Differential activity of MAPK signalling defines fibroblast subtypes in pancreatic cancer

Abstract

Fibroblast heterogeneity is increasingly recognised across cancer conditions. Given their important contribution to disease progression, mapping fibroblasts' heterogeneity is critical to devise effective anti-cancer therapies. Cancer-associated fibroblasts (CAFs) represent the most abundant cell population in pancreatic ductal adenocarcinoma (PDAC). Whether CAF phenotypes are differently specified by PDAC cell lineages remains to be elucidated. Here, we reveal an important role for the MAPK signalling pathway in defining PDAC CAF phenotypes. We show that epithelial MAPK activity promotes the myofibroblastic differentiation of CAFs by sustaining the expression and secretion of TGF-β1. We integrate single-cell profiling of post-perturbation transcriptional responses from mouse models with cellular and spatial profiles of human tissues to define a MAPK^high CAF (mapCAF) phenotype. We show that this phenotype associates with basal-like tumour cells and reduced frequency of CD8⁺ T cells. In addition to elevated MAPK activity, this mapCAF phenotype is characterized by TGF-β signalling, hypoxia responsive signatures, and immunoregulatory gene programs. Furthermore, the mapCAF signature is enriched in myofibroblastic CAFs from various cancer conditions and correlates with reduced response to immune checkpoint inhibition in melanoma. Altogether, our data expand our knowledge on CAF phenotype heterogeneity and reveal a potential strategy for targeting myofibroblastic CAFs in vivo.

Fig. 1. Basal-like PDAC cells bear cancer-associated fibroblasts with elevated MAPK activity.

a Scatter dot plot and boxplots of GSVA scores obtained for the MAPK Biocarta gene set (MSigDB) stratified by Moffitt subtypes. From left to right: CCLE and in house cell lines, PanCuRx, ICGC and TCGA. p values by Wilcoxon test (two-sided). TCGA Classical: Min −0.42, Max 0.35, a median of −0.08, with first quartile (Q1) at −0.15, third quartile (Q3) at 0.08, an interquartile range (IQR) of 0.23, lower whisker (LW) −0.42, upper whisker (UW) 0.35. TCGA Basal-like: Min −0.35, Max 0.35, Med 0.05, Q1 −0.14, Q3 0.19, IQR 0.33, LW −0.35, UW 0.35. ICGC Classical: Min −0.44, Max 0.27, Med −0.06, Q1 −0.18, Q3 0.09, IQR 0.27, LW −0.44, UW 0.27. ICGC Basal-like: Min −0.28, Max 0.38, Med 0.05, Q1 −0.05, Q3 0.19, IQR 0.24, LW −0.28, UW 0.38. PanCuRx Classical: Min −0.31, Max 0.31, Med −0.01, Q1 −0.12, Q3 0.05, IQR 0.17, LW −0.31, UW 0.30. PanCuRx Basal-like: Min −0.30, Max 0.26, Med −0.01, Q1 −0.12, Q3 0.07, IQR 0.19, LW −0.30, UW 0.26. b Representative images of multiplex IF performed on FFPE of human PDAC tissues. The panels represent different areas within the same tumour displaying different molecular phenotypes. GATA6^negKRT81^high, basal-like; GATA6^highKRT81^neg, classical. Scale bars as indicated. See also Supplementary Fig. 2b. c Paired dot plot showing quantification of α−SMA⁺PDPN⁺CAFs with a distance below 100 µm to classical and basal-like cells (n = 15 tissues). p values as determined by paired t-test (two-sided). d Paired dot plot showing the quantification of α−SMA⁺PDPN⁺p-ERK⁺ CAFs with a distance below 100 µm to classical and basal-like cells (n = 15 tissues). p values as determined by paired t-test (two-sided). e Representative images of immunohistochemistry for GATA6 and p-ERK on tissues from two patient derived xenografts. Scale bar, 200 μm. Inserts showed a magnification of selected areas (Scalebar, 25 μm). f Quantification of (e) showing the percentage of stromal p-ERK signal. p values as determined by Mann-Whitney test (two-sided). Results presented as mean values ± SD. n = 7 biological replicates GATA6^high; n = 8 biological replicates GATA6^low.

Fig. 2. scRNA-seq of mouse PDAC tumours treated with MEKi reveals quantitative and qualitative changes in cell subsets.

a Schematic representation of the in vivo experimental setting. Mouse and cell cartoon schematic from Malinova et al. b Images of multiplex immunofluorescence of tumour tissues from mice treated with: vehicle (C), MEKi for 2 (T2), 7 (T7) and 14 (T14) days. Scalebars, 50 µm (main), 25 μm (insets). c Quantification of b showing the percentage of Pan-CK^–α-SMA⁺p-ERK⁺ cells. p values by Mann-Whitney test (two-sided). n ≥ 8 mice/condition. Results as mean values ± SD. d UMAP plot displaying unsupervised clustering of viable cells from 12 mouse tumours samples annotated in 11 different clusters, colour-coded by cell type. e Bubble plot showing selected cell type-specific markers across clusters. Dot size indicates cell percentage; colour intensity shows average expression. f Barplots show cell type percentages in samples from vehicle (C) or MEKi (T) treated mice (2 or 7 days). Numbers indicate total cells per sample. Malignant cells (blue) are shown relative to non-epithelial populations; fibroblasts (brown) are shown relative to other TME cells (grey). p values by χ² test (two-sided). g Barplot showing percentage of cells of the epithelial cluster stratified based on the expression level of the eMEKi signature in vehicle (C2, C7) or MEKi (T2, T7) treated samples for 2 or 7 days. p values by χ² test (two-sided). h Barplot showing the quantification of pan-Cytokeratin⁺ cells in tissues MEKi-treated tumour-bearing mice. p values by Mann-Whitney test (two-sided). n = 6 mice. Results as mean values ± SD. i FACS analysis of CAFs (PDPN⁺ cells) in tumour-bearing mice treated with MEKi for 2 or 7 days, shown as a percentage of total viable cells. p values by Mann-Whitney test (two-sided). n ≥ 6 mice/condition. Results as mean values ± SD. j UMAP plots of epithelial cluster cells from vehicle (C2–C7) and MEKi (T2–T7) treated mice, classified as classical or basal-like per Moffitt’s classification. k Barplots showing percentage of epithelial cells from mice treated with vehicle or MEKi (2 or 7 days), classified by Moffitt’s subtypes. p values by χ² test (two-sided).

Fig. 3. MAPK inhibition alters the myCAFs/iCAFs ratio in mouse PDAC.

a UMAP plot of fibroblast cluster from mice treated with vehicle (C2, C7) or MEKi (T2, T7) for 2 or 7 days, classified as myCAFs or iCAFs according to Elyada’s classification. b Barplots show myCAFs and iCAFs percentages. p values determined by χ² test (two-sided). c UMAP plot of the fibroblast cluster obtained by the integration of vehicle- or MEKi-treated mice for 2 days, colour-coded by Elyada’s subtypes. d UMAP showing velocity (arrows) and pseudotime (colour) for each cell of the fibroblast cluster (annotated in Fig. 3c) from mice treated with either vehicle (C2) or MEKi (T2) for 2 days. Black arrow indicates overall velocity direction. e Paired dot plot of human TGF-β1 levels in conditioned media from human cancer cell lines treated with MEKi for 2 (n = 10 cell lines) or 7 (n = 9 cell lines) days. p values by paired t-test (two-sided). f Representative in situ hybridisation images showing Tnc (red; myCAFs) and Mmp3 (green; iCAFs) expression in PDAC tissues from vehicle- or MEKi-treated mice. Scale bar, 60 μm (main), 15 μm (insets). See also Supplementary Fig. 4j, k. g Paired dot plot displaying the percentage of iCAFs and myCAFs in tissues from 6 tumour-bearing mice shown in (f). p values determined by paired t-test (two-sided). n = 6 biological replicates. h Flow cytometry of myCAFs (PDPN⁺LY6C⁻) and iCAFs (PDPN⁺LY6C⁺) as percentage of total CAFs (PDPN⁺ cells). Each value refers to an individual tumour-bearing mouse. Results presented as mean values ± SD. p values determined by Mann-Whitney test (two-sided). See also Supplementary Fig. 4m. n = 6 mice Vehicle; n = 17 mice MEKi. i Representative images of multiplex IF of tissues from vehicle- or MEKi-treated mice. Scale bar, 100 μm (main), 50 μm (insets). j Paired dot plot shows myCAFs (PDPN⁺α⁻SMA⁺LY6C⁻ cells) and iCAFs (PDPN⁺α⁻SMA⁻LY6C⁺ cells) percentages relative to all CAFs. Each dot represents the average value derived from multiple mice, when available. p values by paired t-test (two-sided). n = 3 biological replicates.

Fig. 4. A MAPK^high signature identifies a subcluster of myCAFs in mouse PDAC.

a UMAP plot of cells from the fibroblast cluster stratified according to sMEKi signature from mice treated with vehicle. b Violin plots showing the enrichment of cells (n = 742 cells) expressing high level of the sMEKi signature in myofibroblastic clusters. Data are presented as mean values and 95% confidence interval (CI). See Supplementary Fig. 4c, d. c Barplot representing the percentage of cells of the fibroblast cluster displaying either high or low levels of the sMEKi signature in vehicles (C) and in MEKi (T) treated samples. p values determined by χ² (two-sided). d Volcano plot representing the differentially expressed genes from the comparison between fibroblasts displaying high vs low MAPK transcriptional activity (based on sMEKi levels). The red dots are some of the genes defining the mapCAF signature (n = 38). e Barplot displaying the frequency of cells expressing the mapCAF signature in the myCAF and iCAF clusters from Elyada et al. p values determined by χ² test (two-sided). f Scatter plot showing the positive correlation between TGFB1 expression and the mapCAF GSVA score for samples of the TCGA (n = 148 sample) cohort. P value from r correlation test. Grey area represents 95% CI. g Heatmap showing expression of pathway-responsive genes in specific CAF phenotypes as assessed by PROGENy analysis.

Fig. 5. The human mapCAF signature identifies basal-like tumours.

a UMAP plot of fibroblast compartment after subclustering. Different cell type clusters are colour coded. b Heatmap showing the relative average expression of the most enriched genes for each cluster. Only two representative genes per cluster are reported. Clusters are colour coded as in UMAP plot of (a) and were annotated by markers and pathway enrichment analysis using GSEA by comparing cells within a cluster to all other cells in the dataset. See also Supplementary Data 6. c Density plot showing enrichment of human myCAF, iCAF and mapCAF signatures in the scRNA-seq dataset d Violin plots showing enrichment of the human myCAF, iCAF and mapCAF signatures in the clusters shown in (a). Data are presented as mean values and 95% CI e Boxplot of the GSVA scores for the human mapCAF and myCAF signatures in samples of TCGA (n = 148 sample) cohort stratified by Moffitt’s subtypes. p values by Wilcoxon test (two-sided). mapCAF Classical: Min −0.73, Max 0.65, Med −0.12, Q1 −0.43, Q3 0.15, IQR 0.58, LW −0.73, UW 0.65. Basal-like: Min −0.55, Max 0.73, Med 0.17, Q1 −0.07, Q3 0.47, IQR 0.54, LW −0.55, UW 0.73. myCAF Classical: Min −0.84, Max 0.84, Med −0.10, Q1 −0.46, Q3 0.40, IQR 0.87, LW −0.84, UW 0.84. Basal-like: Min −0.79, Max 0.77, Med 0.06, Q1 −0.28, Q3 0.50, IQR 0.78, LW −0.79, UW 0.77. f Barplot showing the percentage of cells of the fibroblast cluster stratified according to the expression of mapCAF signature in PDAC samples displaying prevalent basal-like (n = 6) or classical (n = 6) epithelial cells as shown in Supplementary Fig. 6g. p values determined by Wilcoxon test (two-sided).

Fig. 6. Spatial mapping of epithelial and stromal gene programmes reveals specific association between p-ERK⁺CAFs and the mapCAF signature.

a Haematoxylin and Eosin (H&E) staining of 4 primary PDAC tumours selected for VISIUM spatial transcriptomics (ST). Scale bar, 1 mm. b Spatial visualisation of the cell types deconvolved using scRNA-seq data from Peng et al.. See also Supplementary Fig. 7a, b. c Spatial visualisation of gene module score for the classical PDAC epithelial signature. d Distribution of module scores for classical PDAC signature in each section. The red line indicates the median value, while the black dot the average expression value. All pairwise comparisons are statistically significant by Wilcoxon test (two-sided). e Spatial visualisation of gene module score for the basal-like PDAC epithelial signature. f Distribution of module scores for basal-like PDAC signature in each section. The red line indicates the median value, while the black dot the average expression value. All pairwise comparisons are statistically significant by Wilcoxon test (two-sided). g Spatial visualisation of gene module score for the myCAFs and the Hypoxia_Hallmark signature. h Spatial visualisation of gene module score for the mapCAF signature. The red boxes indicate the corresponding regions on the multiplex IHC slide for p-ERK, GATA6 and CD8, conducted on a consecutive section of PDAC tissues. Scale bars as shown in figure. Conducted n = 1. i Distribution of module scores for the mapCAF signature in each section. The red line indicates the median value, while the black dot the average expression value. All pairwise comparisons are statistically significant by Wilcoxon test (two-sided).

Fig. 7. mapCAFs are associated with T cell depleted sub-tumour microenvironment.

a Representative image of multiplex IF performed on FFPE of human PDAC tissues. The panels represent different areas within the same tumour displaying either low or high density of p-ERK⁺ CAFs. Scale bars as indicated. See also Supplementary Fig. 8a. b Paired dot plot showing the quantification of CD8⁺ T cells with a distance below 100 µm to p-ERK⁺ or p-ERK⁻ CAFs (n = 15 tissues) shown in (a). p values as determined by Mann-Whitney test (two-sided). c Immunohistochemical staining for CD8 of tumour tissues from tumour-bearing mice treated with either vehicle or MEKi for 7 and 14 days. Scale bar, 100 µm. Inserts showed a magnification of selected areas (Scale bar, 20 μm). d Quantification shown as total number of CD8⁺ T cells per mm² per sample. p values as determined by Mann-Whitney test (two-sided). n ≥ 4 mice/condition. Results presented as mean values ± SD. e Boxplots showing mapCAF GSVA score in melanoma (n = 28 sample). Samples are separated by binary drug response. CR complete response, PR partial response, SD stable disease, PD progressive disease. p value as determined by Wilcoxon test (two-sided). Hugo CR/PR: Min −0.45, Max 0.57, Med −0.17, Q1 −0.38, Q3 −0.04, IQR 0.34, LW −0.45, UW 0.06. PD: Min −0.41, Max 0.58, Med 0.36, Q1 0.03, Q3 0.41, IQR 0.38, LW −0.41, UW 0.58.