VASCULAR REGULATION OF ANTI-TUMOR IMMUNITY

Abstract

Blood and lymphatic vessels regulate antitumor immunity through direct and indirect mechanisms, providing new opportunities for improving cancer immunotherapy.

Immune checkpoint blockers (ICBs) have transformed cancer treatment. Unfortunately, ICB therapy usually benefits <15 % of patients and causes immune-related adverse events in a significant number of patients. Another immunotherapy—using engineered chimeric antigen receptor (CAR) T-cells to specifically target tumor-associated antigens—has revolutionized the treatment of multiple “liquid” cancers and has the potential for similar success in solid tumors. Emerging data show that the function of vessels associated with tumors is critical in the response to these immunotherapies (1, 2).

Tumor vessels are abnormal, which impairs blood flow and limits the delivery of oxygen, nutrients and therapeutics, including antibodies and immune cells [Figure 1] (3). The resulting hypoxia and low pH can in turn induce the production of immunosuppressive molecules such as tumor growth factor β (TGFβ), vascular endothelial growth factor (VEGF) and adenosine in the tumor microenvironment (TME). Some of these molecules lower the expression of adhesion molecules on tumor vascular endothelium, and thus interfere with the ability of immune cells to adhere to and migrate across the vessel wall, preventing their entry into the tumor. In addition, proangiogenic molecules upregulate Fas ligand on the endothelial cells and thus induce the apoptosis of infiltrating immune cells. VEGF can also hinder maturation of dendritic cells (DCs), which are needed to present tumor-associated antigens and activate naïve immune cells. Furthermore, hypoxia directly upregulates the expression of immune checkpoints (the “brakes” e.g., PD-L1) on various cells in the TME, including myeloid-derived suppressor cells (MDSCs) and DCs, and promotes the recruitment of highly immunosuppressive regulatory T lymphocytes (Tregs). Finally, hypoxia-induced cytokines also reprogram tumor-associated macrophages (TAMs) from an anti-tumor to a pro-tumor phenotype. To make matters worse, the immunosuppressive molecules generated in the TME can enter the systemic circulation and cause systemic immunosuppression.

Excess angiogenic molecules produced by cancer or host cells cause abnormal tumor vasculature. Aberrant ECM deposition by CAFs and uncontrolled tumor growth create mechanical stresses that compress blood vessels, limiting blood perfusion and the delivery of immune cells, oxygen and immunotherapy drugs to the cancer cells. The abnormal, hypoxic and fibrotic microenvironment further upregulates angiogenic molecules such as VEGF, altering endothelial barrier function and adhesion molecules. The abnormal TME also upregulates immune checkpoint molecules such as PD-L1. These alterations further limit the activity of effector T cells, while recruiting immunosuppressive innate immune cells such as MDSCs and reprogramming anti-tumor TAMs to pro-tumor phenotype. Anti-fibrotic drugs can decompress the blood vessels and anti-angiogenesis therapies can reverse many of the TME abnormalities, resulting in improved perfusion, less hypoxia, better immune cell infiltration and creation of an immunostimulatory microenvironment.

Given the consequences of malfunctioning blood vessels, we and others have developed multiple strategies to improve the function of tumor vasculature (4). Since excess VEGF contributes to vascular abnormalities, in 2001 we proposed that the judicious use of anti-VEGF agents could normalize the function of tumor vessels resulting in improved oxygenation and drug delivery. Subsequently, we demonstrated that low dose anti-VEGFR2 antibody enhanced delivery of immune cells to tumors, reprogrammed TAMs from pro-tumor to anti-tumor phenotype and improved the outcome of vaccine therapy in murine breast cancer models (5). A number of pre-clinical studies have now shown the benefit of combining anti-VEGF agents with various immune therapies, including ICBs and adoptive transfer of T cells [reviewed in (3)].

Since anti-VEGF-induced vascular normalization is transient, a number of strategies are emerging to extend the window of normalization. One such strategy is to target angiopoietin-2 (Ang-2), which begins to increase towards the end of the normalization window. Importantly, dual VEGF/Ang-2 inhibition prolonged the duration of normalization and survival in a number of preclinical models, compared to inhibiting either molecule alone. Relevant for immunotherapies, dual VEGF/Ang-2 targeting reprogrammed TAMs from pro-tumor to anti-tumor phenotype (6) and enhanced tumor response to ICBs (7). Additional rationale for the dual blockade of VEGF/Ang-2 pathways comes from the observation that circulating Ang-2 levels are elevated to begin with or increase in melanoma patients with an unfavorable response to ICBs. Moreover, blood Ang-2 levels negatively correlate with response to ICB with or without anti-VEGF therapy in these patients (3).

Beyond VEGF and Ang-2, a number of other agents that target the endothelial cells and/or pericytes have been shown to normalize tumor vessels. Extensively reviewed in (4), these targets include: Tie-2, VE-PTP, semaphorin-3A/neuropilin 1, Notch signaling, regulator of G protein signaling 5 (Rgs5), endothelial glycoprotein L1, R-Ras, lysophosphatidic acid, mechanosensitive ion channels, matrix metalloproteases, endothelial podosome rosettes, endothelial cell metabolism, and oxygen sensors. Other strategies that have shown the ability to induce normalization include restoring perivascular nitric oxide gradients, metronomic chemotherapy, hormone withdrawal from hormone-dependent tumors, some chemotherapeutics such as eribulin, and physical aerobic exercise. Intriguingly, two recent studies showed that ICBs can normalize tumor vessels in breast cancer models in mice (8, 9).

In addition to vascular dysfunction induced by aberrant biochemical signals, physical forces generated by cells and the extracellular matrix in tumors can impair function of blood and lymphatic vessels by compressing (4). This mechanism is dominant in desmoplastic tumors which generally have abundant carcinoma-associated fibroblasts (CAFs), extracellular matrix (ECM) and very poor prognosis (e.g., pancreatic ductal adenocarcinoma, triple-negative breast cancer). Killing cells with cytotoxic agents or depleting specific matrix components using enzymes can re-open these vessels. However, as cancer cells become resistant to cytotoxic agents and begin to proliferate, vessels collapse again. Similarly, enzymes that target a single ECM component (e.g., hyaluronan), become ineffective when other ECM components (e.g., collagen I) contribute to compressive forces.

Fortunately, these problems can be overcome using agents that reprogram CAFs so that they stop producing multiple ECM components. Angiotensin system inhibitors (ASIs) represent one such class of molecules that reprogram CAFs and have been shown to improve the function of tumor vessels as well as the delivery and efficacy of various therapeutics (10). Moreover, these widely used drugs have been shown to activate both innate and adaptive immune pathways in murine and human tumors (10). Based on a successful phase II clinical trial, this approach is now being tested in a randomized clinical trial in locally advanced pancreatic cancer patients ( NCT03563248). Since ASIs can cause hypotension, novel formulations are needed to lower the systemic exposure of these drugs. Our recent study showed that valsartan linked to pH-sensitive polymers can reprogram CAFs to alleviate immunosuppression and improve T lymphocyte activity without adverse hypotension. This approach improved the response to ICBs in mice bearing primary and metastatic breast tumors (2).

Other strategies are also emerging for targeting CAF-mediated desmoplasia and vessel compression. For example, blocking CXCL12/CXCR4 signaling in mouse models of primary and metastatic breast cancer reduces fibrosis, decreases vessel compression and hypoxia, alleviates immunosuppression, and significantly enhances the efficacy of ICBs (1). Similarly, Jiang et al. showed that the activity of tumor-associated immunosuppressive cells and the highly desmoplastic stroma in PDAC tumors are fueled by focal adhesion kinase (FAK) activity. Selective FAK inhibitors were able to normalize the fibrotic TME, improve T cell delivery to the tumor, and increase survival in mouse models (11).

Endothelial cells present in lymphatic vessels and tertiary lymphoid organs within tumors can also regulate antitumor immunity. By judicious combination of anti-VEGFR2 and anti-PD-L1 antibodies, it is possible to induce high endothelial venules (HEVs) in some tumor models (12). Moreover, Treg depletion can induce the formation of HEVs outside of secondary lymphoid organs. When HEVs form in tumors, they can support lymphocyte infiltration and subsequent tumor killing by cytotoxic T cells (13).

Finally, lymphatic vessels are the routes from tissue to lymph nodes and back to the blood circulation. The lymphatic endothelial cells (LECs) are among the first cells to contact antigens that drain from tissue, and lymph contains cytokines and immune cells transiting to lymph nodes. LECs can control dendritic cell maturation and migration as well as directly present antigens to T cells on MHC class I and II molecules (14). They also produce cytokines that modulate the immune response. Multiple investigators have shown that LECs help maintain peripheral tolerance, limit and resolve effector T lymphocyte responses, and modulate leukocyte function (14). Through these mechanisms, LECs promote tolerance to self-antigens, store antigen for later presentation and temper the response of effector immune cells. In cancer, LECs also play a role in suppressing anti-tumor immune response (14). Paradoxically, tumors containing LECs show better responses to ICBs, as the presence of intratumor LECs correlates with T cell infiltration into the tumor, where they become dysfunctional. These dysfunctional, yet antigen-activated, T cells can then be reawakened by ICBs to produce anti-tumor responses.

Immune-related toxicities are a major problem with ICB and CAR-T cell immunotherapy. However, discontinuing or reducing the dose of these agents can reduce or even abrogate these toxicities. Since improving function of tumor’s blood vessels can improve the delivery of antibodies and cells, and thus lower the required dose, such approaches hold promise for reducing toxicities, while improving efficacy. However, one of the biggest challenges in realizing this goal is the lack of validated biomarkers for guiding the dose and schedule of drugs that directly or indirectly target blood vessels. This is further complicated by the recently described “paradoxical effects of obesity”. Obesity is known to fuel tumor progression, desmoplasia, hypoxia, immunosuppression and resistance to various therapies, including chemo- and anti-VEGF therapies (3). Yet, obese mice and patients respond better to ICBs (15). With the emerging epidemic of obesity, a better understanding of the mechanisms underlying these paradoxical effects are likely to yield strategies to improve immunotherapy further.

In summary, combining strategies that improve function of tumor vessels with ICBs holds promise. For example, two recent phase III trials have shown the benefit of combining anti-PD1/PDL1 antibodies with anti-VEGF agents, leading to the FDA approvals for lung and kidney cancers ( NCT02853331, NCT02366143). However, the relative contribution to survival from different effects of VEGF-blockade along with ICBs in these trials is not known. The ongoing trial that aims to test the role of adding losartan – a safe, inexpensive and widely prescribed anti-hypertensive drug shown to decompress tumor blood vessels – to chemo-radiation and anti-PD1 antibody will reveal the potential of this approach in improving the treatment outcome in pancreatic cancer patients ( NCT03563248).