Figure 3 ( A ) Confocal immunofluorescence of muscle biopsy from Pt 01 ( tibialis anterior post - treatment ) stained with anti - dystrophin dys1 antibody ( which recognizes protein fragment encoded by exons 26 - 30 , green signal ) and anti laminin - 2 ( to delineate muscle fibers , red signal ) ; dapi identifies nuclei ( blue signal ) . In the left image only anti dystrophin staining is shown . No dystrophin positive fibers were observed . ( B ) Confocal immunofluorescence of muscle biopsy from Pt 02 ( tibialis anterior post - treatment ) stained with anti - dystrophin dys1 antibody ( green signal ) and anti lamininin - 2 ( red signal ) ; dapi identifies nuclei ( blue signal ) . In the left image only anti dystrophin staining is shown . Some fiber shows mild and discontinuous dystrophin staining . ( C ) Immunofluorescence of muscle biopsy from Pt 05 taken before ( left , gastrocnemius ) and after treatment ( right , gastrocnemius ) stained with anti dystrophin dys2 antibody ( which recognizes exon 77 - 79 ) . The number and intensity of dystrophin - positive fibers is increased in the post - treatment biopsy . ( D ) Immunofluorescence of muscle biopsy from Pt 06 taken before ( left , gastrocnemius ) and after treatment ( right , gastrocnemius ) stained with anti dystrophin dys2 antibody . Few fibers show scattered dystrophin staining , without obvious differences between pre and post - treatment samples . ( E ) Results of western blot analysis involving dystrophin antibodies dys1 , Mandys18 and Manex46e ( recognizing respectively exon 26 - 30 , 17 - 35 , and 46 ) , of total protein extracts ( 20 µg ) obtained from post - treatment biopsy specimens of Pt 01 ( tibialis anterior muscle ) ; Ct was used as positive dystrophin control . Below , bands corresponding to myosin heavy chain are shown as loading control . No bands corresponding to full length dystrophin were observed . A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the western blot . ( F ) Results of western blot analysis involving dystrophin antibodies Mandys18 and Manex46e , of total protein extracts ( 10 - 20 µg ) obtained from pre - treatment ( performed at time of diagnosis ) and post - treatment biopsy specimens of Pt 02 ( tibialis anterior muscle ) ; Ct was used as positive dystrophin control . Below , bands corresponding to myosin heavy chain are shown as loading control . One faint band corresponding to full length dystrophin is observed only in the post - treatment samples with both Mandys18 and Manex46e antibodies . A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the western blot . ( G ) Results of western blot analysis involving dystrophin antibodies Mandys106 ( recognizing exon 43 ) and dys1 , of total protein extracts ( 80 µg , following protein concentration by Amicon Ultra - 0 . 5 centrifugal filter devices , Millipore ) obtained from pre - treatment and post - treatment biopsy specimens of Pt 05 ( gastrocnemius ) ; Ct was used as positive dystrophin control , CtDmd was used as dystrophin negative control . Below , bands corresponding to myosin heavy chain are shown as loading control . Bands corresponding approximately to full length dystrophin are observed in pre and post - treatment samples with both antibodies However , bands in post - treatment sample appeared higher in amount . A schematic representation of the dystrophin point mutation ( black vertical bar ) and the region recognized by used antibodies is depicted above the western blot . ( H ) Results of western blot analysis involving dystrophin antibodies Mandys106 and dys1 , of total protein extracts ( 80 µg , following protein concentration by Amicon Ultra - 0 . 5 centrifugal filter devices , Millipore ) obtained from pre - treatment and post - treatment biopsy specimens of Pt 06 ( gastrocnemius ) ; Ct was used as positive dystrophin control . Below , bands corresponding to myosin heavy chain are shown as loading control . Bands corresponding approximately to full length dystrophin are observed in pre and post - treatment samples with both antibodies Bands in pre - treatment sample appeared higher in amount as compared to post - treatment sample . A schematic representation of the dystrophin point mutation ( black vertical bar ) and the region recognized by used antibodies is depicted above the western blot .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Confocal immunofluorescence of muscle biopsy from Pt 01 (tibialis anterior post-treatment) stained with anti-dystrophin dys1 antibody (which recognizes protein fragment encoded by exons 26-30, green signal) and anti laminin-2 (to delineate muscle fibers, red signal); dapi identifies nuclei (blue signal). In the left image only anti dystrophin staining is shown. No dystrophin positive fibers were observed.(B) Confocal immunofluorescence of muscle biopsy from Pt 02 (tibialis anterior post-treatment) stained with anti-dystrophin dys1 antibody (green signal) and anti lamininin-2 (red signal); dapi identifies nuclei (blue signal). In the left image only anti dystrophin staining is shown. Some fiber shows mild and discontinuous dystrophin staining.(C) Immunofluorescence of muscle biopsy from Pt 05 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti dystrophin dys2 antibody (which recognizes exon 77-79). The number and intensity of dystrophin-positive fibers is increased in the post-treatment biopsy.(D) Immunofluorescence of muscle biopsy from Pt 06 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti dystrophin dys2 antibody. Few fibers show scattered dystrophin staining, without obvious differences between pre and post-treatment samples.(E) Results of western blot analysis involving dystrophin antibodies dys1, Mandys18 and Manex46e (recognizing respectively exon 26-30, 17-35, and 46), of total protein extracts (20 µg) obtained from post-treatment biopsy specimens of Pt 01 (tibialis anterior muscle); Ct was used as positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as loading control. No bands corresponding to full length dystrophin were observed. A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the western blot.(F) Results of western blot analysis involving dystrophin antibodies Mandys18 and Manex46e, of total protein extracts (10-20 µg) obtained from pre-treatment (performed at time of diagnosis) and post-treatment biopsy specimens of Pt 02 (tibialis anterior muscle); Ct was used as positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as loading control. One faint band corresponding to full length dystrophin is observed only in the post-treatment samples with both Mandys18 and Manex46e antibodies. A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the western blot.(G) Results of western blot analysis involving dystrophin antibodies Mandys106 (recognizing exon 43) and dys1, of total protein extracts (80 µg, following protein concentration by Amicon Ultra-0.5 centrifugal filter devices, Millipore) obtained from pre-treatment and post-treatment biopsy specimens of Pt 05 (gastrocnemius); Ct was used as positive dystrophin control, CtDmd was used as dystrophin negative control. Below, bands corresponding to myosin heavy chain are shown as loading control. Bands corresponding approximately to full length dystrophin are observed in pre and post-treatment samples with both antibodies However, bands in post-treatment sample appeared higher in amount. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by used antibodies is depicted above the western blot.(H) Results of western blot analysis involving dystrophin antibodies Mandys106 and dys1, of total protein extracts (80 µg, following protein concentration by Amicon Ultra-0.5 centrifugal filter devices, Millipore) obtained from pre-treatment and post-treatment biopsy specimens of Pt 06 (gastrocnemius); Ct was used as positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as loading control. Bands corresponding approximately to full length dystrophin are observed in pre and post-treatment samples with both antibodies Bands in pre-treatment sample appeared higher in amount as compared to post-treatment sample. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by used antibodies is depicted above the western blot.


Figure 4 ( A ) Axial T1 - weighted images of the right thighs obtained in patients immediately before ( upper panel ) and 18 months after MAB infusion . Please note the progression of fatty degeneration and atrophy except for Pt 05 where no evident modifications can be detected . ( B ) Representative percentile of MRI quantitative parameters in transplanted patients compared to untreated patients . SIR ( left image ) and MVI ( right image ) of the quadriceps . Red line : Pt 01 ; dotted green line : Pt 02 ; dotted purple line : Pt 03 ; dotted dark blue line : Pt 05 ; dotted light blue line : Pt 06 ) . Empty dots correspond to pre - treatment measures . Orange dots correspond to post - transplantation measurement . The black line corresponds to the median , while gray dotted lines to 75 ° , 90 ° and 95 ° percentile . Please note the modification of the trend of Pt 05 after treatment . ( C ) Representative quantile spline regressions of post - transplant trend amelioration of MRI quantitative parameters in Pt 05 . Upper left : SIR of the quadriceps . Lower left : SIR of thigh flexor muscles . Upper right : MVI of the quadriceps . Lower right : MVI of the semitendinosus muscles . Please note the significant modification of the patient trend ( red lines ) compared to median measurements obtained with 85 MRI examinations of untreated patients . Empty dots correspond to pre - treatment measures . Full dots correspond to post - transplantation measurement . The grey dotted line corresponds to the time of the first intra - arterial transplantation .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Axial T1-weighted images of the right thighs obtained in patients immediately before (upper panel) and 18 months after MAB infusion. Please note the progression of fatty degeneration and atrophy except for Pt 05 where no evident modifications can be detected. (B) Representative percentile of MRI quantitative parameters in transplanted patients compared to untreated patients. SIR (left image) and MVI (right image) of the quadriceps. Red line: Pt 01; dotted green line: Pt 02; dotted purple line: Pt 03; dotted dark blue line: Pt 05; dotted light blue line: Pt 06). Empty dots correspond to pre-treatment measures. Orange dots correspond to post-transplantation measurement. The black line corresponds to the median, while gray dotted lines to 75°, 90° and 95° percentile. Please note the modification of the trend of Pt 05 after treatment.(C) Representative quantile spline regressions of post-transplant trend amelioration of MRI quantitative parameters in Pt 05. Upper left: SIR of the quadriceps. Lower left: SIR of thigh flexor muscles. Upper right: MVI of the quadriceps. Lower right: MVI of the semitendinosus muscles. Please note the significant modification of the patient trend (red lines) compared to median measurements obtained with 85 MRI examinations of untreated patients. Empty dots correspond to pre-treatment measures. Full dots correspond to post-transplantation measurement. The grey dotted line corresponds to the time of the first intra-arterial transplantation.


Figure 5 ( A ) North Star scale measurement ( NSAA score ) plotted against age of ambulant DMD patients before , throughout and after MAB clinical trial . Arrows indicate time points of MAB infusion . Dotted line indicates the time period in which Pt05 spontaneously suspended steroids treatment without communication to parents and clinicians . Pt 02 and Pt 05 showed score stabilization throughout eight months of MAB infusions ; Pt05 showed stabilization even in the subsequent period , whereas Pt02 showed progressive deterioration until lost of ambulation . Pt 03 showed progressive deterioration throughout MAB infusion and lost ambulation soon after the end of the trial . ( B ) Six minute walking test ( meters ) plotted against age of ambulant DMD patients before , throughout and after MAB clinical trial . Arrows indicate time points of MAB infusion . Dotted line indicates the time period in which Pt05 spontaneously suspended steroids treatment without communication to parents and clinicians . Pt 02 and Pt 05 showed score stabilization throughout 8 months of MAB infusions ; Pt05 showed stabilization even in the subsequent period , whereas Pt 02 showed progressive deterioration until lost of ambulation . Pt 03 showed progressive deterioration throughout MAB infusion and lost ambulation soon after the end of the trial . ( C ) Time to run 10 meters ( time ) plotted against age of ambulant DMD patients before , throughout and after MAB clinical trial . Arrows indicate time points of MAB infusion . Dotted line indicates the time period in which Pt 05 spontaneously suspended steroids treatment without communication to parents and clinicians . Pt 02 and Pt 05 showed time stabilization throughout 8 months of MAB infusions ; Pt 05 showed stabilization even in the subsequent period , whereas Pt 02 showed progressive increase of time until lost of ambulation . Pt 03 showed progressive increase of time throughout MAB infusion and lost ambulation soon after the end of the trial .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) North Star scale measurement (NSAA score) plotted against age of ambulant DMD patients before, throughout and after MAB clinical trial. Arrows indicate time points of MAB infusion. Dotted line indicates the time period in which Pt05 spontaneously suspended steroids treatment without communication to parents and clinicians. Pt 02 and Pt 05 showed score stabilization throughout eight months of MAB infusions; Pt05 showed stabilization even in the subsequent period, whereas Pt02 showed progressive deterioration until lost of ambulation. Pt 03 showed progressive deterioration throughout MAB infusion and lost ambulation soon after the end of the trial.(B) Six minute walking test (meters) plotted against age of ambulant DMD patients before, throughout and after MAB clinical trial. Arrows indicate time points of MAB infusion. Dotted line indicates the time period in which Pt05 spontaneously suspended steroids treatment without communication to parents and clinicians. Pt 02 and Pt 05 showed score stabilization throughout 8 months of MAB infusions; Pt05 showed stabilization even in the subsequent period, whereas Pt 02 showed progressive deterioration until lost of ambulation. Pt 03 showed progressive deterioration throughout MAB infusion and lost ambulation soon after the end of the trial.(C) Time to run 10 meters (time) plotted against age of ambulant DMD patients before, throughout and after MAB clinical trial. Arrows indicate time points of MAB infusion. Dotted line indicates the time period in which Pt 05 spontaneously suspended steroids treatment without communication to parents and clinicians. Pt 02 and Pt 05 showed time stabilization throughout 8 months of MAB infusions; Pt 05 showed stabilization even in the subsequent period, whereas Pt 02 showed progressive increase of time until lost of ambulation. Pt 03 showed progressive increase of time throughout MAB infusion and lost ambulation soon after the end of the trial.


Figure 6Panel A : viral and donor specific T - cell responses were measured by IFN - γELISpot at different time points : before the beginning of treatment ( PRE ) , prior to each infusion and 2 months after the fourth infusion . Infusions are indicated by arrows . Patient ' s peripheral blood mononuclear cells ( PBMC ) were challenged with irradiated autologous lymphoblastoid cell lines ( EBV line host ) , with cytomegalovirus glycin extract ( CMV antigen ) ( Gehrz et al , 1987 ) and with the following irradiated cells harvested from the donor : untreated mesoangioblasts ( MABdonor ) , MAB activated by 48 hour - exposure to 500 IU / mL IFN - γ ( γMABdonor ) , myotubes differentiated from MAB ( myotubesdonor ) and PBMC ( PBMCdonor ) . Polyclonal stimulation ( phytohemagglutinin , PHA ) was used as positive control . Donor PBMC were challenged with autologous targets as negative controls . Results are expressed as number of specific cells / 105PBMC and calculated according to the following formula : number of spots produced by patient ' s PBMC - number of spots produced by donor ' s PBMC . Results for patientsPt 01 , Pt 02 , Pt 03 , Pt 05 and Pt 06 are shown . Panel B : dystrophin specific T - cell responses were measured by IFN - γ ELISpot at two time points : before the beginning of treatment ( PRE , white bar ) and 2 months after the fourth infusion ( black bar ) . PBMC were challenged with a library of 15 - mer overlapping peptides covering the protein sequence ( pool 1 - 8 ) . Polyclonal stimulation ( phytohemagglutinin , PHA ) was used as positive control . The grey area represents the threshold calculated on a cohort of healthy donors . Results for Pt 01 , Pt 02 , Pt 03 , Pt 05 and Pt 06 are shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Panel A: viral and donor specific T-cell responses were measured by IFN-γELISpot at different time points: before the beginning of treatment (PRE), prior to each infusion and 2 months after the fourth infusion. Infusions are indicated by arrows. Patient's peripheral blood mononuclear cells (PBMC) were challenged with irradiated autologous lymphoblastoid cell lines (EBV line host), with cytomegalovirus glycin extract (CMV antigen)(Gehrz et al, 1987) and with the following irradiated cells harvested from the donor: untreated mesoangioblasts (MABdonor), MAB activated by 48 hour-exposure to 500 IU/mL IFN-γ (γMABdonor), myotubes differentiated from MAB (myotubesdonor) and PBMC (PBMCdonor). Polyclonal stimulation (phytohemagglutinin, PHA) was used as positive control. Donor PBMC were challenged with autologous targets as negative controls. Results are expressed as number of specific cells/105PBMC and calculated according to the following formula: number of spots produced by patient's PBMC - number of spots produced by donor's PBMC. Results for patientsPt 01, Pt 02, Pt 03, Pt 05 and Pt 06 are shown.Panel B: dystrophin specific T-cell responses were measured by IFN-γ ELISpot at two time points: before the beginning of treatment (PRE, white bar) and 2 months after the fourth infusion (black bar). PBMC were challenged with a library of 15-mer overlapping peptides covering the protein sequence (pool 1-8). Polyclonal stimulation (phytohemagglutinin, PHA) was used as positive control. The grey area represents the threshold calculated on a cohort of healthy donors. Results for Pt 01, Pt 02, Pt 03, Pt 05 and Pt 06 are shown.


Figure 1Macrophage - conditioned medium induces MCF10A colony formation in soft agar as compared to control medium . Colonies ≥ 50 μm were counted at 5 weeks . Error bars represent mean ± SEM from 10 independent experiments ( n = 3 per condition ) . Macrophage donors are indicated as D1 - D16 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Representative images of MCF10A spheroids categorized into 4 groups according to the filling of the lumen with cell nuclei ( clear , partially filled , almost filled and filled ) . Spheroids stained for DNA ( Hoechst 33342 in blue ) and F - actin ( Phalloidin in red ) . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Macrophage - conditioned medium induces filling of the spheroid lumen with cell nuclei compared to control . Error bars represent mean ± SEM from 11 independent experiments ( n = 2 per condition ; 50 spheroids each ) . Macrophage donors are indicated as D1 - D16 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Representative images of Ki67 positive nuclei of MCF10A cells . Spheroids stained for DNA ( Hoechst 33342 in blue ) , F - actin ( Phalloidin in red ) and Ki67 ( green ) . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Quantitative analysis of Ki67 positive nuclei per spheroid indicates enhanced proliferation of MCF10A cells upon stimulation with M1 macrophage - conditioned media . Error bars represent mean ± SEM from 3 independent experiments ( 20 - 30 non - empty spheroids per condition ) . Macrophage donors are indicated as D1 - D16 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . NSC23766 ( Rac1 inhibitor ; 50 μM ) reduces the filling of the spheroid lumen with cell nuclei upon macrophage - conditioned medium stimulation compared to control . Filling of the spheroid lumen with cell nuclei categorized into 4 groups ( clear , partially filled , almost filled and filled ) . Error bars represent mean ± SEM from 3 independent experiments ( n = 2 per condition ; 50 spheroids each ) . Partially filled , almost filled and filled MCF10A spheroids were combined together ( non - empty spheroids ) for statistical analysis . Macrophage donors are indicated as D1 - D16 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Invasive protrusions of spheroids into matrigel / collagen mix marked with a white arrow . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . 3D structures of spheroids stained for DNA ( Hoechst 33342 in blue ) , F - actin ( Phalloidin in red ) and laminin V ( green ) . Laminin V staining indicates the loss of basal membrane continuity at invasive protrusions . Invasive protrusions are marked with a white arrow . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Macrophage - conditioned medium induces invasive protrusions in MCF10A spheroids compared to control . Error bars represent mean ± SEM from 11 independent experiments ( n = 2 per condition ; at least 15 spheroids each from at least 2 fields of view ) . Macrophage donors are indicated as D1 - D16 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Macrophage-conditioned medium induces MCF10A colony formation in soft agar as compared to control medium. Colonies ≥ 50 μm were counted at 5 weeks. Error bars represent mean ± SEM from 10 independent experiments (n=3 per condition). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Representative images of MCF10A spheroids categorized into 4 groups according to the filling of the lumen with cell nuclei (clear, partially filled, almost filled and filled). Spheroids stained for DNA (Hoechst 33342 in blue) and F-actin (Phalloidin in red).16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Macrophage-conditioned medium induces filling of the spheroid lumen with cell nuclei compared to control. Error bars represent mean ± SEM from 11 independent experiments (n=2 per condition; 50 spheroids each). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Representative images of Ki67 positive nuclei of MCF10A cells. Spheroids stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and Ki67 (green).16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Quantitative analysis of Ki67 positive nuclei per spheroid indicates enhanced proliferation of MCF10A cells upon stimulation with M1 macrophage-conditioned media. Error bars represent mean ± SEM from 3 independent experiments (20-30 non-empty spheroids per condition). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. NSC23766 (Rac1 inhibitor; 50 μM) reduces the filling of the spheroid lumen with cell nuclei upon macrophage-conditioned medium stimulation compared to control. Filling of the spheroid lumen with cell nuclei categorized into 4 groups (clear, partially filled, almost filled and filled). Error bars represent mean ± SEM from 3 independent experiments (n=2 per condition; 50 spheroids each). Partially filled, almost filled and filled MCF10A spheroids were combined together (non-empty spheroids) for statistical analysis. Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Invasive protrusions of spheroids into matrigel/collagen mix marked with a white arrow.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. 3D structures of spheroids stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and laminin V (green). Laminin V staining indicates the loss of basal membrane continuity at invasive protrusions. Invasive protrusions are marked with a white arrow.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Macrophage-conditioned medium induces invasive protrusions in MCF10A spheroids compared to control. Error bars represent mean ± SEM from 11 independent experiments (n=2 per condition; at least 15 spheroids each from at least 2 fields of view). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


Figure 2Mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice on normal ( ND ) Organoids isolated from ND mice were cultured in matrigel / collagen mix for 2 days followed by macrophage - conditioned or control medium stimulation for 24 h . Representative images of organoids in matrigel / collagen mix . Mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice on normal ( ND ) Organoids isolated from ND mice were cultured in matrigel / collagen mix for 2 days followed by macrophage - conditioned or control medium stimulation for 24 h . Macrophage - conditioned medium induces invasive protrusions in organoids as compared to control . Error bars represent mean ± SEM from 3 independent experiments , with each mouse labelled with a different symbol shape ( n = 2 per condition ; 15 - 25 organoids each ) . Macrophage donors are indicated as D14 , D15 , D17 , D18 . M1A - M1 activated , M2A - M2 activated macrophages . Mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice on normal ( ND ) Organoids isolated from ND mice were cultured in matrigel / collagen mix for 2 days followed by macrophage - conditioned or control medium stimulation for 24 h . 3D structure of organoids stained for DNA ( Hoechst 33342 in blue ) , F - actin ( Phalloidin in red ) and α - SMA ( green ) . Bi - layered structure of internal luminal cells and external basal myoepithelial cells is established for non - invasive organoids . Mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice on normal ( ND ) or high fat diet ( HFD ) ( n = 3 ) . Organoids isolated from ND or HFD mice were cultured in collagen for 2 days . Representative images of organoids cultured in collagen for 2 days . The number of invasive protrusions per organoid is higher for organoids isolated from mice on HFD compared to mice on ND . Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse ( labelled with a different symbol shape ) . Mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice on high fat diet ( HFD ) ( n = 3 ) . Organoids isolated from HFD mice were cultured in collagen for 2 days . 3D structure of organoid stained for DNA ( Hoechst 33342 in blue ) , F - actin ( Phalloidin in red ) and α - SMA ( green ) . α - SMA , myoepithelial cell marker , appeared to be loss at invasive protrusions . Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment . Reformed ducts were then cultured for 7 days in macrophage - conditioned or control medium . Representative images of ductal structures cultured with conditioned medium . Ducts were stained for DNA ( DAPI in blue ) and F - actin ( Phalloidin in red ) . Macrophage - conditioned medium induces filling of the duct lumen with cell nuclei compared to control . Error bars represent mean ± SEM ( A total of 24 structures was counted per each condition : 2 patient ductal structures ( n = 2 ) , each labelled with a different symbol shape , were cultured with macrophage - conditioned media from either donor 17 or donor 18 , 3 ducts each ) . Filling of the lumen was determined as % of luminal space filled with cells . Macrophage donors are indicated as D14 , D15 , D17 , D18 . M1A - M1 activated , M2A - M2 activated macrophages .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from ND mice were cultured in matrigel/collagen mix for 2 days followed by macrophage-conditioned or control medium stimulation for 24h. Representative images of organoids in matrigel/collagen mix.Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from ND mice were cultured in matrigel/collagen mix for 2 days followed by macrophage-conditioned or control medium stimulation for 24h. Macrophage-conditioned medium induces invasive protrusions in organoids as compared to control. Error bars represent mean ± SEM from 3 independent experiments, with each mouse labelled with a different symbol shape (n=2 per condition; 15-25 organoids each). Macrophage donors are indicated as D14, D15, D17, D18. M1A - M1 activated, M2A - M2 activated macrophages.Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from ND mice were cultured in matrigel/collagen mix for 2 days followed by macrophage-conditioned or control medium stimulation for 24h. 3D structure of organoids stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and α-SMA (green). Bi-layered structure of internal luminal cells and external basal myoepithelial cells is established for non-invasive organoids.Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) or high fat diet (HFD) (n=3). Organoids isolated from ND or HFD mice were cultured in collagen for 2 days. Representative images of organoids cultured in collagen for 2 days. The number of invasive protrusions per organoid is higher for organoids isolated from mice on HFD compared to mice on ND. Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse (labelled with a different symbol shape).Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on high fat diet (HFD) (n=3). Organoids isolated from HFD mice were cultured in collagen for 2 days. 3D structure of organoid stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and α-SMA (green). α-SMA, myoepithelial cell marker, appeared to be loss at invasive protrusions.Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium. Representative images of ductal structures cultured with conditioned medium. Ducts were stained for DNA (DAPI in blue) and F-actin (Phalloidin in red). Macrophage-conditioned medium induces filling of the duct lumen with cell nuclei compared to control. Error bars represent mean ± SEM (A total of 24 structures was counted per each condition: 2 patient ductal structures (n=2), each labelled with a different symbol shape, were cultured with macrophage-conditioned media from either donor 17 or donor 18, 3 ducts each). Filling of the lumen was determined as % of luminal space filled with cells. Macrophage donors are indicated as D14, D15, D17, D18. M1A - M1 activated, M2A - M2 activated macrophages.


Figure 3IKKε expression was assessed semi - quantitatively using a 0 - 3 grade scale ( " 0 " : no staining ; " 1 " : weak ; " 2 " : moderate ; " 3 " : strong ) on human breast cancer sections using immunohistochemistry . Representative images according to the IKKε staining grade are shown . Scale bar : 100 μm . Distribution table of IKKε expression in the cohort of 66 human breast carcinomas according to the staining grade . Correlation between IKBKE mRNA levels ( log2 ratio ) and immune signature ( Yoshihara et al , 2013 ) in the METABRIC transcriptomic dataset from 1981 breast cancer patients ( Curtis et al , 2012 ) . Pearson ' s correlation rho coefficient and significance of difference from slope = 0 ( p ) are shown . IKBKE gene copy number and mRNA levels ( log2 ratio ) from the METABRIC transcriptomic dataset ( see D ) are plotted . Estimated + 1 copy number values are shown by the dotted vertical line . No significant correlation by Pearson ' s correlation rho coefficient and significance of difference from slope = 0 ( p ) was detected .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3IKKε expression was assessed semi-quantitatively using a 0-3 grade scale ("0": no staining; "1": weak; "2": moderate; "3": strong) on human breast cancer sections using immunohistochemistry. Representative images according to the IKKε staining grade are shown. Scale bar: 100 μm. Distribution table of IKKε expression in the cohort of 66 human breast carcinomas according to the staining grade.Correlation between IKBKE mRNA levels (log2 ratio) and immune signature (Yoshihara et al, 2013) in the METABRIC transcriptomic dataset from 1981 breast cancer patients (Curtis et al, 2012). Pearson's correlation rho coefficient and significance of difference from slope = 0 (p) are shown. IKBKE gene copy number and mRNA levels (log2 ratio) from the METABRIC transcriptomic dataset (see D) are plotted. Estimated +1 copy number values are shown by the dotted vertical line. No significant correlation by Pearson's correlation rho coefficient and significance of difference from slope = 0 (p) was detected.


Figure 4Amlexanox inhibits colony formation of MCF10A cells grown in soft agar with macrophage - conditioned medium for 5 weeks compared to controls . Colonies ≥ 50 μm were counted . Error bars represent mean ± SEM from 10 independent experiments ( n = 3 per condition ) . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of amlexanox . Representative images showing the filling of the spheroid lumen with cell nuclei . The spheroids were stained for DNA ( Hoechst 33342 in blue ) and F - actin ( Phalloidin in red ) . Scale bar : 50 μm . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of amlexanox . Amlexanox reduces filling of the spheroid lumen with cell nuclei . Filling of the spheroid lumen with cell nuclei categorized into 4 groups ( clear , partially filled , almost filled and filled ) . Error bars represent mean ± SEM from 7 independent experiments ( n = 2 per condition ; 50 spheroids each ) . Partially filled , almost filled and filled spheroids were combined together ( non - empty spheroids ) for statistical analysis . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of amlexanox . Amlexanox reduces filling of the spheroid lumen with cell nuclei . Filling of the spheroid lumen with cell nuclei . Partially filled , almost filled and filled spheroids were combined together ( non - empty spheroids ) . Error bars represent mean ± SEM from 7 independent experiments ( n = 2 per condition ; 50 spheroids each ) . Partially filled , almost filled and filled spheroids were combined together ( non - empty spheroids ) for statistical analysis . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of amlexanox . Amlexanox reduces the number of spheroids with invasive protrusions . Error bars represent mean ± SEM from 8 independent experiments ( n = 2 per condition , at least 15 spheroids each from at least 2 fields of view ) . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . Mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice on normal ( ND ) Organoids isolated from mice on ND were cultured in matrigel / collagen mix for 2 days and then stimulated with macrophage - conditioned or control medium in presence of amlexanox . Amlexanox reduces the number of invasive organoids . Error bars represent mean ± SEM from 3 independent experiments , with each mouse labelled with a different symbol shape ( n = 2 per condition ; 15 - 25 organoids each ) . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages . Mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice on normal ( ND ) or high fat diet ( HFD ) ( n = 3 ) . Organoids were cultured in collagen for 2 days after which the number of invasive protrusions per each organoid was determined microscopically . Amlexanox reduces the number of invasive protrusions in organoids isolated from mice on G ) ND or H ) HFD . Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse ( labelled with a different symbol shape ) . Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment . Reformed ducts were then cultured for 7 days in macrophage - conditioned or control medium in presence of amlexanox . Representative images showing the filling of the ductal lumen with cell nuclei . The ducts were stained for DNA ( DAPI in blue ) and F - actin ( Phalloidin in red ) . Scale bar : 20 μm . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages . Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment . Reformed ducts were then cultured for 7 days in macrophage - conditioned or control medium in presence of amlexanox . Amlexanox inhibits filling of the duct lumen with cell nuclei . Error bars represent mean ± SEM ( A total of 24 structures was counted per each condition : 2 patient ductal structures ( n = 2 ) , each labelled with a different symbol shape , were cultured with macrophage - conditioned media from either donor 17 or donor 18 , 3 ducts each ) . Filling of the lumen was determined as % of luminal space filled with cells . Macrophage donors are indicated as M1A - M1 activated , M2 activated macrophages . IKBKE gene ( encoding for IKKε ) was deleted in MCF10A cells via CRISPR - Cas9 technology . 3 knockout clones were combined for each cell line . IKKε CRISPR - Cas9 knockout MCF10A cells form less colonies when grown in soft agar with macrophage - conditioned medium for 5 weeks compared to CRISPR - Cas9 control ( nontargeting crRNA ) cells . Colonies ≥ 50 μm were counted . Error bars represent mean ± SEM from 3 independent experiments ( n = 3 per condition ) . Media from two macrophage donors were combined in 1 : 1 ratio in each experiment . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages . IKBKE gene ( encoding for IKKε ) was deleted in MCF10A cells via CRISPR - Cas9 technology . 3 knockout clones were combined for each cell line . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . IKKε CRISPR - Cas9 knockout MCF10A cells are less invasive compared CRISPR Control ( nontargeting crRNA ) cells . Error bars represent mean ± SEM from 3 independent experiments ( n = 3 per condition ) . Media from two macrophage donors were combined in 1 : 1 ratio in each experiment . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Amlexanox inhibits colony formation of MCF10A cells grown in soft agar with macrophage-conditioned medium for 5 weeks compared to controls. Colonies ≥ 50 μm were counted. Error bars represent mean ± SEM from 10 independent experiments (n=3 per condition). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Representative images showing the filling of the spheroid lumen with cell nuclei. The spheroids were stained for DNA (Hoechst 33342 in blue) and F-actin (Phalloidin in red). Scale bar: 50 μm. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces filling of the spheroid lumen with cell nuclei. Filling of the spheroid lumen with cell nuclei categorized into 4 groups (clear, partially filled, almost filled and filled). Error bars represent mean ± SEM from 7 independent experiments (n=2 per condition; 50 spheroids each). Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids) for statistical analysis. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces filling of the spheroid lumen with cell nuclei. Filling of the spheroid lumen with cell nuclei. Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids). Error bars represent mean ± SEM from 7 independent experiments (n=2 per condition; 50 spheroids each). Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids) for statistical analysis. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces the number of spheroids with invasive protrusions. Error bars represent mean ± SEM from 8 independent experiments (n=2 per condition, at least 15 spheroids each from at least 2 fields of view). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from mice on ND were cultured in matrigel/collagen mix for 2 days and then stimulated with macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces the number of invasive organoids. Error bars represent mean ± SEM from 3 independent experiments, with each mouse labelled with a different symbol shape (n=2 per condition; 15-25 organoids each). Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) or high fat diet (HFD) (n=3). Organoids were cultured in collagen for 2 days after which the number of invasive protrusions per each organoid was determined microscopically. Amlexanox reduces the number of invasive protrusions in organoids isolated from mice on G) ND or H) HFD. Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse (labelled with a different symbol shape).Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium in presence of amlexanox. Representative images showing the filling of the ductal lumen with cell nuclei. The ducts were stained for DNA (DAPI in blue) and F-actin (Phalloidin in red). Scale bar: 20 μm. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium in presence of amlexanox. Amlexanox inhibits filling of the duct lumen with cell nuclei. Error bars represent mean ± SEM (A total of 24 structures was counted per each condition: 2 patient ductal structures (n=2), each labelled with a different symbol shape, were cultured with macrophage-conditioned media from either donor 17 or donor 18, 3 ducts each). Filling of the lumen was determined as % of luminal space filled with cells. Macrophage donors are indicated as M1A - M1 activated, M2 activated macrophages.IKBKE gene (encoding for IKKε) was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line. IKKε CRISPR-Cas9 knockout MCF10A cells form less colonies when grown in soft agar with macrophage-conditioned medium for 5 weeks compared to CRISPR-Cas9 control (nontargeting crRNA) cells. Colonies ≥ 50 μm were counted. Error bars represent mean ± SEM from 3 independent experiments (n=3 per condition). Media from two macrophage donors were combined in 1:1 ratio in each experiment. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.IKBKE gene (encoding for IKKε) was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line. 16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. IKKε CRISPR-Cas9 knockout MCF10A cells are less invasive compared CRISPR Control (nontargeting crRNA) cells. Error bars represent mean ± SEM from 3 independent experiments (n=3 per condition). Media from two macrophage donors were combined in 1:1 ratio in each experiment. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.


Figure 5PSAT1 expression was assessed semi - quantitatively using a 0 - 3 grade scale ( " 0 " : no staining ; " 1 " : weak ; " 2 " : moderate ; " 3 " : strong ) on human breast cancer sections using immunohistochemistry . Representative images according to the PSAT1 staining grade are shown . Scale bar : 50 μm . Correlation of mRNA levels of the SBP enzyme genes PSAT1 ( E ) , PHGDH ( F ) , PSPH ( G ) with the immune signature ( Yoshihara et al , 2013 ) in the METABRIC transcriptomic dataset from 1981 breast cancer patient ( Curtis et al , 2012 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5PSAT1 expression was assessed semi-quantitatively using a 0-3 grade scale ("0": no staining; "1": weak; "2": moderate; "3": strong) on human breast cancer sections using immunohistochemistry. Representative images according to the PSAT1 staining grade are shown. Scale bar: 50 μm.Correlation of mRNA levels of the SBP enzyme genes PSAT1 (E), PHGDH (F), PSPH (G) with the immune signature (Yoshihara et al, 2013) in the METABRIC transcriptomic dataset from 1981 breast cancer patient (Curtis et al, 2012).


Figure 6NCT502 ( 10 μM ) inhibits colony formation of MCF10A cells grown in soft agar with macrophage - conditioned medium for 5 weeks compared to controls . Colonies ≥ 50 μm were counted . Error bars represent mean ± SEM from 5 independent experiments ( n = 3 per condition ) . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of NCT502 ( 10 μM ) . Representative images showing the filling of the spheroid lumen with cell nuclei . The spheroids were stained for DNA ( Hoechst 33342 in blue ) and F - actin ( Phalloidin in red ) . Scale bar : 50 μm . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of NCT502 ( 10 μM ) . NCT502 reduces the filling of the spheroid lumen with cell nuclei . Filling of the spheroid lumen with cell nuclei categorized into 4 groups ( clear , partially filled , almost filled and filled ) . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of NCT502 ( 10 μM ) . NCT502 reduces the filling of the spheroid lumen with cell nuclei . Filling of the spheroid lumen with cell nuclei . Partially filled , almost filled and filled spheroids were combined together ( non - empty spheroids ) . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of NCT502 ( 10 μM ) . NCT502 reduces the number of spheroids with invasive protrusions ( n = 2 per condition ) . Error bars represent mean ± SEM from 3 independent experiments ( n = 2 per condition ; at least 15 spheroids each from at least 2 fields of view ) . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . Mouse mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice that were either on normal ( ND ) or high fat diet ( HFD ) ( n = 3 ) . Organoids were isolated from mice on ND and cultured in matrigel / collagen mix for 2 days followed by macrophage - conditioned or control medium stimulation for 24h in presence of NCT502 ( 10 μM ) . NCT502 reduces the number of invasive organoids . Error bars represent mean ± SEM from 3 independent experiments , with each mouse labelled with a different symbol shape ( n = 2 per condition ; 15 - 25 organoids each ) . Macrophage donors are indicated as M1A - M1 activated , , M2A - M2 activated macrophages . Mouse mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice that were either on normal ( ND ) or high fat diet ( HFD ) ( n = 3 ) . Organoids were cultured in collagen for 2 days after which the number of invasive protrusions per each organoid was determined microscopically . NCT502 ( 10 μM ) reduces the number of invasive protrusions in organoids isolated from mice that were either on G ) ND or H ) HFD . Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse ( labelled with a different symbol shape ) . Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment . Reformed ducts were then cultured for 7 days in macrophage - conditioned or control medium in the presence of NCT502 ( 10μM ) . Representative images showing the filling of the ductal lumen with cell nuclei . The ducts were stained for DNA ( DAPI in blue ) and F - actin ( Phalloidin in red ) . Scale bar : 20 μm . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages . Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment . Reformed ducts were then cultured for 7 days in macrophage - conditioned or control medium in the presence of NCT502 ( 10μM ) . NCT502 inhibits filling of the duct lumen with cell nuclei . Error bars represent mean ± SEM ( A total of 24 structures was counted per each condition : 2 patient ductal structures ( n = 2 ) , each labelled with a different symbol shape , were cultured with macrophage - conditioned media from either donor 17 or donor 18 , 3 ducts each ) . Filling of the lumen was determined as % of luminal space filled with cells . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages . PHGDH gene was deleted in MCF10A cells via CRISPR - Cas9 technology . 3 knockout clones were combined for each cell line . PHGDH CRISPR - Cas9 knockout MCF10A cells form less colonies when grown in soft agar with macrophage - conditioned medium for 5 weeks compared to CRISPR - Cas9 control ( nontargeting crRNA ) cells . Colonies ≥ 50 μm were counted . Error bars represent mean ± SEM from 3 independent experiments ( n = 3 per condition ) . Media from two macrophage donors were combined in 1 : 1 ratio in each experiment . Macrophage donors are indicated as , M1A - M1 activated , M2A - M2 activated macrophages . PHGDH gene was deleted in MCF10A cells via CRISPR - Cas9 technology . 3 knockout clones were combined for each cell line . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . PHGDH CRISPR - Cas9 knockout MCF10A cells are less invasive compared CRISPR Control ( nontargeting crRNA ) cells . Error bars represent mean ± SEM from 3 independent experiments ( n = 3 per condition ) . Media from two macrophage donors were combined in 1 : 1 ratio in each experiment . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6NCT502 (10 μM) inhibits colony formation of MCF10A cells grown in soft agar with macrophage-conditioned medium for 5 weeks compared to controls. Colonies ≥ 50 μm were counted. Error bars represent mean ± SEM from 5 independent experiments (n=3 per condition). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of NCT502 (10 μM). Representative images showing the filling of the spheroid lumen with cell nuclei. The spheroids were stained for DNA (Hoechst 33342 in blue) and F-actin (Phalloidin in red). Scale bar: 50 μm. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of NCT502 (10 μM). NCT502 reduces the filling of the spheroid lumen with cell nuclei. Filling of the spheroid lumen with cell nuclei categorized into 4 groups (clear, partially filled, almost filled and filled). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of NCT502 (10 μM). NCT502 reduces the filling of the spheroid lumen with cell nuclei. Filling of the spheroid lumen with cell nuclei. Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of NCT502 (10 μM). NCT502 reduces the number of spheroids with invasive protrusions (n=2 per condition). Error bars represent mean ± SEM from 3 independent experiments (n=2 per condition; at least 15 spheroids each from at least 2 fields of view). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.Mouse mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice that were either on normal (ND) or high fat diet (HFD) (n=3). Organoids were isolated from mice on ND and cultured in matrigel/collagen mix for 2 days followed by macrophage-conditioned or control medium stimulation for 24h in presence of NCT502 (10 μM). NCT502 reduces the number of invasive organoids. Error bars represent mean ± SEM from 3 independent experiments, with each mouse labelled with a different symbol shape (n=2 per condition; 15-25 organoids each). Macrophage donors are indicated as M1A - M1 activated, , M2A - M2 activated macrophages.Mouse mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice that were either on normal (ND) or high fat diet (HFD) (n=3). Organoids were cultured in collagen for 2 days after which the number of invasive protrusions per each organoid was determined microscopically. NCT502 (10 μM) reduces the number of invasive protrusions in organoids isolated from mice that were either on G) ND or H) HFD. Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse (labelled with a different symbol shape).Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium in the presence of NCT502 (10μM). Representative images showing the filling of the ductal lumen with cell nuclei. The ducts were stained for DNA (DAPI in blue) and F-actin (Phalloidin in red). Scale bar: 20 μm. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium in the presence of NCT502 (10μM). NCT502 inhibits filling of the duct lumen with cell nuclei. Error bars represent mean ± SEM (A total of 24 structures was counted per each condition: 2 patient ductal structures (n=2), each labelled with a different symbol shape, were cultured with macrophage-conditioned media from either donor 17 or donor 18, 3 ducts each). Filling of the lumen was determined as % of luminal space filled with cells. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.PHGDH gene was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line. PHGDH CRISPR-Cas9 knockout MCF10A cells form less colonies when grown in soft agar with macrophage-conditioned medium for 5 weeks compared to CRISPR-Cas9 control (nontargeting crRNA) cells. Colonies ≥ 50 μm were counted. Error bars represent mean ± SEM from 3 independent experiments (n=3 per condition). Media from two macrophage donors were combined in 1:1 ratio in each experiment. Macrophage donors are indicated as , M1A - M1 activated, M2A - M2 activated macrophages.PHGDH gene was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line. 16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. PHGDH CRISPR-Cas9 knockout MCF10A cells are less invasive compared CRISPR Control (nontargeting crRNA) cells. Error bars represent mean ± SEM from 3 independent experiments (n=3 per condition). Media from two macrophage donors were combined in 1:1 ratio in each experiment. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.


Figure 7A - J ) Ten per group female C57Bl / 6 mice heterozygous for the PyMT - MMTV started high fat diet ( HFD ) when 6 - 7 weeks old ( prior of mammary tumour development ) together with control mice on normal diet ( ND ; n = 10 ) . Wild - type mice on HFD gain more weight compared to ND mice . * p < 0 . 05 by one - tailed students t - test . A - J ) Ten per group female C57Bl / 6 mice heterozygous for the PyMT - MMTV started high fat diet ( HFD ) when 6 - 7 weeks old ( prior of mammary tumour development ) together with control mice on normal diet ( ND ; n = 10 ) . Average adipocytes size ( major diameter ) in mammary fat pads is larger in HFD mice compared to ND mice based on the analysis of at least 100 adipocytes per mice on H & E stained sections . * p < 0 . 05 by two - tailed students t - test . A - J ) Ten per group female C57Bl / 6 mice heterozygous for the PyMT - MMTV started high fat diet ( HFD ) when 6 - 7 weeks old ( prior of mammary tumour development ) together with control mice on normal diet ( ND ; n = 10 ) . The number of F4 / 80 + macrophages in mammary fat pads is increased in HFD mice compared to ND mice . The average of F4 / 80 + macrophages in at least 10 fields of view per mouse is shown . * p < 0 . 05 by two - tailed students t - test . A - J ) Ten per group female C57Bl / 6 mice heterozygous for the PyMT - MMTV started high fat diet ( HFD ) when 6 - 7 weeks old ( prior of mammary tumour development ) together with control mice on normal diet ( ND ; n = 10 ) . Mice were administered amlexanox ( 17 mM ) or vehicle control daily by oral gavage ( Reilly et al , 2013 ) . A control group ( wild - type mice ) was also included to confirm the efficacy of the diet and to control for possible unpredicted interactions of the MMTV - PyMT gene . PyMT - MMTV + / - mice on HFD gain more weight compared to ND mice . * p < 0 . 05 by one - tailed students t - test . Amlexanox does not affect weight gain either in mice on F ) ND or G ) HFD . A - J ) Ten per group female C57Bl / 6 mice heterozygous for the PyMT - MMTV started high fat diet ( HFD ) when 6 - 7 weeks old ( prior of mammary tumour development ) together with control mice on normal diet ( ND ; n = 10 ) . Mice were administered amlexanox ( 17 mM ) or vehicle control daily by oral gavage ( Reilly et al , 2013 ) . A control group ( wild - type mice ) was also included to confirm the efficacy of the diet and to control for possible unpredicted interactions of the MMTV - PyMT gene . Mammary tumours are developing earlier in PyMT - MMTV + / - mice on HFD compared to ND mice . Amlexanox does not affect tumour development in PyMT - MMTV + / - mice on ND , but delays tumour formation in J ) HFD mice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A-J)Ten per group female C57Bl/6 mice heterozygous for the PyMT-MMTV started high fat diet (HFD) when 6-7 weeks old (prior of mammary tumour development) together with control mice on normal diet (ND; n=10). Wild-type mice on HFD gain more weight compared to ND mice. *p < 0.05 by one-tailed students t-test.A-J)Ten per group female C57Bl/6 mice heterozygous for the PyMT-MMTV started high fat diet (HFD) when 6-7 weeks old (prior of mammary tumour development) together with control mice on normal diet (ND; n=10). Average adipocytes size (major diameter) in mammary fat pads is larger in HFD mice compared to ND mice based on the analysis of at least 100 adipocytes per mice on H&E stained sections. *p < 0.05 by two-tailed students t-test.A-J)Ten per group female C57Bl/6 mice heterozygous for the PyMT-MMTV started high fat diet (HFD) when 6-7 weeks old (prior of mammary tumour development) together with control mice on normal diet (ND; n=10). The number of F4/80+ macrophages in mammary fat pads is increased in HFD mice compared to ND mice. The average of F4/80+ macrophages in at least 10 fields of view per mouse is shown. *p < 0.05 by two-tailed students t-test.A-J)Ten per group female C57Bl/6 mice heterozygous for the PyMT-MMTV started high fat diet (HFD) when 6-7 weeks old (prior of mammary tumour development) together with control mice on normal diet (ND; n=10). Mice were administered amlexanox (17 mM) or vehicle control daily by oral gavage (Reilly et al, 2013). A control group (wild-type mice) was also included to confirm the efficacy of the diet and to control for possible unpredicted interactions of the MMTV-PyMT gene. PyMT-MMTV+/- mice on HFD gain more weight compared to ND mice. *p < 0.05 by one-tailed students t-test. Amlexanox does not affect weight gain either in mice on F) ND or G) HFD.A-J)Ten per group female C57Bl/6 mice heterozygous for the PyMT-MMTV started high fat diet (HFD) when 6-7 weeks old (prior of mammary tumour development) together with control mice on normal diet (ND; n=10). Mice were administered amlexanox (17 mM) or vehicle control daily by oral gavage (Reilly et al, 2013). A control group (wild-type mice) was also included to confirm the efficacy of the diet and to control for possible unpredicted interactions of the MMTV-PyMT gene. Mammary tumours are developing earlier in PyMT-MMTV+/- mice on HFD compared to ND mice. Amlexanox does not affect tumour development in PyMT-MMTV+/- mice on ND, but delays tumour formation in J) HFD mice.


Figure 1A ) Upper panels : Representative single z - planes from 3D - SIM image stacks of the DAPI - stained nuclei of AC16 cells kept at 37 ° C or exposed to different cold temperatures for 24h . Lower panels : DAPI signal segmented into chromatin and interchromatin regions according to relative intensity . Scale bar : 5 μm . Arrows mark chromatin concentrated at the nuclear periphery ( P ) and around nucleoli ( N ) . B ) Boxplots of the ratio of chromatin volume to nuclear volume for cells exposed to different temperature conditions . Median ( central line ) , interquartile range ( IQR , box edges ) , most extreme data point no more than 1 . 5x IQR from box edges ( whiskers ) . Number of nuclei : 37 ° C ( 31 ) , 28 ° C 24h ( 26 ) , 18 ° C 5h ( 19 ) , 18 ° C 24h ( 22 ) , 18 ° C 24h then 37 ° C 2h ( 25 ) , 8 ° C 24h ( 23 ) . C ) Left and central panels : representative single z - planes from 3D - SIM image stacks of the DAPI - stained nuclei of cells kept at 37 ° C or exposed to 18 ° C for 24h showing the spatial distribution of the immunofluorescence ( IF ) signal of hnRNPC ( green ) and nuclear pore complexes ( NPCs , red ) . Right panel : DAPI signal overlaid with dots indicating IF signal centroid coordinates for centroids located in the z - plane shown . Scale bar whole nuclei : 5 μm . Scale bar enlarged section : 1 μm . D ) Bar graph of the mean NPC centroid to nearest hnRNPC centroid distance for each temperature condition . Number of distances : 37 ° C ( 112511 ) , 28 ° C 24h ( 94521 ) , 18 ° C 5h ( 72340 ) , 18 ° C 24h ( 74998 ) , 18 ° C 24h then 37 ° C 2h ( 92896 ) , 8 ° C 24h ( 80570 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) Upper panels: Representative single z-planes from 3D-SIM image stacks of the DAPI-stained nuclei of AC16 cells kept at 37°C or exposed to different cold temperatures for 24h. Lower panels: DAPI signal segmented into chromatin and interchromatin regions according to relative intensity. Scale bar: 5 μm. Arrows mark chromatin concentrated at the nuclear periphery (P) and around nucleoli (N). B) Boxplots of the ratio of chromatin volume to nuclear volume for cells exposed to different temperature conditions. Median (central line), interquartile range (IQR, box edges), most extreme data point no more than 1.5x IQR from box edges (whiskers). Number of nuclei: 37°C (31), 28°C 24h (26), 18°C 5h (19), 18°C 24h (22), 18°C 24h then 37°C 2h (25), 8°C 24h (23). C) Left and central panels: representative single z-planes from 3D-SIM image stacks of the DAPI-stained nuclei of cells kept at 37°C or exposed to 18°C for 24h showing the spatial distribution of the immunofluorescence (IF) signal of hnRNPC (green) and nuclear pore complexes (NPCs, red). Right panel: DAPI signal overlaid with dots indicating IF signal centroid coordinates for centroids located in the z-plane shown. Scale bar whole nuclei: 5 μm. Scale bar enlarged section: 1 μm. D) Bar graph of the mean NPC centroid to nearest hnRNPC centroid distance for each temperature condition. Number of distances: 37°C (112511), 28°C 24h (94521), 18°C 5h (72340), 18°C 24h (74998), 18°C 24h then 37°C 2h (92896), 8°C 24h (80570).


Figure 2A ) Number of genes showing significant ( adjusted p < 0 . 05 , Wald test ) up or downregulation in nuclear or cytoplasmic RNA levels upon transfer of AC16 cells from 37 ° C to different cold temperatures for 24h . B ) Relationship between the log2 fold change in RNA level in the nucleus and that in the cytoplasm for all genes upon transfer of AC16 cells from 37 ° C to different cold temperatures for 24h . Points are color - coded from low to high density ( black < blue < red < yellow ) . C ) Relationship between the log2 fold change in RNA level for all genes upon transfer of AC16 cells from 37 ° C to 28 ° C or 18 ° C ( top ) , 28 ° C or 8 ° C ( middle ) and 18 ° C or 8 ° C ( bottom ) for 24h in both the nucleus and cytoplasm . Points are shaded from low to high density ( grey < black ) . Genes that show significant differential expression at a specific temperature or at both temperatures are highlighted with colored circles or triangles , respectively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) Number of genes showing significant (adjusted p < 0.05, Wald test) up or downregulation in nuclear or cytoplasmic RNA levels upon transfer of AC16 cells from 37°C to different cold temperatures for 24h.B) Relationship between the log2 fold change in RNA level in the nucleus and that in the cytoplasm for all genes upon transfer of AC16 cells from 37°C to different cold temperatures for 24h. Points are color-coded from low to high density (black < blue < red < yellow).C) Relationship between the log2 fold change in RNA level for all genes upon transfer of AC16 cells from 37°C to 28°C or 18°C (top), 28°C or 8°C (middle) and 18°C or 8°C (bottom) for 24h in both the nucleus and cytoplasm. Points are shaded from low to high density (grey < black). Genes that show significant differential expression at a specific temperature or at both temperatures are highlighted with colored circles or triangles, respectively.


Figure 3A ) Boxplots of standardized nuclear and cytoplasmic RNA levels at time points during the transfer of AC16 cells from 37 ° C to 18 ° C for 24h and then back to 37 ° C for 24h for the group of 1285 genes showing significant upregulation in nuclear RNA levels at the 18 ° C for 24h time point . Range ( whiskers ) , interquartile range ( IQR , box edges ) , median + / - IQR / [ square root ( number of genes ) ] ( notch ) and median ( central line ) . B ) Log2 fold change in cytoplasmic ( black line , left axis ) and nuclear ( red line , right axis ) RNA level of core circadian clock negative limb genes at time points during the transfer of AC16 cells from 37 ° C to 18 ° C for 24h and then back to 37 ° C for 24h relative to cells kept at 37 ° C . Error bars show the standard error . C ) Nuclear and cytoplasmic full - length RNA - Seq data from AC16 cells kept at 37 ° C or exposed to 18 ° C for 24h for REV - ERBα and CRY2 . Data for each track have been normalized to show counts per million aligned counts ( CPM ) for each nucleotide . Number of 3 ' end RNA - seq sample replicates : 37 ° C ( C : 6 , N : 7 ) , 18 ° C 5h ( 4 ) , 10h ( 4 ) , 24h ( 6 ) , 18 ° C 24h then 37 ° C 2h ( 4 ) , 5h ( 2 ) , 10h ( 2 ) , 24h ( 2 ) . Number of cytoplasmic ( C ) and nuclear ( N ) samples are the same for each condition except 37 ° CWestern blot of Flag - tagged REV - ERBα levels ( short and long exposure ) in whole cell extracts from AC16 cells transferred from 37 ° C to 18 ° C for the time periods indicated and also returned after each of these time periods to 37 ° C for 2h . TP53 was used as a loading control as its transcript levels show minimal changes in response to 18 ° C exposure Ponceau stain is shown as an additional loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) Boxplots of standardized nuclear and cytoplasmic RNA levels at time points during the transfer of AC16 cells from 37°C to 18°C for 24h and then back to 37°C for 24h for the group of 1285 genes showing significant upregulation in nuclear RNA levels at the 18°C for 24h time point. Range (whiskers), interquartile range (IQR, box edges), median +/-IQR/[square root(number of genes)] (notch) and median (central line).B) Log2 fold change in cytoplasmic (black line, left axis) and nuclear (red line, right axis) RNA level of core circadian clock negative limb genes at time points during the transfer of AC16 cells from 37°C to 18°C for 24h and then back to 37°C for 24h relative to cells kept at 37°C. Error bars show the standard error.C) Nuclear and cytoplasmic full-length RNA-Seq data from AC16 cells kept at 37°C or exposed to 18°C for 24h for REV-ERBα and CRY2. Data for each track have been normalized to show counts per million aligned counts (CPM) for each nucleotide. Number of 3' end RNA-seq sample replicates: 37°C (C: 6, N: 7), 18°C 5h (4), 10h (4), 24h (6), 18°C 24h then 37°C 2h (4), 5h (2), 10h (2), 24h (2). Number of cytoplasmic (C) and nuclear (N) samples are the same for each condition except 37°CWestern blot of Flag-tagged REV-ERBα levels (short and long exposure) in whole cell extracts from AC16 cells transferred from 37°C to 18°C for the time periods indicated and also returned after each of these time periods to 37°C for 2h. TP53 was used as a loading control as its transcript levels show minimal changes in response to 18°C exposure Ponceau stain is shown as an additional loading control.


Figure 4Representative maximum intensity z - stack - projected images of DAPI - stained ( blue ) AC16 exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for REV - ERBαRepresentative maximum intensity z - stack - projected images of DAPI - stained ( blue ) AC16 exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for CRY2 ( B ) Representative maximum intensity z - stack - projected images of DAPI - stained ( blue ) AC16 exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for TP53 ( C ) Representative maximum intensity z - stack - projected images of DAPI - stained ( blue ) U2OS cells ( D ) , exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for REV - ERBαMean REV - ERBα per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . AC16 cells ( REV - ERBα ) : 37 ° C ( 65 ) , 18 ° C 5h ( 30 ) , 18 ° C 5h then 37 ° C 2h ( 21 ) , 18 ° C 24h ( 63 ) , 18 ° C 24h then 37 ° C 2h ( 64 ) Mean CRY2 ( F ) per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . ; AC16 cells ( CRY2 ) : 37 ° C ( 43 ) , 18 ° C 24h ( 42 ) , 18 ° C 24h then 37 ° C 2h ( 42 ) Mean TP53 ( G ) transcripts per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . AC16 cells ( TP53 ) : 37 ° C ( 47 ) , 18 ° C 24h ( 45 ) , 18 ° C 24h then 37 ° C 2h ( 48 ) Mean REV - ERBα per U2OS ( H ) cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . U2OS cells ( REV - ERBα ) : 37 ° C ( 61 ) , 18 ° C 5h ( 61 ) , 18 ° C 5h then 37 ° C 2h ( 69 ) , 18 ° C 24h ( 53 ) , 18 ° C 24h then 37 ° C 2h ( 65 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Representative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for REV-ERBαRepresentative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for CRY2 (B)Representative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for TP53 (C)Representative maximum intensity z-stack-projected images of DAPI-stained (blue) U2OS cells (D), exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for REV-ERBαMean REV-ERBα per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. AC16 cells (REV-ERBα): 37°C (65), 18°C 5h (30), 18°C 5h then 37°C 2h (21), 18°C 24h (63), 18°C 24h then 37°C 2h (64)Mean CRY2 (F) per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. ; AC16 cells (CRY2): 37°C (43), 18°C 24h (42), 18°C 24h then 37°C 2h (42)Mean TP53 (G) transcripts per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. AC16 cells (TP53): 37°C (47), 18°C 24h (45), 18°C 24h then 37°C 2h (48)Mean REV-ERBα per U2OS (H) cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. U2OS cells (REV-ERBα): 37°C (61), 18°C 5h (61), 18°C 5h then 37°C 2h (69), 18°C 24h (53), 18°C 24h then 37°C 2h (65)