Depletion of Adipose Stroma-Like Cancer-Associated Fibroblasts Potentiates Pancreatic Cancer Immunotherapy

Abstract

Abstract: Immune checkpoint blockade therapy, transformative in some cancer types, has remained ineffective for patients with pancreatic cancer. The effects of subpopulations of cancer-associated fibroblasts (CAF) on cancer progression and therapy resistance are incompletely understood. In this study, the roles of CAFs expressing platelet-derived growth factor receptor β (Pdgfrb) and of CAFs expressing markers of adipose stromal cells (ASC) were analyzed in mice with pancreatic ductal adenocarcinoma. Ablation of Pdgfrb+ cells resulted in suppression of primary pancreatic tumor growth, reduction of extracellular matrix deposition, and increased cancer cell metastasis to the liver. A peptide D-CAN, which induces apoptosis in ASC-like CAFs, also reduced pancreatic tumor growth and extracellular matrix deposition while promoting metastases. Single-cell RNA sequencing demonstrated that depletion of either Pdgfrb+ or ASC-like CAFs decreased frequencies of tumor endothelial cells and viable cancer cells. However, whereas depletion of Pdgfrb+ CAFs led to stronger induction of cancer cell aggressiveness markers, depletion of ASC-like CAFs had an opposite effect on remaining CAFs. Depletion of ASC-like CAFs using D-CAN also led to higher infiltration of cytotoxic T-lymphocytes and B-lymphocytes. Administration of anti-PDL1 antibody (aPDL1), which inhibits the immune checkpoint, had a stronger suppressive effect on tumor growth when combined with D-CAN in both female and male mice. Liver metastases were also reduced by the D-CAN/aPDL1 combination more effectively than by aPDL1 alone in female mice. We conclude that improved approaches to target ASC-like CAFs may be effective in combination with immunotherapy.

Significance: This study shows that populations of CAFs have distinct effects on pancreatic cancer progression and shows that depletion of CAFs expressing adipose markers potentiates tumor/metastasis suppression effects of immune checkpoint blockade.

Figure 1

The effect of Pdgfrb+ CAF depletion on orthotopic PDAC progression. Tissues resected from Pdgfrb-TK male mice after 25 days of GCV (N = 3) or control PBS (N = 4) treatment were analyzed. A, KPC tumor size upon resection. B, Representative resected KPC tumors. C, Paraffin sections of tumors in B stained with hematoxylin/eosin, trichrome blue, or subjected to αSMA and endomucin (endothelial marker) immunofluorescence. Arrows indicate examples of well-differentiated KPC cells forming cancerous ducts. Scale bar, 50 μm. D, Quantification of KPC cells in livers based on colony counts. Images: representative wells. *, P < 0.05; **, P < 0.01; ***, P < 0.005; Student t test. H&E, hematoxylin and eosin.

Figure 2

The effect of ASC-like CAF depletion on orthotopic PDAC progression. Tissues resected from male mice after 25 days of D-CAN (N = 3) or control PBS (N = 3) treatment were analyzed. A, KPC tumor size upon resection. B, Representative resected KPC tumors. C, Paraffin sections of tumors in B stained with hematoxylin/eosin, trichrome blue, or subjected to αSMA and endomucin immunofluorescence. Arrows indicate examples of well-differentiated KPC cells forming cancerous ducts. Scale bar, 50 μm. D, Quantification of KPC cells in livers based on colony counts. Images: representative wells. *, P < 0.05; **, P < 0.01; ****, P < 0.001, Student t test. H&E, hematoxylin and eosin.

Figure 3

The effects of Pdgfrb+ CAF vs. ASC-like CAF depletion on the TME. Shown are representative analyses of tumors from Figs. 1 and 2. A, t-distributed stochastic neighbor embedding clusters with cell populations identified based on heatmap analysis (not shown) show changes in labeled populations. B, Changes in frequencies of cells corresponding to labeled populations in A. C, IPA analysis of scRNA-seq data of gene expression in KPC cells and CAFs from (A) identifying top genes and pathways induced (orange) or suppressed (blue). D, Quantification of top pathways’ induction (orange) or suppression (blue) in CAFs from A. AP, Antigen Presenting CAF; FC, fold change; MY, myCAFS; Neut, neutrophil.

Figure 4

ASC-like CAF depletion synergizes with immune checkpoint blockade. Following orthotopic grafting of KPC-Luc cells, female (N = 3 per group) and male (N = 3 per group) mice received PBS, D-CAN, aPDL1, or the combination D-CAN + aPDL1. A, IVIS images of two representative mice at day 22 showing the location of pancreatic primary tumor graft (arrow) and extrapancreatic area of tumor invasion and metastases (punctate). B, KPC tumor size upon resection from females and males. C, Paraffin sections of representative tumors subjected to αSMA and endomucin immunofluorescence. Scale bar, 50 μm. D, Quantification of KPC cells in livers based on colony counts. Images: representative wells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, ANOVA. Max, maximum; Min, minimum.