Arginase inhibition suppresses lung metastasis in the 4T1 breast cancer model independently of the immunomodulatory and anti-metastatic effects of VEGFR-2 blockade

Abstract

Tumor angiogenesis promotes tumor growth and metastasis. Anti-angiogenic therapy in combination with chemotherapy is used for the treatment of metastatic cancers, including breast cancer but therapeutic benefits are limited. Mobilization and accumulation of myeloid-derived suppressor cells (MDSC) during tumor progression and therapy have been implicated in metastasis formation and resistance to antiangiogenic treatments. Here, we used the 4T1 orthotopic syngenic mouse model of mammary adenocarcinoma to investigate the effect of VEGF/VEGFR-2 axis inhibition on lung metastasis, MDSC and regulatory T cells (Tregs). We show that treatment with the anti-VEGFR-2 blocking antibody DC101 inhibits primary tumor growth, angiogenesis and lung metastasis. DC101 treatment had no effect on MDSC mobilization, but partially attenuated the inhibitory effect of mMDSC on T cell proliferation and decreased the frequency of Tregs in primary tumors and lung metastases. Strikingly, DC101 treatment induced the expression of the immune-suppressive molecule arginase I in mMDSC. Treatment with the arginase inhibitor N v -hydroxy-nor-Arginine (Nor-NOHA) reduced the inhibitory effect of MDSC on T cell proliferation and inhibited number and size of lung metastasis but had little or no additional effects in combination with DC101.In conclusion, DC101 treatment suppresses 4T1 tumor growth and metastasis, partially reverses the inhibitory effect of mMDSC on T cell proliferation, decreases Tregs in tumors and increases arginase I expression in mMDSC. Arginase inhibition suppresses lung metastasis independently of DC101 effects. These observations contribute to the further characterization of the immunomodulatory effect of anti-VEGF/VEGFR2 therapy and provide a rationale to pursue arginase inhibition as potential anti-metastatic therapy.

Consensus guidelines for the use and interpretation of angiogenesis assays

“…Isolated cells can be used for functional experiments or lysed immediately to measure mRNA/DNA content by RNA/DNA sequencing or PCR or detect proteins by proteomics or antibody arrays, during natural tumor progression or in response to treatments, for example to identify pathways and their alteration by drugs [262, 264, 270]. A critical parameter for successful cell isolation is the relative density of the to-be-sorted cells in the starting population: The lower the frequency (e.g., below 1%) the longer the sorting time (which may affect viability) and the lower the purity (which can pose problems for genetic, genomic, or proteomics studies).…”

Pro-tumoral immune cell alterations in wild type and Shb-deficient mice in response to 4T1 breast carcinomas

“…VEGFA-dependent angiogenesis has been shown to play a role for 4T1 tumor growth and metastasis [ 43 ] although the effects of VEGFR2 inhibition were modest. The vascular plexa shown in Figure 2 are characteristic of VEGFA-dependent angiogenesis [ 44 , 45 ] and their reduction in knockout tumors is in agreement with reduced VEGFA-dependent angiogenesis occurring under these conditions.…”

“…This conforms with what was previously observed in RIP-Tag2 insulinomas [ 32 ], in which VEGFA-dependent angiogenesis was reduced by absence of the Shb gene but compensated for by other mechanisms, such as “inflammation” [ 21 ]. Upon VEGFR2-blocking treatment of 4T1 tumors, an increase in Arg1 gene expression was noted and inhibition of this enzyme reduced metastasis, implicating arginase 1 in metastasis occurring in response to VEGFA inhibition [ 43 ]. The activation of immunosuppressive pre-metastatic niche cells in tumor-bearing conditions results in up-regulated expression of immune suppressive factors such as arginase 1 (encoded by Arg1 ), inducible nitric oxide synthase and Treg cells that inhibit effector T cell function [ 46 ].…”

Chemotherapy-induced immunological breast cancer dormancy: a new function for old drugs?

“…Combining anti-VEGF therapies with check-point inhibitors (e.g., anti-PD-L1) has shown synergy and positive outcomes in phases I to III studies, particularly in patients with high VEGF levels [177] . Using the 4T1 experimental model of TNBC we have demonstrated that inhibition of tumor angiogenesis with the anti-VEGFR-2 antibody DC101, attenuated the inhibitory effect of MDSC on T cell proliferation and decreased the frequency of Tregs in primary tumors and lung metastases [181] . Combined angiopoietin-2 and VEGF inhibition was shown to promote superior vascular regression, tumor necrosis, and enhanced the perivascular recruitment of cytotoxic T lymphocytes, as compared to the single agents in multiple cancer models.…”