Blocking CXCLs-CXCR2 axis in tumor-stromal interactions contributes to survival in a mouse model of pancreatic ductal adenocarcinoma through reduced cell invasion/migration and a shift of immune-inflammatory microenvironment

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense stromal reaction (desmoplasia). We have previously reported that mice with conditional Kras^G12D mutation and knockout of TGF-β receptor type II (Tgfbr2), PKF mice, develop PDAC with desmoplasia modulated by CXC chemokines that are produced by PDAC cells through tumor-stromal interaction. In this study, we further discovered that PDAC and cancer-associated fibroblast (CAF) accelerated each other's invasion and migration through the CXC chemokines-receptor (CXCLs-CXCR2) axis. Heterozygous knockout of Cxcr2 in PKF mice (PKF2h mice) prolonged survival and inhibited both tumor angiogenesis and PDAC microinvasion. Infiltration of neutrophils, myeloid-derived suppressor cells (MDSCs), and arginase-1⁺ M2-like tumor-associated macrophages (TAMs) significantly decreased in the tumors of PKF2h mice, whereas inducible nitric oxide synthase (iNOS)⁺ M1-like TAMs and apoptotic tumor cells markedly increased, which indicated that blockade of the CXCLs-CXCR2 axis resulted in a shift of immune-inflammatory microenvironment. These results suggest that blocking of the CXCLs-CXCR2 axis in tumor-stromal interactions could be a therapeutic approach against PDAC progression.

Fig. 1. Interactions of PDAC cells and cancer-associated fibroblasts (CAFs) mediated by CXCRLs–CXCR2 signaling pathway.

a Top 10 upregulated genes in CAFs after stimulation with conditioned medium (CM) of PDAC cell lines derived from PKF (Ptf1a^cre/+;LSL-Kras^G12D/+;Tgfbr2^flox/flox;Cxcr2^+/+) mice in the microarray analysis. Cxcl genes are red-colored. b qRT-PCR analysis in CAFs (311f, 545f) after addition of CM from mPanIN cells (PK-CM) or PDAC cells (PKF-CM). Data are means ± standard error (SE). **p < 0.01 compared to the control (C) and (PK-CM). c Invasive activity of PDAC with CM of CAFs with/without CXCR2 inhibitor SB225002. Data are means ± SE. *p < 0.05 compared to the CAF-CM. d Migration of CAFs with CM of mPanIN or PDAC. Data are means ± SE. *p < 0.05, **p < 0.01 compared to the control. e Inhibition of CAF migration by CXCR2 inhibitor SB225002. Data are means ± SE. **p < 0.01 compared to the PDAC-CM

Fig. 2. Heterozygous knockout of Cxcr2 is associated with better outcome of PDAC mice with inhibition of PDAC vessel invasion.

a Kaplan–Meier survival analysis of PKF (n = 27) and PKF2h (n = 25; Ptf1a^cre/+;LSL-Kras^G12D/+;Tgfbr2^flox/flox;Cxcr2^hetero+/-) mice. P = 0.0164 by log-rank test. b Gross appearance of pancreatic tumor in PKF and PKF2h mice (7 weeks old (wo)). c Pancreatic tumor weight (7 wo). Data are medians ± standard deviation (SD). d Histopathological findings including staining for Ki67, CD31, and LYVE-1 in pancreatic tumor in 7-wo PKF (n = 7) and PKF2h mice (n = 6). Scale bars, 100 μm. Insets: 400× magnification. e Histogram of histopathological status of the pancreatic tumor (7 wo). ADM acinar ductal metaplasia. f Count of stained cells in the tumor ductal lumen, juxtatumoral, or dense interlobular stroma with desmoplastic reaction (desmoplastic stroma). Data are medians ± SD. *p < 0.05; **p < 0.01. g Microscopic invasion of PDAC (arrows) into veins or lymph vessels analyzed by double staining with epithelial marker K-19 and CD31 or LYVE-1

Fig. 3. Heterozygous knockout of Cxcr2 inhibits infiltration of MPO⁺ and CD11b⁺/Ly-6G⁺ granulocytes in pancreatic tumor.

a Immunostaining for MPO and double staining for CD11b and Ly-6G in pancreatic tumor of PKF and PKF2h mice (7 wo). Scale bars, 100 μm. Insets: 400× magnification. b Quantification of MPO⁺ and CD11b⁺/Ly-6G⁺ cells. Data are medians ± SD. *p < 0.05; **p < 0.01

Fig. 4. Inhibition of Cxcr2 induces iNOS⁺ macrophage infiltration and TUNEL⁺ apoptotic cells.

a Detection of macrophage markers (F4/80, iNOS, and arginase-1) by immunohistochemistry or apoptotic cells using TUNEL (brown) and K-19 double staining in PKF and PKF2h mice (7 wo). Scale bars, 100 μm. Insets: 400× magnification. b Quantification of F4/80⁺, iNOS⁺ or arginase-1⁺ macrophages and TUNEL⁺ apoptotic cells in the tumor location. Data are medians ± SD. *p < 0.05; **p < 0.01

Fig. 5. Cxcr2-heterozygous knockout inhibits infiltration of CD8⁺ and FoxP3⁺ lymphocytes in the tumor lesion.

a Immunohistochemical analyses for lymphocytic markers in PKF and PKF2h. Scale bars, 100 μm. Insets: 400× magnification. b Quantification of stained lymphocytes in pancreatic tumors. Data are medians ± SD. *p < 0.05; **p < 0.01

Fig. 6. Hypothetical mechanism of PDAC progression/inhibition according to the Cxcr2-heterozygous knockout PKF2h model.

a Secretion of CXCLs from PanIN/PDAC and CAF stimulates CXCL expression and migration/invasion activity via CXCR2. Induction of CXCLs in these cells also promotes tumor angiogenesis. Dotted line represents unclear functions. b In the Cxcr2-heterozygous knockout pancreatic tumor of PKF2h mice, recruitment of MPO⁺ neutrophils, CD11b⁺Ly-6G⁺ MDSCs and arginase-1⁺ M2 macrophages is inhibited compared to PKF mice. In contrast, iNOS⁺ M1 macrophages and TUNEL⁺ apoptotic cells significantly increase in the pancreatic tumor. Micro-vessel density of CD31⁺ vessels is decreased and PDAC microinvasion into the vein and lymph vessels is inhibited in PKF2h pancreatic tumor