Cholecystokinin receptor antagonist alters pancreatic cancer microenvironment and increases efficacy of immune checkpoint antibody therapy in mice

Abstract

Advanced pancreatic ductal adenocarcinoma (PDAC) has typically been resistant to chemotherapy and immunotherapy; therefore, novel strategies are needed to enhance therapeutic response. Cholecystokinin (CCK) has been shown to stimulate growth of pancreatic cancer. CCK receptors (CCKRs) are present on pancreatic cancer cells, fibroblasts, and lymphocytes. We hypothesized that CCKR blockade would improve response to immune checkpoint antibodies by promoting influx of tumor-infiltrating lymphocytes (TILs) and reducing fibrosis. We examined the effects of CCKR antagonists or immune checkpoint blockade antibodies alone or in combination in murine models of PDAC. Monotherapy with CCKR blockade significantly decreased tumor size and metastases in SCID mice with orthotopic PDAC, and in C57BL/6 mice, it reduced fibrosis and induced the influx of TILs. Immune-competent mice bearing syngeneic pancreatic cancer (Panc02 and mT3-2D) that were treated with the combination of CCK receptor antagonists and immune checkpoint blockade antibodies survived significantly longer with smaller tumors. Tumor immunohistochemical staining and flow cytometry demonstrated that the tumors of mice treated with the combination regimen had a significant reduction in Foxp3+ T-regulatory cells and an increase in CD4+ and CD8+ lymphocytes. Masson's trichrome stain analysis revealed 50% less fibrosis in the tumors of mice treated with CCKR antagonist compared to controls and compared to checkpoint antibody therapy. CCKR antagonists given with immune checkpoint antibody therapy represent a novel approach for improving survival of PDAC. The mechanism by which this combination therapy improves the survival of PDAC may be related to the decreased fibrosis and immune cells of the tumor microenvironment.

Keywords: Cholecystokinin; Fibrosis; Tumor microenvironment; Tumor-infiltrating lymphocytes.

Fig. 1

Monotherapy with CCKR antagonist inhibits primary tumor growth and alters the pancreas microenvironment. a Panc02 pancreatic cancer growth is increased in SCID mice compared to C57BL/6 mice (**p < 0.01). Tumor growth is suppressed in SCID mice when the animals are treated with a CCK receptor antagonist (****p < 0.0001) but not in the immune-competent mice. The percentage of mice with metastases is shown above each respective column. CCKR antagonist therapy reduced metastases in C56BL/6, mice and there were no metastases in SCID mice treated with the CCKR antagonist. b CD3+ tumor-infiltrating lymphocytes significantly increase in the pancreatic cancer microenvironment with CCKR antagonist treatment in both C57BL/6 mice bearing Panc02 tumors and in the mutant KRAS transgenic mice (**p < 0.01). c Trichrome stain of Panc02 tumor from a vehicle-treated mouse demonstrating marked fibrosis. d Trichrome stain of a Panc02 tumor from a mouse treated with the CCKR antagonist proglumide demonstrates marked decreased fibrosis. e Quantitation of fibrosis from Panc02 tumors of untreated mice (control) is significantly greater than tumors of mice treated with proglumide (***p < 0.001). f Trichrome stain of a pancreas from the control mutant KRAS transgenic mouse shows extensive fibrosis. g Trichrome stain from an age-matched mutant KRAS transgenic mouse that was treated with the CCK receptor antagonist proglumide, showed significantly diminished fibrosis. (Figures f and g were reproduced with permission from Pancreas (2014); [21])

Fig. 2

Combination therapy with PD-1 antibody and CCKR antagonist inhibits pancreatic cancer growth. a Tumor volumes were not significantly smaller in immune-competent mice treated with PD-1 antibody or CCKR antagonist monotherapy (L-364,718) compared to PBS-treated controls. In contrast, tumor volumes of mice receiving both the CCKR antagonist and the immune checkpoint blockade antibody were significantly smaller (*p < 0.05). b The number of CD8+ cells significantly increased in mice treated with the combination therapy. c CD4+ cells in the tumors also increased in number with combination therapy. d Immunohistochemistry of mouse Panc02 tumors shows positive immunoreactivity for CD8+ cells with each treatment and the greatest in the combination group. e Immunohistochemistry of tumors demonstrates CD4+ stained cells for each treatment (mag 20×)

Fig. 3

Effects of CCKR blockade with L364,718 and CTLA-4 antibody on survival and tumor immune cells. a Kaplan–Meier survival curve for immune-competent C57BL/6 mice bearing Panc02 subcutaneous tumors show that all control animals (PBS) had died by day 50 when 42% of the CCKR antagonist (CCK) treated, 42% of the CTLA4-Ab (CTLA4) treated, and 71% of the combination-treated mice were still alive (*p < 0.05). b Foxp3+ (Tregs) immune cells decrease in the tumor microenvironment with combination therapy. c CD8+ cell numbers increased in pancreatic cancer treated with the combination of CTLA-4 immune checkpoint blockade antibody and CCKR antagonism. d CD4+ cell numbers were increased also in tumors of mice treated with the combination of the CCKR antagonist and a CTLA-4 immune checkpoint blockade antibody. e CD4+ and CD8+ tumor-infiltrating lymphocytes analysis by flow cytometry. Although both populations of T cells increased with therapy, the increase in the number of infiltrating CD8+ cells was greater. The CD4:CD8 ratio decreased significantly with the combination therapy using both the CCK receptor antagonist and the CTLA-4 antibody

Fig. 4

Effects of CCKR blockade with proglumide and PD-1 antibody on growth of mT3 murine pancreatic cancer. a Growth curves over time shows that CCKR antagonist monotherapy (**p < 0.01), PD-1 monotherapy (***p < 0.001), combination therapy (***p < 0.001) significantly slowed tumor growth of mT3 tumors compared to PBS controls. b CD4+ tumor-infiltrating lymphocytes increased with each of the treatments compared to PBS control-treated mice. c CD8+ tumor-infiltrating lymphocytes increase with each of the treatments compared to PBS. d Foxp3+ (Tregs) decrease with each of the treatments compared to PBS control-treated mice. e Control-treated mice mT3 tumors also exhibited dense fibrosis as shown by Masson’s trichrome stain (e-1). Fibrosis was decreased in tumors of mice treated with the CCKR antagonist (e-2) but not in tumors of mice treated with PD-1 antibody monotherapy (e-3). Combination treatment decreased tumor-associated fibrosis similar to that of proglumide-treated mice (e-4). f Quantitative analysis of intratumoral fibrosis demonstrated significant differences in mice treated with proglumide by Kruskal–Wallis analysis (**p < 0.01)