EMBO/soda-nel huggingface dataset:
The AhR-SRC axis as a therapeutic vulnerability in BRAFi-resistant melanoma. (2022)
Figure 5

Figure 5. Inhibition of the SRC pathway increases BRAFi sensitivity and prevents invasive phenotype of melanoma cells.

A. Heatmap of the percentage of cell viability in SKR (left) or SKR KO or SKR KO CA-AhR (right) cells treated with a combination of increasing doses of vemurafenib (y) and bosutinib or dasatinib (1 µM). IC50 values are represented by black squares.

B. Three-dimensional spheroid growth of a 50%/50% mix of SK28R WT (in red) and KO cells (in green) over five days. Cells were treated or not with SRC inhibitors: bosutinib (1 µM) or dasatinib (1 µM) every two days (n = 3 independent technical experiments, mean ± s.d.). Comparisons with control (DMSO) were performed using unpaired t-tests with the Sidak-Bonferroni method, p<0.001 ***.

C. Three-dimensional spheroid growth of SK28R WT (in red) and KO cells (in green) over five days. Cells were treated or not with dasatinib (1 µM) in combination with ITE (5 μM) or TCDD (20 nM) every two days (n = 3 independent technical experiments, mean ± s.d.). Comparisons with control (DMSO) were performed using unpaired t-tests with the Sidak-Bonferroni method, p<0.05 *, p<0.01 **.

Comparisons were performed using unpaired t-tests with the Sidak-Bonferroni method.

D. Protein levels of AhR, p-SRC (Y416), and SRC in the SK28R cell line after 24 h of treatment with ITE (5 μM) or SRC inhibitor (dasatinib) (1 μM).

E. Evaluation of AhR transcriptional activity related to AhR/ARNT binding sites (XRE) using p3xXRE-luciferase constructs. HaCat keratinocytes cells were exposed or not to 10 nM TCDD alone or in combination with increasing concentrations of dasatinib O/N (n = 3 independent technical experiments, mean ± s.d.). Comparisons with control (0) were performed using unpaired t-tests with the Sidak-Bonferroni method, p<0.05 *, p<0.001 ***.

F. Expression heatmap of the median of gene expression for the various signatures (left) and AhR target genes (right) from RNAseq datasets for SK28R cells treated or not for 24 h with SRC inhibitors: bosutinib (Bos, 1 μM) or dasatinib (Das, 1 μM). The scale corresponds to the Z scores.
The AhR-SRC axis as a therapeutic vulnerability in BRAFi-resistant melanoma. (2022)
Figure 6

Figure 6. Inhibition of SRC sensitizes melanoma to BRAFi treatment in a PDX model.

A. PDX model MEL006R (BRAFi resistant) was implanted in NMRI nude mice. Mice with tumors reaching 200mm³ were treated daily with vehicle (n=6), dabrafenib alone (Dab, Biorbyt, 30 mg/kg, n=5) dasatinib alone (Das, Selleckchem, 30 mg/kg, n=9) or in combination dabrafenib + dasatinib (Das before combo, 30 mg/kg, n=12).

B. PDX tumor volumes were measured every 2 days until reaching 1500mm3. Values correspond to the mean +/- sem. Statistical analysis (2way ANOVA) has been performed between the different experiments (Das or Das before combo vs Dab) at different times. Stars represent time from when tumor size is significantly lower than group treated with BRAFi alone (Dabrafenib) p<0.05 *, p<0.01 **.

C. Kaplan-Meier survival curve for MEL006R mice treated with the different drugs. Comparison of Survival Curves have been performed using the nonparametric Log-rank (Mantel-Cox) test.

D. Number of days to reach max tumor volume (endpoint point size). Values correspond to the median +/- sd. Non-parametric Mann-Whitney t test for the different treatments was performed compared to vehicle *** p<0.001.
Autophagy variation within a cell population determines cell fate through selective degradation of Fap-1 (2013)
figf1

(a,b) BJAB lymphoma cells stably expressing mCherry–GFP–LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP (a). The high and low 20% were sorted (a), re-plated and treated with lysosomal protease inhibitors pepstatin and E-64d for 1 h; lysates were then immunoblotted for the indicated proteins (b). (c) Densitometry of LC3-II and p62 western blots (normalized to actin and hour 0, mean ± s.e.m., n = 3 blots from 2 independent experiments, *P = 0.051, **P = 0.0091). (d,e) HeLa mCherry–GFP–LC3 cells were sorted as in a, cytospun on to slides, fixed and visualized by confocal microscopy (d); autophagic LC3 puncta were assessed by quantitative microscopy (e; punctate area per cell, mean ± s.e.m., n = 50 fields, *P = 0.010, **P = 0.053). (f) Electron micrographs of HeLa mCherry–GFP–LC3 cells sorted for autophagic flux as in a. Yellow arrows denote autophagosomes; red arrows indicate autolysosomes. (g) Quantification of autophagosomes and autolysomes from electron micrographs (mean ± s.e.m., n = 50 fields). Uncropped images of blots are shown in Supplementary Fig. 6.
Autophagy variation within a cell population determines cell fate through selective degradation of Fap-1 (2013)
figf2

(a–c) BJAB lymphoma cells stably expressing mCherry–GFP–LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP. Cells were then re-plated in growth medium and autophagic flux was again measured by flow cytometry at the indicated time points (median ratio of mCherry/GFP fluorescence ± s.e.m., n = 3 wells). Lysates from cells collected at the indicated time points were immunoblotted with the indicated antibodies (b). Representative flow cytometry histograms for cells at 0 h and 24 h after sorting (c). (d) BJAB mCherry–GFP–LC3 cells were sorted for autophagic flux as in a (top and bottom 20%). Following treatment with Fas ligand (1.5 ng ml−1) or TRAIL (4 ng ml−1), cell viability was determined by MTS assay (a tetrazolium reduction-based metabolic measurement) at 24 h (percentage of untreated control, mean ± s.e.m., n = 3 wells, *P = 2.3×10−4, **P = 0.0036). (e) Long-term growth of BJAB mCherry–GFP–LC3 cells sorted for autophagic flux followed by treatment with Fas ligand (4 ng ml−1) or TRAIL (15 ng ml−1) for 24 h. Cells were then re-plated and allowed to recover for 5 (Fas ligand) or 6 (TRAIL) days then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.012). (f) BJAB mCherry–GFP–LC3 cells were sorted for autophagic flux as above and apoptosis was measured at 1 h by flow cytometry using AnnexinV and DAPI (mean ± s.e.m., n = 3 wells, *P = 0.0046). (g) Cells sorted as in a, were re-plated and, starting at the indicated times following sorting, treated with Fas ligand (4 ng ml−1) for 24 h; cell viability was then determined by MTS (percentage of untreated control, mean ± s.e.m.,
n = 3 wells). Uncropped images of blots are shown in Supplementary Fig. 6.
Autophagy variation within a cell population determines cell fate through selective degradation of Fap-1 (2013)
figf3

(a) BJAB and Jurkat cells were treated with vehicle or chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (1.5 ng ml−1). Cell viability was determined by MTS 24 h later (percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 0.0058). (b) Immunoblots of cells in a,c probed for the indicated antibodies. CQ, chloroquine. (c) BJAB and Jurkat cells were treated with chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by treatment with Fas ligand (4 ng ml−1) for 24 h (same technical replicate as a). Cells were then re-plated at low density in growth media and allowed to recover for 5 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m.,
n = 3 wells, *P = 0.0024). (d) BJAB and Jurkat cells were transduced with the indicated lentiviral shRNA constructs, followed by 3 days of puromycin selection. Selected cells were plated and treated with Fas ligand (1.5 ng ml−1) or TRAIL (4 ng ml−1) for 24 h and viability was assessed by MTS (percentage of control (no ligand), mean ± s.e.m., n = 3, *P = 1.7×10−4, **P = 0.024). (e) Immunoblots for Atg5, Atg7 and p62 confirm protein depletion and autophagy inhibition in d,f. (f) BJAB and Jurkat cells expressing control or Atg5 shRNA were treated with Fas ligand (15 ng ml−1) or TRAIL (12.5 ng ml−1) for 24 h (same technical replicate as d). Cells were then re-plated at low density and allowed to recover for 5–6 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0089). Atg7 knockdown data were not included for long-term viability owing to the growth suppressive effect of Atg7 knockdown in the absence of drug treatment. Uncropped images of blots are shown in Supplementary Fig. 6.
Autophagy variation within a cell population determines cell fate through selective degradation of Fap-1 (2013)
figf4

(a,b) BJAB and Jurkat cells stably expressing mCherry–GFP–LC3 were flow sorted for high and low autophagic flux, treated with Fas ligand (1.5 ng ml−1) for 24 h and viability was assessed by MTS (a; percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0046). (b) Immunoblots following sorting of the samples in a. (c,d) The indicated cell lines were treated with chloroquine (BJAB, CEM 20 μM; SKW6.4, 25 μM; Jurkat, 10 μM) for 16 h, followed by Fas ligand (BJAB, 12.5 ng ml−1; SKW6.4, 50 ng ml−1; Jurkat, 0.4 ng ml−1; CEM, 40 ng ml−1). Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies (c). Cell viability was assessed by MTS 24 h following Fas ligand treatment (d; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 2.7×10−4, **P = 1.6×10−4). (e,f) BJAB and Jurkat cells were transduced with control, Atg5, Atg7 or Vps34 shRNA lentiviruses, followed by three days of puromycin selection. Cells were then treated with Fas ligand (1.25 ng ml−1) or TRAIL (1.25 ng ml−1) for 24 h and viability was assessed by MTS (e; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 1.4×10−6, **P = 2.3×10−5, ***P = 3.9×10−5, §P = 9.2×10−6,
§§P = 3.4×10−6,
§§§P = 1.7×10−5). Immunoblots demonstrate Atg5, Atg7 and Vps34 knockdown, autophagy inhibition and altered Fap-1 levels. The blots are separated because different exposures were required to detect the proteins in one cell line without overexposing the lanes for the other; the Fap-1 blot is not separated to demonstrate the lack of Fap-1 protein in Type II Jurkat cells (f). Fap-1 blots (b,c,f) were run on separate gels owing to the quantity of protein required for detection (see
Methods). Uncropped images of blots are shown in Supplementary Fig. 6.
Autophagy variation within a cell population determines cell fate through selective degradation of Fap-1 (2013)
figf5

(a,b) BJAB and Jurkat cells expressing the indicated Fap-1 wild-type (WT) and catalytically inactive (ΔCD) constructs were treated with chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (BJAB, 1.5 ng ml−1; Jurkat 15 ng ml−1) for 24 h. Cell viability was determined by MTS (a; percentage of control (no ligand), mean ± s.e.m.,
n = 3 wells, *P = 2.7×10−4, **P = 2.4×10−5, ***P = 0.0024). Cell lysates were blotted and probed with the indicated antibodies (b). (c–e) BJAB cells expressing control or Fap-1 shRNAs were treated with 20 μM chloroquine for 16 h followed by Fas ligand (12.5 ng ml−1) or TRAIL (25 ng ml−1) for 24 h; cell viability was then determined by MTS (c; percentage of control (no ligand), mean ± s.e.m.,
n = 3 wells, *P = 4.8×10−6, **P = 8.1×10−5, ***P = 8.0×10−4,
§P = 0.013, NS
P>0.05). (d) Immunoblots of lysates collected following chloroquine treatment. Long-term growth was assessed in cells from c by re-plating them at low density and allowing them to recover for 5 days, followed by viability assay (percentage of untreated control, mean ± s.e.m., n = 3 wells, *P = 0.043; e). Fap-1 blots (b,d) were run on separate gels owing to the quantity of protein required for detection (see Methods). Uncropped images of blots are shown in Supplementary Fig. 6.
Autophagy variation within a cell population determines cell fate through selective degradation of Fap-1 (2013)
figf6

(a,b) Cell viability was determined by MTS assay following Fas ligand (4 ng ml−1) treatment in BJAB cells expressing control or p62 lentiviral shRNAs (a; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 3.2×10−5, **P = 1.8×10−6). Immunoblots confirm p62 depletion and Fap-1 levels (b). (c) Co-immunoprecipitation of endogenous p62 and Fap-1 in BJAB cells treated with 20 μM chloroquine for 16 h, followed by treatment with Fas ligand (50 ng ml−1) for 2 h at 4 °C. (d) Model of the mechanism for autophagy promotion of Fas apoptosis in Type I cells. MOMP, mitochondrial outer membrane permeabilization. Fap-1 blots (b,c) were run on separate gels owing to the quantity of protein required for detection (see Methods). Uncropped images of blots are shown in Supplementary Fig. 6.
FAK activity in cancer-associated fibroblasts is a prognostic marker and a druggable key metastatic player in pancreatic cancer (2020)
Figure 1

Figure 1: Increased FAK activation in human pancreatic adenocarcinoma-associated fibroblasts.

(A-B) Representative Hematoxylin and Eosin (H&E) staining, and immunohistochemistry of phosphorylated FAK on tyrosine (Y) 397 (pY397 FAK), alpha-Smooth Muscle Actin (α-SMA), and Fibroblast Activated Protein alpha (FAP-α) in serial sections of human normal pancreas (Ct1), of human normal adjacent PDAC tissue (Ct2) and of human PDAC tissues (PDAC1-3); scale bar, 100 μm (A), and 300 µm (B).

(C) Quantification of pY397 FAK level in cancer-associated fibroblasts from human PDAC microarray: 40 different PDAC patient samples and 10 normal human pancreas have been stained for pY397 FAK and double blinded quantified (based on intensity and positive cell number) on a scale between 0 (no staining) and 6 (Y397 FAK highly positive) in whole cell (left), cytoplasm (middle) and nucleus (right). Values are means ± SEM. **p<0.01, by unpaired two-tailed Student's t-test.
FAK activity in cancer-associated fibroblasts is a prognostic marker and a druggable key metastatic player in pancreatic cancer (2020)
Figure 2

Figure 2: Increased FAK activation in human pancreatic adenocarcinoma-associated fibroblasts is an independent prognostic marker.

(A) Representative images of Definiens® Tissue studio multi-resolution segmentation algorithm on two PDAC TMA examples (X, Y). From left to right: IHC for pY397 FAK; ROI detection of tumour cells (dark blue), stroma (orange) and other (light blue/grey); Nuclear/ cytoplasm detection in blue and green respectively; IHC staining classification for pY397 FAK intensity signal (dark red for high signal, orange for medium signal, yellow for low signal and white for negative signal).

(B-C) Disease Free Survival (B) and Overall Survival (C) from 120 patient cohort according to pY397 FAK expression in CAFs; HR: Hazard Ratio.

(D-E) Disease Free Survival (D) and Overall Survival (E) from 120 patient cohort according to pY397 FAK expression in tumour cells.

(B-E) Survival curves were estimated with the Kaplan-Meier method and compared using log-rank test, non-parametric tests (chi2-test) to compare independent groups for categorical data.
FAK activity in cancer-associated fibroblasts is a prognostic marker and a druggable key metastatic player in pancreatic cancer (2020)
Figure 3

Figure 3: Fibroblastic FAK inactivation prevents tumour metastasis without impacting tumour growth.

Tumour cells were syngeneically and orthotopically co-grafted with mouse FAK-WT or FAK-KD fibroblasts. Thirty-eight days after injection, mice were euthanized.

(A) Primary tumour mass quantification. Values are means ± SEM from at least 7 mice per group. NS: not significantly different using one-way ANOVA test with Tukey's method for multiple comparison.

(B) Representative images from primary tumour stained for H&E, cytokeratin 19 (CK19), α-SMA, Sirius Red and polarized Sirius Red. Scale bar, 100 μm.

(C) Quantification of fibrillar collagen I (Yellow-Orange) and III (Green) based on polarized Sirius Red images from B. Each dot represents a mouse (n=11 per group). Values are means ± SEM, *p<0.05 using unpaired two-tailed Student's t-test.

(D-E) IHC representative images (left) and quantification (right) the expression of CD31 (D) and two hypoxia markers CaIX and HIF1α (E) from primary tumour; scale bar, 100 μm. Values are means ± SEM from 7 mice per group. NS: not significantly different using unpaired two-tailed Student's t-test.

(F) Representative images of H&E, CK19 and α-SMA IHC staining performed on lung sections. Arrow identified lung metastasis.

(G) Quantification of lung metastasis size and number based on CK19 positive staining using NanoZoomer software. Values are means ± SEM of 6 mice per group. * p<0.05, **p<0.01, ***p<0.001, using one-way ANOVA test with Tukey's method for multiple comparison.. NS: not significantly different.
FAK activity in cancer-associated fibroblasts is a prognostic marker and a druggable key metastatic player in pancreatic cancer (2020)
Figure 4

Figure 4: Fibroblastic FAK inactivation reduces M2 macrophage polarization, migration and correlates with M2 macrophage number in human PDAC samples

(A-B) Relative frequencies of tumour-infiltrating M1 macrophages and M2 macrophages analyzed by flow cytometry at 21 days (A) and 38 days (B) after grafting. Values are means ± SEM from 5-10 mice per group, *p<0.05, **p<0.01 using unpaired two-tailed Student's t-test.

(C) Schematic of BMDM isolation and macrophage polarization assay.

(D) Impact of conditioned media (CM) secreted by activated mouse fibroblasts expressing FAK-WT or FAK-KD on mouse BMDM-derived macrophage polarization. Relative frequencies of macrophages expressing CD80, CD86, Nos2, CD206, CMH2, Dectin, and CD163 markers acquired by flow cytometry and processed using FlowJo software. * p <0.05, by unpaired two-tailed Student's t-test; values are mean +/- SEM.

(E) Impact of soluble factors secreted by CAFs on macrophage migration. Left: schematic of BMDM derived M1 or M2-like macrophages. Right: Representative pictures and quantification of M1 and M2 macrophage migration at 24 and 48h towards CM secreted by CAFs (previously treated or not with FAK inhibitor for 48h). Cells were stained using Cristal Violet and percentage of cristal violet positive area quantified using Cell Observer videomicroscope (Zeiss) and analyzed with ImageJ. * p <0.05, by paired t-test; values are mean +/- SEM from 3 experiments.

(F) Quantitative real-time PCR conducted with gene-specific primers (CCL2, CCL5 and M-CSF) on four different FAK-I-treated (16h) and non-treated CAFs for gene expression. The normalized fold change ± SEM was calculated using the delta-delta Ct method with RSP16 as a control gene; values are mean +/- SEM. Each individual patient-derived activated fibroblasts was assigned to a specific symbol (see table EV2).

(G) Elisa quantification of CCL2 (MCP1) concentration (µg/ml) in conditioned medium from hCAFs treated or not with FAK-I during 72h. * p <0.05, by paired t-test; values are mean +/- SEM. Each individual patient-derived activated fibroblasts was assigned to a specific symbol (see table EV2).

(H) Left: Representative immunohistochemistry pictures of phosphorylated FAK on tyrosine (Y) 397 and of CD206 in serial sections of human PDAC tissues. Scale bar: 200µm Middle: Correlation plot between fibroblastic pY397 FAK score and CD45 or CD206+ cell number within human PDAC samples. Fibroblastic pY397 FAK score and percentage of CD206 positive cell quantifications were performed on a 47 patient cohort based on IHC staining. Spearman r = 0.4145, p=0.0019. Right: Percentage of CD206 positive cells in PDAC patient expressing either low or high fibroblastic FAK activity score. Values (blue bars) are means +/- SEM; **p<0.01 using unpaired two-tailed Student's t-test.
FAK activity in cancer-associated fibroblasts is a prognostic marker and a druggable key metastatic player in pancreatic cancer (2020)
Figure 5

Figure 5: Fibroblastic FAK activity controls tumour cell migration and invasion.

(A) Representative images of three independent scratch wound assays of co-cultivated red-labelled pancreatic tumour cells with either FAK-WT (wild-type) or FAK-KD (Kinase-dead) green-labelled fibroblasts at time zero and 46 hours (corresponding to the wound closure of co-cultured FAK-WT fibroblasts with tumour cells). Scale bar, 150 μm.

(B-H) Cell migration (has been analyzed in detail by tracking the location of cells (n >16 cells per condition) over time for fibroblasts or tumour cells (B), and measuring fibroblast velocity (C), directionality (a cell with a directionality equal to 1 is moving straightforward) (D) and migration distance (E) or tumour cell velocity (F), directionality (G) and distance (H). Values are means ± SEM of 16 tracked cells from two independent experiments. * indicates p<0.05, **p<0.01, ***p<0.001, by one-way ANOVA with Tukey's method for multiple comparison.

(I) 3D reconstitution of cell invasion into collagen matrix from spheroid composed of green-labelled FAK-WT or FAK-KD fibroblasts plus red-labelled pancreatic tumour cells (ratio 1/1). Shown are representative 3D reconstitution of spheroid invasion into 2mg/ml collagen I matrix obtained using Zen software.

(J-K) Quantification of fibroblast (J) or tumour cell (K) invasion within collagen matrix. Values are means ± SEM of 60 tracked cells from three independent experiments. *p<0.05, ***p<0.001, by unpaired two-tailed Student's t-test.
Wnt and Src signals converge on YAP-TEAD to drive intestinal regeneration (2021)
Figure 1

Figure 1. YAP/TAZ double knockouts exhibit defects in the transverse folds of the ascending colon.

A) Murine small intestines immunostained for the proliferation marker Ki67, apoptosis marker cleaved caspase 3 (Cas3), or YAP from 4 control (Yapfl/fl Tazfl/fl) and 4 YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to a moderate increase in apoptosis (Cas3+) in crypt base stem cells, quantified on the right, but no overall effect on the morphology of the small intestine. Arrows point to apoptotic cells.

B) Murine large intestines immunostained for proliferation marker Ki67, apoptosis marker cleaved caspase 3 (Cas3), or YAP from control (Yapfl/fl Tazfl/fl) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to an increased number of apoptotic (Cas3+) cells within the transverse folds of the ascending colon.

C) Murine small intestines immunostained for YAP or cleaved caspase 3 (Cas3) from control (Villin-CreERt) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to a mild increase in apoptosis (Cas3+) in crypt base stem cells, but no overall effect on the morphology of the small intestine. Arrows point to stem cells (YAP nuclear staining), while arrows point to apoptotic cells (Cas3 staining). N > 10 animals per genotype.

D) Murine large intestines immunostained for YAP or cleaved caspase 3 (Cas3) from control (Villin-CreERt) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to increased apopotosis (Cas3+) and severe morphological defects in the transverse folds of the ascending colon, but does not affect morphology of the remaining colon regions. Quantitatively, 75% of n = 4 animals showed this severe phenotype.