Pharmacologic Normalization of Pancreatic Cancer-Associated Fibroblast Secretome Impairs Prometastatic Cross-Talk With Macrophages

Abstract

Background & aims: Cancer-associated fibroblasts (CAFs) from pancreatic adenocarcinoma (PDA) present high protein synthesis rates. CAFs express the G-protein-coupled somatostatin receptor sst1. The sst1 agonist SOM230 blocks CAF protumoral features in vitro and in immunocompromised mice. We have explored here the therapeutic potential of SOM230, and underlying mechanisms, in immunocompetent models of murine PDA mimicking the heavy fibrotic and immunosuppressive stroma observed in patient tumors.

Methods: Large-scale mass spectrometry analyses were performed on media conditioned from 9 patient PDA-derived CAF primary cultures. Spontaneous transgenic and experimental (orthotopic co-graft of tumor cells plus CAFs) PDA-bearing mice were longitudinally ultrasound-monitored for tumor and metastatic progression. Histopathology and flow cytometry analyses were performed on primary tumors and metastases. Stromal signatures were functionally validated through bioinformatics using several published, and 1 original, PDA database.

Results: Proteomics on the CAF secretome showed that SOM230 controls stromal activities including inflammatory responses. Among the identified secreted proteins, we validated that colony-stimulating factor 1 (CSF-1) (a macrophage growth factor) was reduced by SOM230 in the tumor and plasma of PDA-harboring mice, alongside intratumor stromal normalization (reduced CAF and macrophage activities), and dramatic metastasis reduction. In transgenic mice, these SOM230 benefits alleviate the chemotherapy-induced (gemcitabine) immunosuppressive stroma reshaping. Mechanistically, SOM230 acts in vivo on CAFs through sst1 to disrupt prometastatic CAF production of CSF-1 and cross-talk with macrophages. We found that in patients, stromal CSF-1 was associated with aggressive PDA forms.

Conclusions: We propose SOM230 as an antimetastatic therapy in PDA for its capacity to remodel the fibrotic and immunosuppressive myeloid stroma. This pharmacotherapy should benefit PDA patients treated with chemotherapies.

Keywords: Antimetastatic Therapy; Cancer-Associated Fibroblasts; Macrophages; Pancreatic Adenocarcinoma; Somatostatin Receptor; Stroma Normalization; Stromal Cell Cross-Talk.

Graphical abstract

Figure 1

SOM230 decreases CAF production of secreted proteins enriched in PDA activated stroma signatures. (A) Experimental design of CAF secretome analysis. (B) Graphic representation of quantitative proteomics data. Proteins are ranked in a volcano plot according to their statistical P value (y-axis) and their relative abundance ratio (log fold change) between untreated (NT) and SOM230-treated CAFs (x-axis). (C) SOM230-modulated protein secretion. Heatmap showing the differentially secreted proteins in 9 CAF cultures treated (SOM) or not (NT) with SOM230. Proteins are ordered by the level of significance of the differential test. The first 2 columns indicate if each protein was associated previously with an activated form of stroma in human primary tumors (Moffit et al or Puleo et al databases). The 2 main heatmaps show the level of quantification, normalized by pairs of treated/untreated CAFs as log ratios, in each experiment. Upper box plots show the global levels of these differentially secreted proteins. The 2 right panels illustrate the P value of the differential analysis and the log fold change (FC). (D) Box plot of the average of all secreted protein quantification measured in each sample. Pairs between SOM230-treated and untreated conditions are indicated by a gray segment. The Wilcoxon matched-pairs signed rank test was used to generate P values. ∗∗P < .01. (E) Gene set enrichment analysis for down-regulated pathways in SOM230-treated CAF secretomes, as compared with untreated CAFs. (F) Venn diagram of the overlap of down-regulated proteins in secretome analysis and activated stroma signatures from the Moffitt et al or Puleo et al databases. Common proteins are listed in the right panel. (G) Gene set enrichment analysis comparing secretome genes and activated stroma signatures from Moffitt et al and Puleo et al. (H) CAF primary cultures (n = 5) were transfected with a small interfering RNA targeting sst1 (siSst1) or a control nontargeting siRNA (siCTR), and treated or not (NT) with SOM230. Biglycan, connective tissue growth factor (CTGF), and thrombospondin (THBS 2) were quantified in the CAF conditioned media by ELISA (n = 5, Kruskal–Wallis test followed with the Dunn multiple comparison post-test was used to generate P values, ∗∗∗P < .001). Representative Western blot showing expression of sst1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (internal loading control for protein expression in each condition) in CAFs. LC, liquid chromatography; NES, normalized enriched score; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Figure 2

SOM230 reduces metastasis dependently on CAF-expressed sst1 in a murine PDA model. (A–D) Characterization of WT and KOsst1 murine PSCs. (A) The abundance of sstr1 mRNA was analyzed by real-time quantitative PCR in WT or KOsst1 PSCs, and in spleen as a positive control, and normalized to the housekeeping rps16 mRNA. (B) Western blot analyses of the indicated proteins in PSCs, either WT, heterozygous (Het sst1), or KO sst1. β-actin was used as an internal loading control. (C) Western blot analyses of the indicated proteins in PSCs freshly isolated from mouse pancreata and cultured for 5, 9, and 13 days. (D) Western blot analyses of the indicated proteins in WT or KOsst1 PSCs, treated or not with SOM230 (10^-7 mol/L; 24 h). β-actin was used as an internal loading control. (E) Experimental design of the study. Cancer cells (CC) were syngeneically and orthotopically co-grafted with murine sst1-WT or sst1-KO PSCs (PSC KOsst1). After 3 weeks, mice were randomized and treated or not (NT) with SOM230–long-acting releasing form (LAR) for 3 weeks. After 6 weeks, mice were killed (n = 4–6 mice/group). (F) Tumor volume evolution from treatment day 1 to day 21, monitored weekly by ultrasound, in mice grafted with CCs plus WT or KOsst1 PSCs. (G) Metastasis incidence in mouse lungs and distribution of mice according to their metastatic lung load (cumulative metastatic lung area less than or greater than 0.1% of total lung area). The chi-squared test was used to generate P values. ∗P < .05. (H) Representative H&E staining of mouse lungs. Dashed lines encircle metastases. Scale bars: 100 μm.

Figure 3

SOM230 reduces tumoral and plasmatic CSF-1 expression dependently on CAF-expressed sst1, explaining its antimetastatic effect in murine PDA models. (A) Membrane antibody arrays using plasma from mice grafted with cancer cells (CCs) plus PSCs, and treated or not (NT) with SOM230 (pool of n = 5 mice/membrane). (B) Cytokine array quantification of plasma proteins in CCs plus PSC grafted mice and treated with SOM230 as compared with untreated (NT), showing down-regulated proteins (pool of n = 5 mice/group). (C) Venn diagram of the overlap of down-regulated plasma proteins from grafted mice identified in cytokine arrays (A, B) and from CAF secretome analysis (Table 1). Common proteins are listed underneath. (D and H) CSF-1 ELISA quantification in (D) conditioned media of human CAF (n = 5 CAF primary cultures), or (H) murine PSC overexpressing CSF-1 or not (mock) (performed in quadruplicate), treated or not (NT) with SOM230, and expressed relative to NT. The paired or unpaired Student t test was used. (E) Immunofluorescence confocal analysis of CSF-1 (green), sst1 (red), and cytokeratin-19 (CK-19; purple) expression in 2 patient-derived PDA tissues, showing co-localization in the stroma (at distance from tumor epithelial CK-19–positive cells) of sst1 and CSF-1. Scale bars: 50 μm. (F) Plasma CSF-1 ELISA quantification at death of ungrafted mice (first condition), or grafted mice treated or not with SOM230 (from Figure 2, n = 4 per group). Each dot represents the CSF-1 plasma concentration measured in 1 mouse. Analysis of variance followed by the Bonferroni multiple comparison post-test was used to generate P values. (G) Western blot analysis of tumor lysates from grafted mice (from Figure 2, mouse tumors 1-to-6) treated or not (NT) with SOM230 (n = 6 mice/treatment condition), with anti–CSF-1 or Akt (loading control) antibody, and quantification (densitometric analysis) of CSF-1 normalized to Akt expression (A.U., arbitrary unit). The unpaired Student t test was used to generate P values. (I–K) CCs were syngeneically and orthotopically co-grafted with murine PSCs overexpressing or not (MOCK) a secreted CSF-1 form (PSC–CSF-1). After 3 weeks, mice were randomized and treated or not (NT) with SOM230-LAR for 3 weeks. After 6 weeks, mice were killed (n = 6 mice/group). (I) Tumor volume evolution from treatment day 1 to 21, monitored weekly by ultrasound. (J) Metastasis incidence in mouse lungs and distribution of mice according to their metastatic lung load (cumulative metastatic lung area less than or greater than 0.1% of the total lung area). The chi-squared test was used to generate P values. (K) Representative H&E staining of mouse lungs. Dashed lines encircle metastases. Scale bars: 100 μm. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001

Figure 4

SOM230 reduces TAM presence. (A and B) Experimental design of the study. (A) Cancer cells were syngeneically and orthotopically co-grafted with murine WT or KOsst1 PSCs. One week after graft, mice were randomized and treated or not with SOM230-LAR. Three weeks after graft, mice were killed. (B) Gating strategy to identify viable cells, total CD45-positive cells, total macrophages, CD206high macrophages, and myeloid-derived suppressor cell immune populations (i, red boxes), or total CD45-positive cells, total lymphocytes, helper CD4 lymphocytes, and cytotoxic CD8 lymphocytes immune populations (ii, blue boxes). (C–I) Proportions of (C) total immune cells (CD45+ cells), (D) total macrophages (CD45+; F4/80+; CD11b+ cells), (E) CD206hi macrophages, (F) myeloid-derived suppressor cell (CD45+; CD11b; lymphocyte antigen 6 complex locus G [Ly6g]+ cells), (G) T lymphocytes (CD45+; CD3+ cells), (H) helper T lymphocytes (CD4+ T lymphocytes), and (I) cytotoxic T lymphocytes (CD8+ T lymphocytes) were quantified (in % of total cells) by flow cytometry on tumors from cancer cells + PSC-injected mice (n = 4–5 mice/group). The Kruskal–Wallis test followed by the Dunn multiple comparison post-test was used to generate P values. ∗P < .05.

Figure 5

SOM230 delays the growth and metastasis of spontaneous pancreatic tumors generated in the immunocompetent KPC mouse model. (A) Representative immunostaining of αSMA and sst1 receptor in serial sections of a KPC primary tumor (untreated mice) showing expression of sst1 receptor in αSMA-positive cell areas. Scale bars: 200 μm. (B–H) Experimental design. (B) KPC mice had an ultrasound weekly from 8 weeks of age until tumor size enrollment was reached (homogenous treatment group tumor volumes, 31–200 mm³; mean, 103.9 mm³; analysis of variance followed by the Bonferroni multiple comparison post-test was used). (C) Mice were randomized into treatment groups (PBS-treated [NT], SOM230-long-acting release [LAR], gemcitabine [Gem], or gemcitabine plus SOM230-LAR [Gem-SOM230], n = 8 mice per group). (D–H) Tumor volume was quantified by ultrasound until ethical end point (when maximal-authorized volume of the primary tumor, or clinical end points, were reached). Tumor volume progression from treatment day 1 to 21. Analysis of variance followed by the Bonferroni multiple comparison post-test was used to generate P values. (D) Representation of tumor volume progression from treatment day 1 until death for each mouse in the 4 treatment conditions. (E–H) KPC mouse identity numbers are indicated, according to Table 3. (I and J) Pdx1-Cre; LSL-KrasG12D; Ink4a^fl/fl mice (KIC) were treated as indicated. (I) Tumor weight was measured at euthanasia (n = 4–5 mice/group) (analysis of variance followed by the Bonferroni multiple comparison post-test was used to generate P values). (J) Survival analysis (Kaplan–Meier) was analyzed using a log-rank test. (K–M) KPC liver metastasis and mouse survival analyses. (K) Representative H&E staining. Arrows indicate metastases. Scale bars: 2.5 mm. Metastasis incidence in KPC livers and distribution of mice according to their metastatic liver load (cumulative metastatic liver area less than or greater than 3% of the total liver area). (L) Significance of liver metastatic overload (>3% of the total liver area) was determined with the chi-squared test. (M) Survival analysis of KPC mice treated as indicated. Kaplan–Meier survival data were analyzed using a log-rank test. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001.

Figure 6

Macrophage polarization, ECM deposit, and angiogenesis are decreased in primary KPC tumors upon combination treatment as compared with gemcitabine. Quantification and representative pictures of markers analyzed by immunohistochemistry using the indicated antibodies in KPC primary tumors (Tum.). Each value represents the marker quantification in each tumor (n = 8 mice/group). (A and B) Phospho-γH2AX–positive cells (in % of total cells). Scale bars: 50 μm. (C–F) Quantification of (C) Ki-67–positive cells (in % of total cells), and of (D) CD8-positive cells, (E) lymphocyte antigen 6 complex locus G [Ly6G]-positive cells, or (F) myeloperoxidase (MPO)-positive cells (number/field). (G and H) CD206 mean fluorescence intensity (in intensity of each field). Scale bars: 100 μm. (I and J) αSMA-positive area (in % of each total field area). (K and L) Turquoise blue/green-positive area from Movat pentachrome staining (in % of each total field area). (M) Quantification of phospho-STAT3–positive cells (in number/mm²). (N) Representative pictures. (O) Quantification of MECA32-positive cells (in % of 4′,6-diamidino-2-phenylindole [DAPI]-positive cells) (n = 4–6 mice/group). (P) Quantification of NG2/MECA32 double-positive cells (% of NG2-positive pericytic cells in the Meca32-positive cell population, as in (n = 4–6 mice/group). Kruskal–Wallis test followed by the Dunn multiple comparison post-test was used to generate P values. ∗P < .05, ∗∗P < .01. p-STAT3, phospho-signal transducer and activator of transcription.

Figure 7

Stroma analyses of KPC metastases, and quantification of tumor and plasmatic CSF-1 expression according to mouse treatment and metastasis size. (A–D) Quantification and representative immunohistochemistry pictures of (A and B) CD206-positive cells (number/field), and of (C and D) αSMA-positive area (in % of each metastasis area). Each value represents the marker quantification per liver metastasis (n = 6–38 metastases/group). L, liver; M, metastasis. Kruskal–Wallis test followed by the Dunn multiple comparison post-test was used to generate P values. (E) Representation of αSMA-positive area (in % of each metastasis area) for each KPC liver metastasis (Met.), and according to metastasis size (<0.7 mm² or >0.7 mm²). The unpaired Student t test was used to generate P values. (F and G) Cytokine array quantification of plasma proteins in KPC mice treated with the combination (gemcitabine plus SOM230) compared with gemcitabine alone (pool of n = 5 mouse plasma/treatment condition), (F) showing the membranes, and (G) down-regulated proteins (pool of n = 5 mice/group). (H) Venn diagram of the overlap of SOM230 down-regulated plasma proteins from KPC (treatment Gem-SOM230 vs Gem) (Table 4), from grafted mice (Figure 3A and B, Table 2), both identified using cytokine arrays, and from CAF secretome analysis (Figure 1, Table 1). Common proteins are listed in the right panel. (I) Plasma CSF-1 ELISA quantification in KPC mice treated or not (NT) with gemcitabine (Gem) or SOM230 or gemcitabine + SOM230 (n = 8 mice/treatment condition), at death. Each dot represents CSF-1 plasma concentration measured in 1 mouse. Analysis of variance followed by the Bonferroni multiple comparison post-test was used to generate P values. (J) Western blot analysis of tumor lysates from KPC mice, treated or not (NT) with gemcitabine (Gem), SOM230, or gemcitabine + SOM230 (n = 5–6 mice/treatment condition, randomly chosen from the n = 8 mice enrolled in each treatment group), with anti–CSF-1 and Akt (loading control) antibody, and underneath, quantification (densitometric analysis) of CSF-1 normalized to Akt expression (A.U., arbitrary unit). Analysis of variance followed by the Bonferroni multiple comparison post-test was used to generate P values. (K) Correlation between plasma CSF-1 concentrations and metastasis liver area in KPC mice (untreated mice, n = 12). (L and M) Immunofluorescence confocal images of αSMA (red), CSF-1 (green), and CK-19 (white) on formalin-fixed, paraffin-embedded tissue slides of (L) KPC primary tumors, scale bar: 20 μm (except for the lower-right panel, which is a magnification of the lower-left panel [dashed-lined box], scale bar: 10 μm), and (M) in liver metastases, either stroma-rich (3 upper panels) or stroma-poor (3 lower panels), scale bar: 50 μm. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. IL, interleukin.

Figure 8

Stromal CSF-1 is a factor of poor prognosis in human PDA. (A–G) Analyses of stromal and of tumor epithelial CSF-1 mRNA expression in correlation with functional molecular signatures of PDA stroma and tumor cells, in the Maurer et al data set (RNAseq analyses of laser-microdissected PDA lesions [n = 65 tumors] of the tumor epithelium [tumor Epith.] and of the stroma). (A) Differential expression of CSF-1 mRNA in laser-microdissected PDA lesions of the stroma or of the tumor epithelium (tumor Epith.) (n = 65 tumors) from Maurer et al. The unpaired t test with Welch correction was used to generate P values. (B and E) GSEA for activated stromal signature, as defined by Moffitt et al, or for (C and F) epithelial cancer basal-like signature, or for (D and G) epithelial cancer classic-like signature, in (B–D) high vs low stromal or (E–G) tumor epithelial CSF-1 mRNA-expressing lesions from Maurer et al. (H–L) Analyses of stromal and tumor epithelial CSF-1 mRNA expression in correlation with functional molecular signatures of PDA stroma and tumor cells, in the Nicolle et al data set (PaCaOmics, n = 30 PDX). (H) Differential expression of CSF-1 mRNA in stromal (of murine origin) or tumor epithelial (of human origin) cells. The unpaired t test with Welch correction was used to generate P values. (I and J) Correlation between (I) stromal or (J) tumor epithelial level of CSF-1 mRNA with resectability of patients. The Mann–Whitney test was used to generate P values. (K and L) GSEA for pancreatic cancer cell basal signature in high vs low (K) stromal or (L) tumor epithelial CSF-1 mRNA PDX. (M–Y) Analyses of stromal and of tumor epithelial CSF-1 protein expression, in correlation with clinical data of patients, with pathologic data of PDA tumors, and with functional molecular signatures of PDA stroma and tumor cells (RNAseq analyses), in a personal PDA cohort (n = 38 primary tumors). (M) Representative images of CSF-1 and αSMA expression analyzed by immunohistochemistry on formalin-fixed, paraffin-embedded tissue slides of 2 PDA patient primary tumors (PDA1 and PDA2). (N–W) Quantification using Definiens Tissue Studio of CSF-1 immunohistochemistry (IHC) scores in the stromal (left panels) or in the tumor epithelial (right panels) compartment, and (N) plotted in correlation with a high vs low (based on the median) positive lymph node ratio (LNR), (O) with pathologic markers of the tumors quantified by IHC, that is, activated stroma index (ASI), (P) CD163-positive cells, or (Q) CD8-positive cells. (R–W) RNAseq analyses of the n = 38 PDA tumors generated transcriptomic signature index, as defined by, of the (R) activated stroma index, (S) activated/inflamed stroma index, (T) inactivated/structured stroma index, and (U) immune stroma index, and of the tumor epithelium, (V) classic-like index or (W) basal-like index, which were compared in CSF-1^high vs CSF-1^low (based on the median) IHC scores quantified in the stroma (left panel) or in the tumor epithelium (right panel). The unpaired t test was used to generate P values. (X and Y) Survival analysis of patients presenting a (X) high vs low stromal or (Y) tumor epithelial CSF-1 immunohistochemistry score. Kaplan–Meier survival data were analyzed using a log-rank test. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001