John M. L. Ebos

Angiogenesis and the Tumor Microenvironment: Vascular Endothelial Growth Factor and Beyond

Our understanding of the dynamic tumor microenvironment (TME) has improved exponentially over the last few decades. In addition to traditional cytotoxic agents, anti-cancer strategies now include numerous molecular-targeted drugs that modulate distinct elements of the TME. Angiogenesis is an underlying promoter of tumor growth, invasion, and metastases. From traditional and emerging angiogenic cytokines and their receptors to novel immune checkpoint inhibitors, regulation of the tumor microenvironment is potentially key in countering tumor progression. In this article, an overview of the architecture of the TME and the orchestration of angiogenesis within the TME is provided. Additionally, traditional and novel angiogenic targets of current interest within the TME are reviewed.

Preclinical recapitulation of antiangiogenic drug clinical efficacies using models of early or late stage breast cancer metastatis

Historically, preclinical tumor therapy models in mice have frequently been deficient in predicting subsequent clinical activity; over-prediction of clinical anti-tumor efficacy is common. Several approaches are being made in an attempt to improve the clinical relevance of preclinical models, and include the use of genetically engineered mouse models (GEMMs) of cancer or patient derived xenografts (PDXs). Here we summarize, in the context of breast cancer, another approach, namely, the development of postsurgical models of either macroscopic or microscopic metastatic disease to mimic metastatic or adjuvant therapy. To do so we used in vivo selected metastatic variants of established human breast cancer cell lines such as MDA-MB-231. Testing antiangiogenic drugs such as the oral tyrosine kinase inhibitor (TKI) sunitinib alone or combined with chemotherapy in models involving treatment of established primary tumors invariably resulted in demonstrable anti-tumor activity. In contrast, identical treatments of postsurgical mice with advanced metastatic disease did not: survival times were not prolonged. This reflects multiple failed phase III trials of sunitinib based therapies in metastatic breast cancer patients. However, using a VEGF pathway targeting antibody drug instead of a TKI, with (paclitaxel) chemotherapy, resulted in anti-tumor activity in the metastatic setting, partially reflecting prior clinical results of the E2100 phase III trial of weekly paclitaxel plus bevacizumab. Other experiments involving postsurgical adjuvant treatment of early stage disease foreshadowed the phase III clinical trial failures of adjuvant bevacizumab in colorectal or breast cancer. In contrast, some investigational metronomic oral chemotherapy protocols alone or in combination with an antiangiogenic drug demonstrated potent activity in the advanced metastatic setting; these encouraging results have yet to be validated in randomized phase III clinical trials, which are underway based on some encouraging phase II clinical trial results. We have also observed circumstances where mice with advanced systemic disease, when successfully treated so as to prolong survival, sometimes relapse with brain metastases, reflecting a similar clinical phenomenon. Given our overall findings, we suggest that using preclinical mouse tumor models which mimic postsurgical adjuvant or metastatic therapy may be a promising strategy to help improve the ability to predict subsequent clinical outcomes.

Prodding the Beast: Assessing the Impact of Treatment-Induced Metastasis

The arsenal of treatments for most cancers fit broadly into the categories of surgery, chemotherapy, radiation, and targeted therapy. All represent proven and successful strategies, yet each can trigger local (tumor) and systemic (host) processes that elicit unwanted, often opposing, influences on cancer growth. Under certain conditions, nearly all cancer treatments can facilitate metastatic spread, often in parallel (and sometimes in clear contrast) with tumor reducing benefits. The paradox of treatment-induced metastasis (TIM) is not new. Supporting preclinical studies span decades, but are often overlooked. With recent evidence of prometastatic effects following treatment with targeted agents blocking the tumor microenvironment, a closer inspection of this literature is warranted. The TIM phenomena may diminish the impact of effective therapies and play a critical role in eventual resistance. Alternatively, it may simply exemplify the gap between animal and human studies, and therefore have little impact for patient disease and treatment. This review will focus on the preclinical model systems used to evaluate TIM and explore the mechanisms that influence overall treatment efficacy. Understanding the role of TIM in established and emerging drug treatment strategies may help provide rationales for future drug combination approaches with antimetastatic agents to improve outcomes and reduce resistance.

Neoadjuvant antiangiogenic therapy reveals contrasts in primary and metastatic tumor efficacy

Thousands of cancer patients are currently in clinical trials evaluating antiangiogenic therapy in the neoadjuvant setting, which is the treatment of localized primary tumors prior to surgical intervention. The rationale is that shrinking a tumor will improve surgical outcomes and minimize growth of occult micrometastatic disease—thus delaying post‐surgical recurrence and improving survival. But approved VEGF pathway inhibitors have not been tested in clinically relevant neoadjuvant models that compare pre‐ and post‐surgical treatment effects. Using mouse models of breast, kidney, and melanoma metastasis, we demonstrate that primary tumor responses to neoadjuvant VEGFR TKI treatment do not consistently correlate with improved post‐surgical survival, with survival worsened in certain settings. Similar negative effects did not extend to protein‐based VEGF pathway inhibitors and could be reversed with altered dose, surgical timing, and treatment duration, or when VEGFR TKIs are combined with metronomic ‘anti‐metastatic’ chemotherapy regimens. These studies represent the first attempt to recapitulate the complex clinical parameters of neoadjuvant therapy in mice and identify a novel tool to compare systemic antiangiogenic treatment effects on localized and disseminated disease.

Sunitinib Stimulates Expression of VEGFC by Tumor Cells and Promotes Lymphangiogenesis in Clear Cell Renal Cell Carcinomas

Sunitinib is an antiangiogenic therapy given as a first-line treatment for renal cell carcinoma (RCC). While treatment improves progression-free survival, most patients relapse. We hypothesized that patient relapse can stem from the development of a lymphatic network driven by the production of the main growth factor for lymphatic endothelial cells, VEGFC. In this study, we found that sunitinib can stimulate vegfc gene transcription and increase VEGFC mRNA half-life. In addition, sunitinib activated p38 MAPK, which resulted in the upregulation/activity of HuR and inactivation of tristetraprolin, two AU-rich element-binding proteins. Sunitinib stimulated a VEGFC-dependent development of lymphatic vessels in experimental tumors. This may explain our findings of increased lymph node invasion and new metastatic sites in 30% of sunitinib-treated patients and increased lymphatic vessels found in 70% of neoadjuvant treated patients. In summary, a therapy dedicated to destroying tumor blood vessels induced the development of lymphatic vessels, which may have contributed to the treatment failure. Cancer Res; 77(5); 1212-26. ©2017 AACR.

Androgens modulate male-derived endothelial cell homeostasis using androgen receptor-dependent and receptor-independent mechanisms

BackgroundSex-related differences in the role of androgen have been reported in cardiovascular diseases and angiogenesis. Moreover, androgen receptor (AR) has been causally involved in the homeostasis of human prostate endothelial cells. However, levels of expression, functionality and biological role of AR in male- and female-derived human endothelial cells (ECs) remain poorly characterized. The objectives of this work were (1) to characterize the functional expression of AR in male- and female-derived human umbilical vein endothelial cell (HUVEC), and (2) to specifically analyze the biological effects of DHT, and the role of AR on these effects, in male-derived HUVECs (mHUVECs).ResultsImmunohistochemical analyses of tissue microarrays from benign human tissues confirmed expression of AR in ECs from several androgen-regulated and non-androgen-regulated human organs. Functional expression of AR was validated in vitro in male- and female-derived HUVECs using quantitative RT-PCR, immunoblotting and AR-mediated transcriptional activity assays. Our results indicated that functional expression of AR in male- and female-derived HUVECs was heterogeneous, but not sex dependent. In parallel, we analyzed in depth the biological effects of DHT, and the role of AR on these effects, on proliferation, survival and tube formation capacity in mHUVECs. Our results indicated that DHT did not affect mHUVEC survival; however, DHT stimulated mHUVEC proliferation and suppressed mHUVEC tube formation capacity. While the effect of DHT on proliferation was mediated through AR, the effect of DHT on tube formation did not depend on the presence of a functional AR, but rather depended on the ability of mHUVECs to further metabolize DHT.Conclusions(1) Heterogeneous expression of AR in male- and female-derived HUVEC could define the presence of functionally different subpopulations of ECs that may be affected differentially by androgens, which could explain, at least in part, the pleiotropic effects of androgen on vascular biology, and (2) DHT, and metabolites of DHT, generally thought to represent progressively more hydrophilic products along the path to elimination, may have differential roles in modulating the biology of human ECs through AR-dependent and AR-independent mechanisms, respectively.

Modeling Spontaneous Metastasis following Surgery: An In Vivo-In Silico Approach

Rapid improvements in the detection and tracking of early-stage tumor progression aim to guide decisions regarding cancer treatments as well as predict metastatic recurrence in patients following surgery. Mathematical models may have the potential to further assist in estimating metastatic risk, particularly when paired with in vivo tumor data that faithfully represent all stages of disease progression. Herein, we describe mathematical analysis that uses data from mouse models of spontaneous metastasis developing after surgical removal of orthotopically implanted primary tumors. Both presurgical (primary tumor) growth and postsurgical (metastatic) growth were quantified using bioluminescence and were then used to generate a mathematical formalism based on general laws of the disease (i.e., dissemination and growth). The model was able to fit and predict pre/postsurgical data at the level of the individual as well as the population. Our approach also enabled retrospective analysis of clinical data describing the probability of metastatic relapse as a function of primary tumor size. In these data-based models, interindividual variability was quantified by a key parameter of intrinsic metastatic potential. Critically, our analysis identified a highly nonlinear relationship between primary tumor size and postsurgical survival, suggesting possible threshold limits for the utility of tumor size as a predictor of metastatic recurrence. These findings represent a novel use of clinically relevant models to assess the impact of surgery on metastatic potential and may guide optimal timing of treatments in neoadjuvant (presurgical) and adjuvant (postsurgical) settings to maximize patient benefit.

Mind the Gap: Potential for Rebounds during Antiangiogenic Treatment Breaks

VEGF pathway inhibitors have shown benefits in many cancers, yet many (often controversial) questions remain about whether vascular and tumor regrowth can occur when therapy is stopped. Marked increases in endothelial cell proliferation could play a role in putative rebounds, potentially influencing overall efficacy, dosing schedules, and presurgical intervention strategies. Clin Cancer Res; 18(14); 3719–21. ©2012 AACR.

The challenges of modeling drug resistance to antiangiogenic therapy.

Drug resistance remains an ongoing challenge for the majority of patients treated with inhibitors of the vascular endothelial growth factor (VEGF) pathway, a key regulator of tumor angiogenesis. Preclinical models have played a significant role in identifying multiple complex mechanisms of antiangiogenic treatment failure. Yet questions remain about the optimal methodology to study resistance that may assist in making clinically relevant choices about alternative or combination treatment strategies. The origins of antiangiogenic treatment failure may stem from the tumor vasculature, the tumor itself, or both together, and preclinical methods that define resistance are diverse and rarely compared. We performed a literature search of the preclinical methodologies used to examine resistance to VEGF pathway inhibitors and identified 109 papers from more than 400 that use treatment failure as the starting point for mechanistic study. We found that definitions of resistance are broad and inconsistent, involve only a small number of reagents, and derive mostly from in vitro and in vivo methodologies that often do not represent clinically relevant disease stages or progression. Together, this literature analysis highlights the challenges of studying inhibitors of the tumor microenvironment in the preclinical setting and the need for improved methodology to assist in qualifying (and quantifying) treatment failure to identify mechanisms that will help predict alternative strategies in patients.

Development of Orthotopic and Spontaneous Metastatic Human Tumor Xenograft Models for Experimental Therapeutics

We summarize a decade of experience in developing pre-clinical models in mice of locally-advanced primary tumor or postsurgical spontaneous metastatic disease. These models were developed for both early- and late-stage cancer to use for experimental therapeutics, employing orthotopic transplantation of established human tumor cell lines. The emphasis has mainly been on breast cancer though other tumor types have been studied as well, including melanoma and renal cell, colorectal, ovarian, and hepatocellular carcinomas (HCC). The in vivo serial selection methodology is described for generating these metastatic models, as well as a number of key findings that have been uncovered using them. These include (1) development of heritable spontaneous brain metastatic models discovered as a result of therapeutically prolonging survival of mice with systemic metastatic disease, (2) recapitulation of prior randomized Phase III clinical trial failures involving patients with advanced metastatic breast cancer treated with anti-angiogenic tyrosine kinase inhibitor (TKI) therapies, (3) discovery that most anti-angiogenic-drug resistant lung metastases are non-angiogenic and instead depend primarily on vessel co-option, (4) obtaining results foreshadowing the phase III clinical trial failures of adjuvant antiangiogenic drug therapy, and (5) evidence for the potent efficacy of metronomic chemotherapy regimens in the advanced metastatic setting, findings which have helped contribute to clinical trial assessment of this therapeutic concept. As a result of these studies, a preliminary attempt was made at establishing models of orthotopic/metastatic patient-derived xenografts (PDXs) showing overt metastases using triple-negative breast cancers. Minimal success was achieved; various factors are discussed which may account for this finding as well as potentially serve to acquire a better understanding of host-mediated control and therapeutic responsiveness of metastatic disease.