results .
|
PMC11699213_p3
|
PMC11699213
|
Introduction
| 3.87073 |
biomedical
|
Review
|
[
0.9924246072769165,
0.002244804287329316,
0.005330507643520832
] |
[
0.038380395621061325,
0.0008221991010941565,
0.9605700969696045,
0.00022724605514667928
] |
en
| 0.999996 |
Usually, inequalities in attendance are documented based on independent social dimensions. To explore correlations between social dimensions and BCS attendance, most studies incorporate variables deemed relevant (i.e. based on specific assumptions) into statistical models and then, in multivariate analyses, estimate the independent effect of each social dimension with the effects of other covariates held constant. However, as no individual can be defined by a single social dimension alone , it is unlikely that examining the independent effect of each social dimension will provide a comprehensive understanding of the inequalities in accessing cancer screenings.
|
PMC11699213_p4
|
PMC11699213
|
Introduction
| 3.640365 |
biomedical
|
Study
|
[
0.9949877262115479,
0.0003706666175276041,
0.004641667474061251
] |
[
0.9497860074043274,
0.008065477013587952,
0.0420067124068737,
0.00014173243835102767
] |
en
| 0.999999 |
Subsets and Splits
SQL Console for rntc/test-pp-aa
The query retrieves a sample of documents that are clinical cases with an educational score above 3, providing limited analytical value.
Clinical Cases Sample
Returns a sample of 100 clinical case documents, providing a basic overview of the document type's content.