Pancreatic ductal adenocarcinomas (PDAC) have a dense stroma -- rich in type I collagen and hyaluronan -- which compresses blood vessels thus reducing vascular perfusion, and the delivery and efficacy of drugs. In parallel, the PDAC microenvironment is immunosuppressive. Therefore, one major goal of our current studies is to determine how the targeting of tumor stroma and immune modulation affects the efficacy of pancreatic cancer therapy. CD40 activation by agonist antibodies can increase the infiltration of tumoricidal macrophages in PDAC models and patients. PDAC desmoplasia is driven by tumor-associated fibroblasts, which are activated by downstream effectors of angiotensin II. Our group has previously shown that the angiotensin II type I receptor (AT1R) blocker losartan reduces desmoplasia and improves the delivery and effectiveness of cytotoxic agents in PDAC models. Moreover, the AT1R blocker telmisartan has potent anti-fibrotic effects at doses that do not affect the mean arterial blood pressure in mice. Also compared to losartan, telmisartan has a greater volume of distribution in tissue. In the present study we determined in a murine model of PDAC how the CD40 agonist antibody FGK45 and telmisartan affect the tumor stroma, the infiltration of immune cells and tumor growth.
We first analyzed the effects of CD40-activation by the agonist antibody FGK45 (100 µg / mouse, iv every other day for 8 days) and telmisartan (1 or 5 mg / kg / day) on the tumor stroma in the orthotopic PDAC model AK4.4. FGK45 did not modify the tumor area occupied by collagen, but significantly reduced the tumor area occupied by hyaluronan. To assess vessel perfusion, we injected fluorescent-lectin intravenously then sacrificed mice 5 min later. In comparison to vehicle-treated tumors there was a trend for a lower density of CD31-positive vessels in telmisartan-treated tumors, however the difference was not significant. Telmisartan did not affect the fraction of lectin-perfused vessels. FGK45 did not change the density of CD31-positive vessels or the number of lectin-perfused vessels. We also determined the effects of a higher dose of telmisartan (10 mg / kg /day) and telmisartan combined with FGK45 on tumor perfusion and hypoxia. Pimonidazole and biotin-lectin were administered respectively 1 hour and 5 min before tumor resection. The higher dose of telmisartan alone or combined with FGK45 significantly increased vascular perfusion. In tumors treated with telmisartan alone or combined with FGK45 there was also a trend for a decrease in hypoxia. We analyzed the composition of the tumor immune infiltrate by flow cytometry. Both FGK45 and FGK45 combined with telmisartan significantly increased the infiltration of macrophages in tumors and reduced the recruitment of monocytic myeloid derived suppressor cells. Interestingly, FGK45 induced a significant increase in B cell infiltration in AK4.4 tumors.
We then determined the effects of the FGK45 antibody and telmisartan on tumor growth. We treated mice with AK4.4 tumors with four doses (100 µg / mouse, iv every other day) of FGK45. In mice treated with FGK45 the AK4.4 tumors were significantly smaller than in the control group. To determine the effects of telmisartan on tumor growth, mice with AK4.4 tumors were surgically implanted with ALZET osmotic pumps and continuously dosed with telmisartan (1 or 5 mg / kg / day) for 12 days. Interestingly, both the dose of 1 and 5 mg / kg / day significantly reduced the growth of AK4.4 tumors. In contrast, in our previous studies with the same tumor model higher doses of losartan (20 or 40 mg / kg / day) did not affect tumor growth. We also evaluated the effect of FGK45 combined with telmisartan. Mice were continuously dosed with telmisartan (10 mg / kg / day) or the DMSO vehicle by osmotic pumps. Two days after the start of telmisartan treatment, 3 doses of FGK45 or a control IgG2a antibody were administered (100 µg / mouse, IV, every other day). We observed tumor regression after FGK45 treatment in 1 of 8 mice and after FGK45 combined with telmisartan treatment in 2 of 7 mice. We also found that both FGK45 alone or combined with telmisartan significantly reduced the bloody ascites, a hallmark of metastasis.
In conclusion, our findings show that CD40 activation improves the infiltration of macrophages and B cells in PDAC lesions. While CD40 activation significantly reduced the accumulation of hyaluronan, it did not affect vascular perfusion or hypoxia. In contrast, telmisartan alone or combined with FGK45 increased vascular perfusion and reduced hypoxia, and did not affect the immune infiltration caused by CD40 activation. FGK45-activation of CD40 alone or combined with telmisartan induced tumor regressions. Currently we are evaluating the effects of FGK45 combined with telmisartan on mice survival.
Citation Format: Shiwei Han, Jelena Grahovac, Trupti Vardam, Yves Boucher. Combination of AT1R blockade with CD40 activation provides enhanced therapeutic efficacy for mouse pancreatic adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B70.