Fig 2: Expression of IL-34 and its two receptors on CP-stimulated MRPTEpiC.Scheme for the in vitro analysis using cultured MRPTEpiC stimulated with CP (A). MRPTEpiC and the culture supernatants were harvested after stimulation with CP (2 μg/mL) for 0 to 24 h. Real-time RT-PCR for Kim-1 (B), IL-34 (C), and CSF-1 (D) in MRPTEpiC after stimulation with CP or TNF-α for 0 to 24 h. IL-34 and CSF-1 protein levels evaluated by ELISA in the culture supernatants of MRPTEpiC after stimulation with CP or TNF-α for 0 to 24 h (E and F). Real-time RT-PCR for cFMS (G) and PTP-ζ (H) in MRPTEpiC after stimulation with CP or TNF-α for 0 to 24 h. In the analysis of real-time RT-PCR, the values were normalized to the β-actin transcript, and are expressed as a relative ratio. Data are expressed as the mean ± SEM. The Mann-Whitney U test was used for statistical analysis.

Fig 3: 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.

Fig 4: 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

Fig 5: 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 al27 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.27 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,4 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.27 (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).5 (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 by6, 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-1high vs CSF-1low (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