EMBO/soda-nel huggingface dataset:
Functional systemic CD4 immunity is required for clinical responses to PD-L1/PD-1 blockade therapy (2019)
Figure 2

Figure 2. Differential systemic CD4 immunity and responses to PD-1/PD-L1 blockade in NSCLC patients. (A) The scatter plot shows PD-1 expression after co-culture of CD4 T cells from healthy donors or NSCLC patients, as indicated, with A459-SC3 lung cancer cells. Relevant statistical comparisons with the test of Mann-Whitney are indicated. (B) Upper graphs, flow cytometry density plots of PD-1 and LAG-3 co-expression in CD4 T cells from healthy donors, a G1 responder (G1 R), a G1 non-responder (G1 NR) and a G2 non-responder as indicated, following stimulation with A549-SC3 cells. Percentage of expressing cells are indicated within each quadrant. Below, same as in the upper graphs but as a scatter plot of the percentage of CD4 T cells that simultaneously co-express PD-1 and LAG-3. Relevant statistical comparisons are shown with the test of Mann-Whitney. (C) Upper flow cytometry histograms of Ki67 expression in CD4 T cells from the representative subjects as indicated on the right, after stimulation with A549-SC3 cells. Vertical dotted line indicates the cut-off value of positive vs negative Ki67 expression. The percentage of Ki67-expressing CD4 T cells is shown within the histograms. Below, same data represented as a scatter plot from a sample of G1 and G2 donors as indicated, with relevant statistical comparisons with the test Mann-Whitney. (D) Proliferation of CD4 T cells stimulated by A549-SC3 cells from the indicated patient groups. CD28 expression is shown together with the proliferation marker ki67. Percentages of cells within each quadrant are shown. (E) Same as in (D) but in the presence of an isotype control antibody or an anti-PD-1 antibody with the equivalent sequence to pembrolizumab. The effects on CD4 T cells from a G1 and a G2 patient are shown, divided in CD28 high or low/negative subsets as indicated. Relevant statistical comparisons are shown with paired Student´s t test. (F) Top, flow cytometry density plots of Ki67 expression in CD4 T cells from representative G1 or G2 patients after three cycles of therapy, activated by incubation with A549-SC3 cells. Below, same as above but as a dot-plot graph. A comparison between proliferating CD4 T cells before and after therapy is shown in unpaired patient samples. G1 R, G1 objective responder patient. G1 NR, G1 patient with no objective responses; Green, objective responders (OR) and red, no OR; Iso, treatment with an isotype antibody control; α-PD-1, treatment with anti-PD-1 antibody; Statistical comparisons were performed with the test of Mann-Whitney. *, **, *** represents significant (P<0.05), very significant (P<0.001) and highly significant differences (P<0.0001), respectively.
Functional systemic CD4 immunity is required for clinical responses to PD-L1/PD-1 blockade therapy (2019)
Figure 3

Figure 3. Lung cancer antigen-specific CD4 T cells in NSCLC patients. (A) Scatter plot graph with the percentage of lung cancer-specific systemic CD4 T cells quantified by an autologous DC-based antigen presentation assay (See Materials and Methods), in a sample of G1 and G2 patients as indicated. Objective responses (OR) are shown in green. In red, patients with no OR. (B) The scatter plot graph on the left represents the percentage of CD4 THD cells within lung-cancer specific CD4 T cells in a sample of patients from the indicated G1/G2 groups. On the right, same as left but representing the percentage of CD28+ CD4 T cells within lung-cancer specific CD4 T cells. Objective responders (OR) are shown in green. In red, patients with no OR. Relevant statistical comparisons are shown within the graphs with the test of Mann-Whitney. N, number of biological replicates (independent patients); Ns, no significant differences (P<0.05).
Functional systemic CD4 immunity is required for clinical responses to PD-L1/PD-1 blockade therapy (2019)
Figure 4

Figure 4. Systemic circulating CD4 T cells in NSCLC patients are proficient in cytokine production with an overall Th17 profile. (A) Column graphs representing the percentage of CD4 T cells from NSCLC patients or age-matched healthy donors as represented in the graph, expressing the indicated cytokines after T cell stimulation with anti-CD3/anti-CD28 antibodies. Data on total CD4 (left graph), CD28+ subsets (center graph) and CD28negative subsets (right graph) are shown. Error bars correspond to standard deviations and bars represent means from 5 independent biological replicates (patients). (B) as in (A) but representing CD4 T cells expressing only one cytokine, two (C) or three cytokines simultaneously (D).Relevant statistical comparisons are shown within the graphs by the test of Kruskall-Wallis. *, **, ***, indicate significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences.
Functional systemic CD4 immunity is required for clinical responses to PD-L1/PD-1 blockade therapy (2019)
Figure 5

Figure 5. CD8 dysfunctionality recovers in G1 patients undergoing immunotherapy. (A) Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549-SC3 cells from the indicated patient groups, before the start of immunotherapy. Relevant statistical comparisons are shown with the test of Mann-Whitney. (B) Dot plots of the percentage of Ki67+ proliferating CD8 T cells after ex vivo activation by A549-SC3 cells. CD8 T cells were obtained from samples of G1 or G2 patients before immunotherapy and after three cycles of anti-PD-1 therapy. Relevant statistical comparisons are shown with the test of Mann-Whitney. Green, objective responders (OR) and red, no ORs. (C) Same as in (A) but in the presence of an isotype control antibody or an anti-PD-1 antibody molecularly equivalent to pembrolizumab. Relevant statistical comparisons are shown with comparisons carried out with paired Student´s t test. (D) Change in CD8 CD28+ T cells from baseline to post-therapy in G1 patients (left) or in G2 patients (right). Statistical comparisons were carried out with paired Student´s t test.
Functional systemic CD4 immunity is required for clinical responses to PD-L1/PD-1 blockade therapy (2019)
Figure 6

Figure 6. PD-1/LAG-3 co-blockade recovers proliferative capacities of CD4 and CD8 T cells from G2 patients. (A) Scatter plots of PD-1/LAG-3-expressing CD8 T cells after activation by A459-SC3 cells in a sample of G1 and G2 patients within CD28+ and CD28negative populations as indicated in the figure. (B) Dot-plot representing the percentage of proliferating CD4 T cells from a sample of G2 patients before starting immunotherapy, activated ex vivo by A549-SC3 cells in the presence of the indicated antibodies. "Dual" represents the addition of both anti-PD-1 and anti-LAG-3 antibodies. Appropriate statistical comparisons are shown within the graph with two-way paired ANOVA. Data from CD28+ and CD28negative subsets are represented as indicated. (C) as in (B) but for CD8 T cells.*, ** indicate significant and very significant differences, respectively. Ns, indicates non-significant differences (P>0.05).
Pharmacological targeting of the protein synthesis mTOR/4E-BP1 pathway in cancer-associated fibroblasts abrogates pancreatic tumour chemoresistance. (2015)
Figure 1

CAFs, but not PaSCs, secrete soluble proteins providing pancreatic cancer cell resistance to gemcitabine that is inhibited by CAF pre-treatment with SOM230

A CAFs isolation from human pancreatic tumour resections (left panel) and in vitro primo-culture (right panel).

B Isolated CAFs and PaSC characterization in vitro by immunofluorescence using anti-vimentin (left panel) or anti-α-SMA (right panel) antibody (one representative field of n = 3).

C Experimental method schematic representation. CAFs or PaSC was treated or not with SOM230 (10−7 M) for 48 h. Conditioned media (CM) were collected. Pancreatic cancer cells were incubated for 48 h with the indicated CM, in the presence or not of gemcitabine (100 μg/ml).

D, E Panc-1 cell viability was assessed by MTT. Results (mean ± SD) are presented for each treatment (CM from PaSC, or from CAF, or from CAF ± SOM230, SOM230) as a percentage of the respective gemcitabine-untreated cells (= 100%) (n = 4; from left to right: **P = 0.007, ##P = 0.006 in D; *P = 0.032, #P = 0.041, #P = 0.047 in E).

F Apoptosis induced by gemcitabine was evaluated by Western blot using an anti-cleaved caspase-3 or anti-PARP antibody (representative of n = 3).

G Panc-1 was analysed by flow cytometry. Percentages (mean ± SD) of annexin V-positive cells are indicated (n = 3; from left to right: **P = 0.005, ##P = 0.004, **P = 0.006).

Data information: * effect of treatment (gemcitabine or SOM230) versus untreated cells; #CM-incubated versus non-incubated gemcitabine-treated cells. Source data are available online for this figure.
Pharmacological targeting of the protein synthesis mTOR/4E-BP1 pathway in cancer-associated fibroblasts abrogates pancreatic tumour chemoresistance. (2015)
Figure 2

High protein synthesis in CAFs mediates chemoprotective effect on pancreatic cancer cells—reversion through inhibition of protein synthesis with SOM230

Immunoblotting of protein extracts from PaSCs or CAFs treated (+) or not with SOM230 (10−7 M) for 48 h, using the anti-puromycin antibody (representative of n = 3).

Polysomes profiles of CAFs treated or not with SOM230 for 48 h (representative of n = 3).

Immunoblotting using an anti-P-Akt, anti-P-S6 or anti-β-actin (loading control) antibody of protein extracts from PaSCs or CAFs treated (+) with SOM230 for 30 min (representative of n = 3).

Immunoblotting of protein extracts from CAFs treated (+) or not with SOM230 (10−7 M) for the indicated times, using anti-P-Akt, anti-P-S6 or anti-4E-BP1 antibody (representative of n = 3).

Protein concentration in untreated or SOM230-treated CAF-CM or extracts normalized per 1 × 106 cells. Results (mean ± SD) are presented as a percentage of the untreated CAFs (= 100%) (n = 3; from left to right: ##P = 0.008, ##P = 0.007).

Immunoblotting using an anti-4E-BP1 or anti-GAPDH (loading control) antibody of protein extracts from siCTR- or si4E-BP1-transfected CAFs (representative of n = 3).

Immunoblotting of equal amounts of protein from siCTR- or si4E-BP1-transfected CAFs treated (+) or not with SOM230 for 48 h, using the anti-puromycin antibody (representative of n = 3).

Panc-1 cell viability was assessed by MTT. Panc-1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230-treated CAFs transfected with the siCTR or si4E-BP-1. Results (mean ± SD) are presented as a percentage of the untreated CAFs (= 100%) (n = 3; **P = 0.003, §§P = 0.002).

Caspase-3 and PARP cleavage induced by gemcitabine in Panc-1 was evaluated by Western blot using the respective antibodies (representative of n = 3). Arrow indicates cleaved forms of PARP.

Data information: * gemcitabine-treated versus gemcitabine-untreated cells; # SOM230-treated versus SOM230-untreated cells; § si4E-BP1-transfected versus siCTR-transfected cells. Source data are available online for this figure.
Pharmacological targeting of the protein synthesis mTOR/4E-BP1 pathway in cancer-associated fibroblasts abrogates pancreatic tumour chemoresistance. (2015)
Figure 3

CAFs express the somatostatin receptor subtype 1 sst1 that mediates SOM230 inhibitory effect on CAF-induced chemoprotection

A Expression of the somatostatin receptor subtype 1 (sst1) analysed by RT–qPCR in the human pancreatic endocrine cell line BON and in CAFs isolated from 11 different patients (mean ± SD).

B Immunoblotting of protein extracts from PaSCs and CAFs, using anti-sst1, anti-α-SMA, anti-P-S6 or anti-GAPDH (loading control) antibody (representative of n = 3).

C Immunoblotting of protein extracts from BON, CAFs and indicated pancreatic cancer cell lines, using anti-sst1 or anti-GAPDH (loading control) antibody (representative of n = 3).

D Immunofluorescence confocal microscopy analyses of α-SMA and sst1 in CAFs. Co-localization of α-SMA and sst1 is shown (merge) (one representative field of n = 3) (scale bar = 100 μm).

E–G Representative immunofluorescence confocal microscopy analyses of sst1 co-localization with cytokeratin-19 (n = 42 PDAC samples) (E), α-SMA (n = 42 PDAC samples) (F) or with P-4E-BP1 (n = 25 PDAC samples) (G) in serial slides (scale bar = 100 μm). Dashed white lines represent the limits between stroma and tumour epithelial glands.

H, I Immunoblotting of protein extracts from siCTR- or sisst1-transfected CAFs treated (+) or not with SOM230 for 48 h (sst1, puromycin and GAPDH as loading control, H) or for 30 min (P-Akt, P-S6, 4E-BP1 and GAPDH as loading control, I) (representative of n = 3).

J Panc-1 cell viability was assessed by MTT. Panc-1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230-treated CAFs transfected with siCTR or si-sst1. Results (mean ± SD) are presented as the percentage of the untreated cells (= 100%) (n = 3; **P = 0.003, §§P = 0.002).

Data information: * gemcitabine-treated versus gemcitabine-untreated cells; § si-sst1-transfected versus siCTR-transfected cells. Source data are available online for this figure.
Pharmacological targeting of the protein synthesis mTOR/4E-BP1 pathway in cancer-associated fibroblasts abrogates pancreatic tumour chemoresistance. (2015)
Figure 4

Molecular mechanisms of SOM230 action through sst1 for inhibition of Akt-4E-BP1 signalling pathways in CAF

A–D Immunoblotting using an anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAFs pre-treated (+) or not overnight with PTX (100 ng/ml) (A), gallein (10 μM) (B), NSC87877 (10 μM) (C), Src inhibitor (5 μM) (D), treated (+) or not with SOM230 (10−7 M) for 30 min (representative of n = 3).

E, F Immunoblotting using anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAF siCTR- or siSHP2- (E) or siPTPε- (F) transfected CAF treated or not with SOM230 for 30 min (representative of n = 3).

G, H Immunoblotting using an anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAFs treated with the indicated molecules (PDGF receptor inhibitor, 5–10 μM; Jak1/2 inhibitor ruxolitinib, 5 or 10 μM; EGFR inhibitor, 150 or 300 nM and recombinant PDGF, 5 μg/ml) (representative of n = 3).

Source data are available online for this figure.
Pharmacological targeting of the protein synthesis mTOR/4E-BP1 pathway in cancer-associated fibroblasts abrogates pancreatic tumour chemoresistance. (2015)
Figure 5

SOM230 increases in vivo sensitivity to gemcitabine of tumour xenograft (MIA PaCa-2-Luc cells and CAFs or human PDAC resection)

A–C MIA PaCa-2-GLuc cells were injected with or without CAFs into the pancreas of nude mice. Mice were treated with each indicated treatment (SOM230-LAR at day 7 and gemcitabine at days 7, 10, 14 and 17), and the plasmatic luciferase activity was measured (mean ± SD) at days 7, 14 and 21 (n = 5) (P = 0.003 for gemcitabine + SOM230LAR versus untreated) (A). Tumours were excised, photographed (scale bars are 1 cm) (B), weighted (P = 0.0008) (C) and paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (E).

D Human tumours were subcutaneously xenografted in nude mice and tumour volumes (mean ± SD) measured (at day 37, P = 0.0009 for gemcitabine + SOM230LAR versus untreated and P = 0.021 for gemcitabine + SOM230LAR versus gemcitabine). Mice were treated with the indicated treatments (SOM230-LAR at days 3 and 31, and gemcitabine at day 3 and twice a week thereafter).

E, F Tumours excised from mice as in (A-C) were paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (scale bar = 100 μm) (E). Cleaved caspase-3 quantification was performed by counting the mean number of positive cells per field in five independent tumours (mean ± SD) (F) (*P = 0.036, **P = 0.009).

G, H Tumours excised from mice as in (D) were paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (scale bar = 100 μm) (G). Cleaved caspase-3 quantification was performed by counting the mean number of positive cells per field in five independent tumours (mean ± SD) (H) (*P = 0.027).

Data information: * treated versus untreated xenograft. # SOM230-LAR + gemcitabine-treated versus gemcitabine-treated xenograft. *P < 0.05; **P < 0.01; ***P < 0.001.
Pharmacological targeting of the protein synthesis mTOR/4E-BP1 pathway in cancer-associated fibroblasts abrogates pancreatic tumour chemoresistance. (2015)
Figure 6

Identification of proteins differentially secreted by CAFs upon SOM230 treatment and involved in CAF-CM-induced chemoresistance on pancreatic cancer cells

A Membrane antibody array assay using CM from SOM230-treated or not CAFs (representative of n = 3). Controls are circled in blue dashed line and IL-6 in red square.

B, C Anti-IL-6 ELISA assay (mean ± SD) using CM or protein extracts from SOM230-treated or not CAF (from left to right: §§§P = 0.0002, §§§P = 0.0002) (B), or CM from siCTR- or si4E-BP1-transfected CAFs treated or not with SOM230 (from left to right: ***P = 0.0003, $$P = 0.004) (C) (n = 3).

D Panc-1 cell viability was assessed by MTT. Results (mean ± SD) are presented for each treatment as the percentage of the respective untreated cells (= 100%) (n = 3; from left to right: **P = 0.004, ##P = 0.001, §P = 0.023, §P = 0.019).

E, F Immunoblotting using an anti-cleaved caspase-3, anti-PARP or anti-GAPDH (loading control) antibody of protein extracts from Panc-1 cells incubated with CAF-CM pre-incubated or not with an anti-IL-6 neutralizing antibody (E), or with human recombinant IL-6 (representative of n = 3) (F).

G, H Immunoblotting using an anti-survivin or anti-GAPDH (loading control) antibody of protein extracts from Panc-1 cells stimulated or not with CAF-CM pre-incubated or not with anti-IL-6 neutralizing antibody (G), or with human recombinant IL-6 (representative of n = 3) (H).

I Immunoblotting using an anti-P-Akt, anti-P-S6 or anti-β-actin (loading control) antibody of protein extracts from SOM230-treated or SOM230-untreated CAF incubated or not with IL-6 neutralizing antibody (representative of n = 3).

Data information: * gemcitabine-treated versus gemcitabine-untreated cells; # CM-incubated versus CM-non-incubated cells; § effect of treatment (SOM230 or anti-IL-6 neutralizing antibody, or recombinant IL-6) versus untreated cells; $ si4E-BP1-transfected versus siCTR-transfected cells. Source data are available online for this figure.
Pharmacological targeting of the protein synthesis mTOR/4E-BP1 pathway in cancer-associated fibroblasts abrogates pancreatic tumour chemoresistance. (2015)
Figure 7

IL-6 concentration correlated with α-SMA-expressing CAFs abundance in human and mouse pancreatic tumours and plasma

A–C Immunohistofluorescence using an anti-α-SMA or anti-IL-6 antibody in paraffin-embedded sections from MIA PaCa-2-Gluc and CAF cells orthotopic co-xenografted tumours from Fig5A (A) or from human tumour subcutaneously xenografted from Fig5D (B) in nude mice treated or not with gemcitabine + SOM230 (representative of five different tumours). Representative co-localization of α-SMA with IL-6 in human PDAC samples (C) (n = 15). Scale bar = 100 μm.

D, E Plasma was collected from MIA PaCa-2GLuc and CAFs (*P = 0.034, **P = 0.007) (D), or human pancreatic tumour (*P = 0.028, **P = 0.004) (E), xenografts in nude mice and human IL-6 plasmatic concentrations (mean ± SD) were quantified by ELISA (n = 5 mice / group). * treated versus untreated mice.
Hair follicle stem cell progeny heal blisters while pausing skin development (2021)
Figure 1

Figure 1. Healing of subepidermal blisters in neonatal mice.

A Schematic diagram of suction blistering and sample collection. A blister produced on C57BL/6 wild-type (WT) mouse dorsal skin at P1. BM: basement membrane.

B-D Blistered samples at P1. (B) Hematoxylin and eosin (H&E, top) and alkaline phosphatase (AP, bottom) staining. Hair follicles (HFs) remaining in the dermis (indicated by arrows). Scale bar: 500 μm. (C) α6 Integrin (ITGA6, indicated by arrowheads) and type IV collagen (COL4, arrows) labeling (left). Laminin 332 (L332, arrows) staining (right). Scale bar: 100 μm. (D) Ultrastructural findings of blistered skin (left image: blister roof, right image: blister bottom) . Hemidesmosomes (white arrowheads) and lamina densa (arrows) are indicated. Scale bar: 1 μm.

E H&E (left), pan-cytokeratin (PCK, middle), and ITGA6 staining (right) at P2. The regenerated epidermis is indicated by arrowheads. Scale bar: 200 μm.

F Keratin 14 (K14) and keratin 10 (K10) staining of the nonlesional (intact) and lesional (blistered) skin at P2 (upper images and inlets: sections, lower images: whole-mount imaging). HFs are indicated by arrows in the whole-mount images. Scale bar: 30 μm.

G H&E (top) and loricrin (LOR, bottom) staining at P3. Scale bar: 200 μm.

H H&E (left), PCK (middle), and LOR staining (right) at P4. Scale bar: 200 μm.

Data information: Blisters are indicated by stars. Representative images are shown from three or more replicates in each group.
Hair follicle stem cell progeny heal blisters while pausing skin development (2021)
Figure 3

Figure 3. Predominant contribution of HF-derived keratinocytes to subepidermal blister healing.

A (Top) BrdU labeling of blistered samples at P2. Scale bar: 100 μm. BrdU-positive cells are indicated by arrows. Blisters are indicated by stars. (Bottom) Quantification of BrdU-positive cells in the epidermis (left) and HFs (right) (n=4 biological replicates). The data are shown as the mean ± SE. *0.01<p<0.05, one-way ANOVA test, followed by Tukey's test. NS, no significance.

B α5 integrin (ITGA5) labeling at P2 (left image: section, right image: whole-mount). Scale bar: 100 μm. Blister edges (epidermal tongue) and HFs are indicated by arrowheads and arrows, respectively. Blisters are indicated by stars.

C Lineage tracing strategy.

D (Top) Sections of K14CreER:H2B-mCherry mice at P4. Scale bar: 100 μm. (Bottom) Quantification of mCherry-positive cells (n=3).. The data from individual mice are connected by lines. Student's t-test. NS, no significance.

E Whole-mount imaging of K14CreER:R26R-confetti samples at P4. Scale bar: 200 μm.

F (Top) Sections of Lrig1CreER:H2B-mCherry mouse skin at P4. Scale bar: 100 μm. (Bottom) Quantification of mCherry-positive cells (n=3). The data from individual mice are connected by lines. *0.01<p<0.05, Student's t-test.

G Whole-mount imaging of Lrig1CreER:R26R-confetti mouse samples at P4. Scale bar: 200 μm.

Data information: Representative images are shown from three or more replicates in each group.