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Hypoxia develops in germinal centers (GCs) induced by model antigens; however, it is unknown whether tumor-reactive GCs are also hypoxic. We identified GC hypoxia in lymph nodes (LNs) draining murine mammary tumors and lethally irradiated tumor cells, and found that hypoxia is associated with the levels of antibody-secreting B cells. Hypoxic culture conditions impaired the proliferation of activated B cells, and inhibited class-switching to IgG1 and IgA immunoglobulin isotypes in vitro . To assess the role of the hypoxic response in tumor-reactive GCs in vivo , we deleted von Hippel-Lindau factor (VHL) in class-switched B cells and found decreased GC B cells in tumor-draining LNs, reduced class-switched and tumor-specific antibodies in the circulation, and modified phenotypes of tumor-infiltrating T cells and macrophages. We also detected the hypoxia marker carbonic anhydrase IX in the GCs of LNs from breast cancer patients, providing evidence that GC hypoxia develops in humans. We conclude that GC hypoxia develops in TDLNs, and that the hypoxic response negatively regulates tumor-induced humoral immune responses in preclinical models.

Poorly oxygenated (hypoxic) cells are key components of the solid tumor microenvironment and are well-established contributors to poor therapeutic outcome. Patients with tumors that contain hypoxic cells have worse outcome after radiotherapy, chemotherapy, and surgery, underscoring the therapeutic relevance of hypoxia in driving more aggressive tumor phenotypes. Angiotensin II type 1 receptor (AT1R) blockers (ARBs) are widely prescribed anti-hypertensive agents that inhibit the activity of angiotensin II derived from the renin-angiotensin system. ARBs have also been shown to exhibit anti-fibrotic activity by inhibiting AT1R expressed on myofibroblasts. We recently reported that the ARB telmisartan inhibits collagen I (Col1) deposition in solid tumors, increasing net tumor perfusion, stabilizing microregional tumor blood flow, and decreasing tumor hypoxia. Cancer-associated fibroblasts (CAFs) produce Col1, although recent clinical data indicate the presence of multiple, functionally-diverse CAF subsets in tumors from patients with breast or ovarian cancer. To further explore how ARBs and CAFs affect the solid tumor microenvironment, we were interested in whether murine CAF subsets are present in human tumor xenografts and, if so, whether telmisartan preferentially influences collagen-producing CAF subsets in solid tumors. We were also interested in whether telmisartan affects collagen deposition, hypoxia, and radiation response in head & neck and cervix tumors that are commonly treated with ionizing radiation therapy. We then asked whether head & neck cancer patients taking ARBs as anti-hypertensive medications have quantifiably different tumor microenvironments and improved therapeutic outcome. Using a panel of six human head & neck and cervix tumor xenografts, we identified four unique subsets (S1-S4) of CAFs that are present at varying levels in the different tumor lines. Telmisartan significantly altered the CAF content of tumors while also reducing CAF-mediated collagen deposition in the tumor microenvironment. We found that telmisartan also decreased tumor hypoxia and improved radiation response in our pre-clinical models, indicating that CAF subsets help to create hypoxia and contribute to radioresistance of solid tumors in an AT1R-dependent manner. Using a retrospective cohort of over 1,100 oropharyngeal cancer patients, we found that patients taking ARBs had dramatically improved therapeutic outcomes after radiation therapy, validating our pre-clinical data and supporting the further development and testing of ARBs as neoadjuvant therapies for patients with hypoxic tumors. Understanding how the solid tumor microenvironment influences cancer therapy is central to improving treatment outcome, and our work identifies CAFs as key contributors to the development of hypoxia in solid tumors. Repurposing clinically approved, anti-hypertensive ARBs represents a novel therapeutic strategy to inhibit CAF activity, modify the solid tumor microenvironment, reduce tumor hypoxia, and improve radiation response. Citation Format: Brennan J. Wadsworth, Che-Min Lee, Ryan Urban, Sarah N. Hamilton, Kevin L. Bennewith. Angiotensin II receptor blockers modify the solid tumor microenvironment and improve radiation therapy response [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-001.

Poorly oxygenated (hypoxic) tumor cells are resistant to radiation and associated with poor patient outcomes. “Chronically” hypoxic cells develop from limited oxygen diffusion through tumor tissue. Changes in blood vessel perfusion cause fluctuations in oxygen delivery resulting in “transiently” hypoxic cells. Transiently hypoxic cells are often near vasculature and more metastatic than chronically hypoxic cells, identifying transient hypoxia as an important therapeutic target. Angiotensin II receptor (AT1R) type 1 blockers (ARBs) are anti-hypertensive agents with potential anti-fibrotic activity. ARB treatment reduces collagen 1 deposition (Col1) which is thought to occur by targeting AT1R-expressing cancer associated fibroblasts (CAFs) in solid tumors. AT1R signaling is activated through its ligand, angiotensin II (Ang II), which ARBs compete with to inhibit AT1R. It is unclear whether systemic sources of Ang II or localized tumor-derived Ang II contributes to CAF AT1R signaling and collagen deposition. Regardless, ARB-mediated inhibition of Col1 deposition was shown to reduced blood vessel compression and improved perfusion. We recently found that treatment with the ARB telmisartan substantially reduced Col1 deposition, stabilized vascular perfusion, and inhibited the development of transient hypoxia in a human tumor xenograft model. However, it only exhibited a modest reduction in CAF activation. Therefore, we hypothesized that only certain subsets of CAFs are responsible for the development of transient hypoxia in solid tumors, and that AT1R signaling between CAFs and cancer cells may be crucial for initiating the Col1 deposition leading to transient hypoxia development. Recent work by Dr. Mechta-Grigoriou’s group reliably identified four functionally distinct CAF subsets in patient breast and ovarian tumor samples using with a multi-color flow cytometry panel of CAF markers. To distinguish CAF subsets in a murine model, we used a similar flow cytometry panel and indeed, found distinguished subsets of CAFs in various human xenograft murine models. Additionally, we show that crosstalk between cancer cells and cancer associated fibroblasts (CAFs) exists in complementary secretion of Ang II and AT1R by cancer cells and cultured fibroblasts, respectively. This indicates that disruption of this localized Ang II may result in changes in CAF secretion and processing of Col1. We predict that this collaboration of CAFs and tumor cells is responsible for collagen deposition, which leads to blood vessel compression, hypoxia, and reduced radiation response. Citation Format: Che-Min Lee, Brennan J. Wadsworth, Kevin L. Bennewith. Changes in cancer associated fibroblast subsets following angiotensin II type I receptor blocker (ARB) treatment reduces transient hypoxia and radiation resistance [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-034.