Amanda Tracz

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.

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.

Histone 2B-GFP Label-Retaining Prostate Luminal Cells Possess Progenitor Cell Properties and Are Intrinsically Resistant to Castration

Summary The existence of slow-cycling luminal cells in the prostate has been suggested, but their identity and functional properties remain unknown. Using a bigenic mouse model to earmark, isolate, and characterize the quiescent stem-like cells, we identify a label-retaining cell (LRC) population in the luminal cell layer as luminal progenitors. Molecular and biological characterizations show that these luminal LRCs are significantly enriched in the mouse proximal prostate, exhibit relative dormancy, display bipotency in both in vitro and in vivo assays, and express a stem/progenitor gene signature with resemblance to aggressive prostate cancer. Importantly, these LRCs, compared with bulk luminal cells, maintain a lower level of androgen receptor (AR) expression and are less androgen dependent and also castration resistant in vivo. Finally, analysis of phenotypic markers reveals heterogeneity within the luminal progenitor cell pool. Our study establishes luminal LRCs as progenitors that may serve as a cellular origin for castration-resistant prostate cancer.

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.

Linking prostate cancer cell AR heterogeneity to distinct castration and enzalutamide responses.

Expression of androgen receptor (AR) in prostate cancer (PCa) is heterogeneous but the functional significance of AR heterogeneity remains unclear. Screening ~200 castration-resistant PCa (CRPC) cores and whole-mount sections (from 89 patients) reveals 3 AR expression patterns: nuclear (nuc-AR), mixed nuclear/cytoplasmic (nuc/cyto-AR), and low/no expression (AR−/lo). Xenograft modeling demonstrates that AR+ CRPC is enzalutamide-sensitive but AR−/lo CRPC is resistant. Genome editing-derived AR+ and AR-knockout LNCaP cell clones exhibit distinct biological and tumorigenic properties and contrasting responses to enzalutamide. RNA-Seq and biochemical analyses, coupled with experimental combinatorial therapy, identify BCL-2 as a critical therapeutic target and provide proof-of-concept therapeutic regimens for both AR+/hi and AR−/lo CRPC. Our study links AR expression heterogeneity to distinct castration/enzalutamide responses and has important implications in understanding the cellular basis of prostate tumor responses to AR-targeting therapies and in facilitating development of novel therapeutics to target AR−/lo PCa cells/clones.The functional significance of the observed heterogeneity of androgen receptor (AR) expression in prostate cancer is unknown. Here the authors show AR expression heterogeneity is associated with distinct castration/enzalutamide responses and identify BCL-2 as a potential therapeutic target in castration-resistant prostate cancer.

Tumor-Independent Host Secretomes Induced By Angiogenesis and Immune-Checkpoint Inhibitors

The levels of various circulating blood proteins can change in response to cancer therapy. Monitoring therapy-induced secretomes (TIS) may have use as biomarkers for establishing optimal biological effect (such as dosing) or identifying sources of toxicity and drug resistance. Although TIS can derive from tumor cells directly, nontumor “host” treatment responses can also impact systemic secretory programs. For targeted inhibitors of the tumor microenvironment, including antiangiogenic and immune-checkpoint therapies, host TIS could explain unexpected collateral “side effects” of treatment. Here, we describe a comparative transcriptomic and proteomic analysis of host TIS in tissues and plasma from cancer-free mice treated with antibody and receptor tyrosine kinase inhibitors (RTKI) of the VEGF, cMet/ALK, and PD-1 pathways. We found that all cancer therapies elicit TIS independent of tumor growth, with systemic secretory gene change intensity higher in RTKIs compared with antibodies. Our results show that host TIS signatures differ between drug target, drug class, and dose. Notably, protein and gene host TIS signatures were not always predictive for each other, suggesting limitations to transcriptomic-only approaches to clinical biomarker development for circulating proteins. Together, these are the first studies to assess and compare “off-target” host secretory effects of VEGF and PD-1 pathway inhibition that occur independent of tumor stage or tumor response to therapy. Testing treatment impact on normal tissues to establish host-mediated TIS signatures (or “therasomes”) may be important for identifying disease agnostic biomarkers to predict benefits (or limitations) of drug combinatory approaches. Mol Cancer Ther; 17(7); 1602–12. ©2018 AACR.

Abstract 5599: Soluble PD-L1 as a surrogate biomarker of metastatic progression and resistance to antiangiogenic therapy

Immune-checkpoint inhibitors are now approved for the treatment of early- and late-stage cancers. These include agents that block the T-cell regulatory protein programmed cell death 1 (PD-1) from being activated by the PD-1 ligand 1 (PD-L1) expressed on cancer cells. There is an urgent need to identify biomarkers of PD-1 pathway inhibition that would predict patient populations responsive to treatment and/or serve as surrogates for drug activity and resistance. PD-L1 expression on tumors is currently a biomarker candidate, but reliable detection and quantification methodologies have proven challenging to standardize. Recently, a soluble PD-L1 (sPD-L1) fragment was identified that can derive from cell-bound PD-L1. Retrospective clinical examinations of sPD-L1 levels in cancer patients suggest a potential use as a surrogate for disease progression and response to treatment; but few preclinical studies have been performed to test this predictive value. We undertook experiments to evaluate plasma sPD-L1 in mouse tumor models during localized primary tumor growth (after orthotopic cell implantation) and spontaneous metastatic disease progression (after surgical removal of the primary). Mouse syngeneic and human xenograft implantation models included breast, kidney, colon, and melanoma cell systems. Our results show that circulating plasma sPD-L1 can correlate with primary and metastatic progression in a stage and model specific manner. Next, we evaluated sPD-L1 following treatment with neutralizing antibodies to PD-1 and PD-L1 in tumor-free mice and found significant dose-dependent sPD-L1 increases, suggesting systemic changes may have utility as a measurement of target saturation and dosing independent of tumor growth. Finally, with current approvals of PD-1 inhibitors in renal cell carcinoma (RCC) patients previously treated with antiangiogenic agents that block vascular endothelial growth factor (VEGF), we evaluated plasma in mouse models of sunitinib resistance - a VEGF receptor tyrosine kinase inhibitor (RTKI). Our results demonstrate that VEGF pathway resistance yields changes in sPD-L1 and may be useful in predicting response to PD-1 pathway inhibition in the refractory setting. Together, these investigations suggest that circulating sPD-L1 changes during disease progression (both local and disseminated) may serve as a potential predictive biomarker for immune-checkpoint and antiangiogenic therapy. Citation Format: Michalis Mastri, Amanda Tracz, Yuhao Shi, Georg Bjarnason, Tran Nguyen, Brian Rini, John M.L. Ebos. Soluble PD-L1 as a surrogate biomarker of metastatic progression and resistance to antiangiogenic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5599. doi:10.1158/1538-7445.AM2017-5599

Abstract 3251: A Senescence-like phenotype associates with rapid metastasis promotion following antiangiogenic drug resistance and therapy withdrawal

Few studies have investigated mechanisms of antiangiogenic drug resistance in mouse models that faithfully recapitulate clinically relevant spontaneous metastatic disease or evaluate the impact of therapy cessation on tumor and stromal cell growth. Generation of such models may be critical to study reported instances of antiangiogenic therapy-induced metastasis in animals that have often proved challenging to confirm in patients. Here we describe the derivation of several human and mouse tumor cell lines obtained from spontaneous metastatic lesions present after surgical removal of primary tumors and following long-term in vivo treatment with sunitinib or axitinib. Selected drug-resistant metastatic (kidney, breast, and melanoma) and non-malignant stromal cell variants (endothelial and fibroblast) were continuously exposed to drug in vitro and then evaluated following both short- and long-term treatment removal (48 hours and 6 months, respectively). Our results show that metastatic drug-resistant cells receiving sustained treatment were re-sensitized to therapy upon orthotopic re-implantation into treatment-naive animals, suggesting a predominant host-mediated role in therapy failure. However, significant increases in tumor growth and metastatic potential were observed in all models when cells were re-implanted and therapy stopped, suggesting a tumor-dependent pro-metastatic mechanism activated by therapy withdrawal. Whole genome expression analysis for resistant cells on and off treatment revealed reversible and irreversible gene changes implicating a senescence-like phenotype capable of influencing metastatic potential. Senescent-like characteristics included increased cell size, decreased proliferation, cell cycle check-point protein alteration, and therapy-induced SA-β-galactosidase expression, depending on the cell line. Critically, antiangiogenic therapy could induce a senescence associated secretory phenotype (SASP) in both tumor and stroma cells which reversed or persisted following therapy cessation in certain instances. Importantly, we found that interleukin-6 (IL-6) - a major component of the SASP - was upregulated in resistant tumor and stroma cells, but only remained upregulated in stromal cells following therapy withdrawal. These results suggest that antiangiogenic treatment-induced senescence-like changes may contribute to treatment failure and contribute to pro-metastatic growth depending on cell origin (i.e., tumor or stroma) and whether treatment is sustained or stopped. Citation Format: Michalis Mastri, Amanda Tracz, Biao Liu, Christina R. Lee, John ML Ebos. A Senescence-like phenotype associates with rapid metastasis promotion following antiangiogenic drug resistance and therapy withdrawal. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3251.

Abstract 2996: Sunitinib withdrawal uncovers complementary stromal- and tumor-mediated mechanisms of resistance and rebound growth in metastatic mouse models

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Despite the clinical approval of VEGF pathway targeted agents in the treatment of late-stage metastatic disease, sustained benefits are not seen in the majority of patients, and eventual relapse occurs. Understanding mechanisms of intrinsic and acquired drug resistance has taken on increasing importance as both host- and tumor-mediated pathways may contribute to treatment failure. However, few studies have aimed to assess resistance in clinically-relevant spontaneous metastatic disease after surgical resection of the primary tumor. Methods: Here we describe the derivation of spontaneous metastatic human kidney, breast, and melanoma cell lines following long-term in vivo sunitinib treatment. Re-exposure of selected tumor and stromal cells to drug in vitro for extended periods allowed evaluation of reversible and irreversible gene expression changes. Cells were implanted orthotopically and then surgically removed to assess primary and metastatic growth potential following treatment cessation. Results: Our results show that metastatic sunitinib-resistant cells retained treatment sensitivity when re-implanted orthotopically regardless of in vitro drug exposure conditions, suggesting a predominant host-mediated role in treatment failure. However, sunitinib treatment withdrawal elicited significant increases in tumor growth and metastatic potential, a finding which was enhanced when non-tumor bearing mice were ‘conditioned’ with drug prior to i.v. tumor cell inoculation. Parallel studies in vitro show increased proliferation and migration following therapy removal. Results from gene and protein expression analysis of cells in different sunitinib treatment conditions will be presented showing that coordinated tumor and host reactions can contribute to changes in metastatic phenotype upon therapy removal. Conclusions: Taken together, clinically relevant models of drug-resistant spontaneous metastatic disease may have the potential to distinguish tumor- and stromal-responses to treatment that can alter in vivo disease progression both on (and off) therapy. Citation Format: Michalis Mastri, Amanda Tracz, Christina R. Lee, Derya Deveci, John M. L. Ebos. Sunitinib withdrawal uncovers complementary stromal- and tumor-mediated mechanisms of resistance and rebound growth in metastatic mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2996. doi:10.1158/1538-7445.AM2014-2996