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Background: Previous studies have shown that rapid autopsies (RA) provide a unique opportunity for tissue collection from patients who succumb to the disease. Because cancer patients are unable to donate their organs to other people, this program provides the patient an opportunity to leave a legacy by donating their body to research. These donations are vital for advancing breast cancer research. The UPMC/Pitt RA group revamped an existing program in 2018 through the formation of a larger multidisciplinary team that includes breast cancer laboratory and clinical researchers, pathologists, nurses, bioinformaticians, and tissue bankers. Because recruitment to the RA program was a challenge, we recently added patient advocates to the team to provide their essential perspective, and a dedicated research coordinator who serves as an ambassador for the program. Methods: Autopsy is performed by the Autopsy and Forensic Pathology Center of Excellence/Decedent Affairs Service of UPMC. Samples are banked in the Pitt Biospecimen Core (PBC), in addition to immediate processing including preparing of samples for sequencing and growing of organoids in the laboratory. Immunohistochemical (IHC) analysis is performed by UPMC/Magee Pathology. Results: The research coordinator quickly became an integral part of the program and closely interacts with care providers, patients and their families, pathologists on call, and manages interactions with transport services. Five breast cancer advocates have been instrumental in advising on additional changes to the program. The advocates attend regular team meetings and have formulated patient considerations for the the RA program, including appropriate and sensitive recruitment of patients, the role of physicians in decision making by the patient, registration for more than one RA program, potential issues with transporting a body across state lines and more. The advocates also developed the name for the program - “Hope for OTHERS” with Others standing for “Our Tissue Helping Enhance Research & Science”. As of June 2022, the team has completed 26 autopsies, and an additional 20 patients have consented to the program. The completed autopsies include patients with breast tumors representing different molecular and histological classes, ethnicities, and genders. The average disease-free survival and overall survival of patients that underwent autopsy was 81.6 and 127.8 months, respectively. Most patients passed outside the hospital (86%), with 62% in home hospice and 24% in inpatient hospice. Average time between death and start and end of autopsy was 4.56 hrs and 7.09 hours, respectively. The most common metastatic sites from which specimens are collected are liver, lung and lymph nodes. Per patient we collect on average specimens from 4 different organs. In addition to the metastatic lesions, we have access to primary tumor tissue and liquid biopsies obtained during the breast cancer disease progression for 44% and 73% of the patients, respectively. For a subset of the patients, tissue has been grown as patient-derived organoids or xenograft models. Preliminary IHC and sequencing analysis has provided insight into inter- and intra-patient and intra-tumor heterogeneity. Further molecular studies are ongoing. Conclusion In summary, over the last 5+ years, we have established a successful post-mortem tissue collection program, by addressing a series of barriers through the formation and work of a multi-disciplinary well-coordinated team. We are currently expanding our omics studies using state-of-the-art technologies to improve understanding how intra- and inter-tumor heterogeneity play a role in the clinical course of advanced breast cancer, to increase diversity of the patients enrolled in the RA program, and to support the successful implementation of other RA programs nationwide and worldwide. Citation Format: Steffi Oesterreich, Lori Miller, Margaret Q. Rosenzweig, Tanner L. Bartholow, Megan Yates, Ashuvinee Elangovan, Laura Savariau, Allison N. Casey, Nolan Priedigkeit, Kai Ding, Abdalla Wedn, Jie Bin Liu, Daniel D. Brown, Tara Hyder, Geoffrey Pecar, Neil Carleton, Humberto Trejo Bittar, Daniel Geisler, Oscar Lopez-Nunez, Amanda M. Clark, Alan Wells, Partha Roy, Shannon Puhalla, Naomi Howard, Christine Needles, Susan Trent, Stephanie Walker, Christine Hodgdon, Rohit Bhargava, Jennifer M. Atkinson, Adrian V. Lee. Hope for OTHERS – An organ donation program for metastatic breast cancer research [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-02.

Background: Micrometastases from breast cancer (BC) might undergo quiescence following adjuvant therapies. Oftentimes they reactivate, prompting metastatic relapse and aggressive, uncurable disease. It is necessary to find alternative, safe therapies that could be used as adjuvants for delaying the dormancy process and reduce metastatic breast cancer (MBC) progression. Prior epidemiological studies found prolonged overall survival from chronic statin use. However, the effectiveness of statin use through different stages of progression, including advanced MBC, has not yet been defined. Methods: A real-world, retrospective analysis of 1,016 deceased patients with MBC treated from 1999-2019 at the University of Pittsburgh Medical Center (UPMC). Lipophilic statin (atorvastatin and simvastatin) use and dosing were retrieved from medical records through an IRB-approved R3 request and merged with already available clinical variables. A total of 122 patients were included in the analysis as the statin use group. This included patients who were prescribed statins through the course of their breast cancer. For measuring time to relapse, only patients with statin prescription before being diagnosed with MBC were selected. Log-ranks were used to compute survival. Uni- and multi-variate Cox hazard models were also applied. Statistical analysis was computed using the package ggpubr on R v3.6.3.Results: Women who used statins were older at the time of detection of primary (60 vs 52 years of age, respectively) and MBC (66 vs 56 years of age, respectively). An almost 2-fold reduction was found in the onset of liver metastasis as first relapse in the statin group, compared to general population (5.7% vs 10.4%, respectively). Increased overall survival (OS) was found among statin users who had ER (+) (p Citation Format: Juan Luis Gomez Marti, Alan Wells. Lipophilic statins for prolonging dormancy and overall survival: A real-world retrospective investigation [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-22.

Even the most therapy responsive melanomas eventually evade immune checkpoint inhibition. Interestingly, initial responses seem to benefit from localized inflammation, but this can become prohibitive when treating advanced disease. Subsequently, there is an urgent need to elucidate how inflammation impacts the tumor microenvironment (TME). Here, inhibition of EGFR and Ras/Raf/MEK is shown to drive the release of a pro-inflammatory secretome that activates the TME to promote progression. This secretome was enriched with immune recruiting cytokines and members of the transforming growth factor beta (TGFβ) superfamily. Both healthy and cancer associated fibroblasts (CAFs) were hyperactivated in response to the inflamed secretome with induction of the inflammatory marker, alpha smooth muscle actin (αSMA). Pharmacologic inhibition of TGFβ receptor type I (TGFβRI) using SB431542 or clinically relevant TEW-7197, diminished CAF αSMA phenotype, disrupted TGFβ and phospo-SMAD2/3 signaling, and blunted CAF expression of tumor promoting extracellular matrix and adhesion molecules. Invasion of melanoma spheroids was enhanced ~2.5-fold in CAF coculture transwells. However, treatment with MEK inhibitor AZD6244 and TEW-7197 only limited invasion of mixed melanoma and CAF multi-cell spheroids in vitro. Prompted by this, the extracellular matrix of patient-derived and melanoma xenograft tumors was analyzed for inflammatory protein expression. Micrometastatic foci were flanked by peri-tumor αSMA+ CAFs and enriched for inflammatory Tenascin-C (TNC) at the invasive front. The analysis of human melanoma tissue microarrays corroborated the experimentally observed expression of αSMA CAFs and TNC which statistically increase with malignant tumor grade. Conversely, the anti-invasive Decorin (DCN), decreased in malignant melanoma while collagen-1 increased during late stage tumor fibrosis. These findings prompted the use of ex vivo human skin organ cultures (SOCs) to define how cell matrix impacted melanoma. Addition of DCN to the SOC eliminated melanoma vertical invasion whereas pathological TNC promoted progression. Thus, a BRAFV600E resistant patient-derived metastatic cell line was used to establish intrasplenic xenografts that were treated after one week using AZD6244 and TEW-7197. Both monotherapy and combination therapy were well tolerated and resulted in reduced splenic tumor burden, αSMA staining, and halted metastasis to the liver. Citation Format: Andrew M. Bradshaw, Erica Kuo, Jelena Grahovac, Kyle Sylakowski, Cindy Sander, Howard Edington, John M. Kirkwood, Alan Wells. The therapeutically inflamed tumor microenvironment drives melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3169.

How microenvironmental inflammation impacts melanoma progression remains controversial. Inflammation appears beneficial during interferon stimulated expression of checkpoint targets. However, when tipped too far, therapy-driven inflammation has been linked to debilitating autoimmune disease in cancer patients. One unanswered question is whether this inflammation also promotes melanoma dissemination. Here, drug-stressed melanomas are shown to promote progression via a pro-inflammatory secretome that activates the tumor microenvironment. Analyses showed the drug specific activation of immune recruiting cytokines and the transforming growth factor beta (TGFβ) superfamily. In coculture, TGFβ from drug-stressed melanomas resulted in the paracrine hyperactivation of cancer associated fibroblasts (CAFs) as noted by the inflammatory marker alpha smooth muscle actin (αSMA). Pharmacologic inhibition of TGFβ using SB431542 or clinically relevant EW-7197, blunted the αSMA phenotype as well as downstream phospho-SMAD activation. Invasion of melanoma spheroids in CAF cocultures resulted in enhanced invasion which could suppressed by targeted MEK inhibition (AZD6244) and TGFβ receptor blockade. Engrafted melanomas in vivo presented with the rapid expansion of peri-tumoral αSMA+ CAFs surrounding invasive micrometastatic nodules in mouse livers. We have previously reported that extracellular matrix (ECM) protein tenascin-C (TNC) is enriched at the invasive fronts of melanoma, which was recapitulated in vivo and correlated to human melanomas. Human tissue microarrays were assayed for pro-inflammatory TNC, healthy ECM marker Decorin (DCN), αSMA CAFs, and fibrotic Collagen (COL1A1) expression. We detected statistically significant increases in TNC, αSMA and COL1A1 correlating to progression and a concomitant decrease in DCN. Evidence from drug treated spheroid cocultures did not show complete suppression single cell invasion. Prompted by this, ex vivo organotypic skin organ cultures containing TNC and DCN were developed to show that the ECM provides physiological pro-and-anti invasive signals. Expression of DCN therapeutically inhibited collective and single cell invasion in this model. We also observed transcriptomic suppression of TNC and key inflamed matrisome proteins in anti-TGFβ treated CAFs, even when challenged with TGFβ conditioned media. Informed by this, we used a TNC+ vemurafenib resistant patient-derived metastatic melanoma in our xenograft model. Cohorts treated with MEK inhibitor and combination therapy with EW-7197 showed drug efficacy and tolerance with detectable decreases in αSMA and tumor burden of the spleen. Treated animals also showed reduced liver metastases compared to control. Taken together, combination therapy that blocks off-target inflammation and cancer proliferation represent an underappreciated but potentially successful strategy to halt melanoma progression. Citation Format: Andrew Bradshaw, Erica Kuo, Jelena Grahovac, Cindy Sander, Howard Edington, John M. Kirkwood, Alan Wells. The therapeutically inflamed tumor microenvironment drives melanoma progression [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT002.

Breast cancer (BrCa) mortality continues to result predominately from distant metastases that can emerge years after successful treatment of the primary disease. Metastatic resistance to agents that eradicate the primary mass is likely due to protection from the metastatic microenvironment and the quiescent state of dormant BrCa cells. Advancements for the treatment of metastatic tumors have been made, but significant progress has been hampered by the lack of relevant model systems, particularly for dormancy. We address this gap with an innovative all-human 3D liver microphysiological system (MPS). The liver is both a major site for BrCa metastasis (and other solid tumors) and the primary site of drug metabolism and limiting toxicities, an important consideration in evaluating cancer therapy efficacy and availability. Primary hepatocytes and non-parenchymal cells (NPC) from human liver resections were seeded into the MPS. Following tissue formation on day 3, tagged BrCa cells were seeded and allowed a minimum of 4 days to integrate into the tissue before interventions were initiated. On day 7, chemotherapy treatment of micrometastases was initiated for 72h. Cultures were allowed 3 days to recover before the MPS was challenged with inflammatory factors (LPS/EGF) for 48h. BrCa cells were then re-treated with chemotherapy (either the same or alternate therapy) on day 21 for 72h. Hepatocyte function and injury were measured by urea, AST, ALT, A1AT, fibrinogen and CYP P450 assays. BC proliferation was monitored by quantification, Ki67 staining, and EdU incorporation. Communication networks within the metastatic microenvironment during different stages of metastatic BrCa progression were identified using Luminex assays (55 analytes). The metastatically aggressive MDA-MB-231 BrCa cells demonstrated growth attenuation after 12d of culture in a subpopulation of cells (Ki67-/EdU-). Treatment of BrCa cells with doxorubicin for 72h eradicates the cycling cells, leaving behind a dormant cell population (Ki67-/EdU-) that can be subsequently stimulated to cycle by addition of inflammatory stimuli. A second dose of doxorubicin or cisplatin reduced the BrCa load but did not eradicate the BrCa. Luminex analysis of culture supernatants identified signaling molecules potentially involved in metastatic progression. In addition, we present the use of adjuvant therapy in the MPS to prevent this outgrowth of the dormant tumor cells. In parallel, we have piloted hydrogel scaffolds that better support tissue formation and produce signals consistent with a healthier liver physiology. Hydrogels enhanced MDA-MB-231 cell entry into dormancy, resulting in reduced efficacy of doxorubicin treatment with greater persistence of tumor load. The MPS provides a mechanism to close the gap in understanding metastatic dormancy. We demonstrate spontaneous dormancy for the first time in an all-human system and mimicked the dormancy and outgrowth observed in patients. Namely, that dormant BrCa are resistant to chemotherapy and can be stimulated to reemerge following an inflammatory insult. The completion of these studies will provide insights into the tumor biology of metastatic seeding, dormancy, and re-emergence and provide an accessible tool for testing therapeutics against metastatic BrCa in a metabolically competent system capable of evaluating dose-limiting toxicity. Citation Format: Beckwitt C, Wheeler SE, Clark AM, Pillai VC, Young CL, Stolz DB, Lauffenburger DA, Venkataramanan R, Griffith LG, Wells A. Breast cancer dormancy, re-emergence, and treatment. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-19.

Most breast cancer (BrCa) mortality results from distant metastases. Current evidence strongly suggests that in some instances these disseminated cells remain dormant for long periods of time. Both the non-proliferative state and protective microenvironment of the metastatic niche likely contribute to the observed resistance of metastases to chemotherapies that are otherwise effective against the primary tumor. Although significant interventional progress has been made on primary tumors, the lack of relevant accessible model systems for metastases has hindered the development of therapies against this stage. To address this gap, we developed an innovative all-human 3D ex vivo hepatic microphysiological system (MPS) to faithfully reproduce human physiology and thereby facilitate the investigation of BrCa behavior in a micrometastatic niche. The liver is a major site of metastasis for carcinomas and is also the primary site of drug metabolism (activation and/or detoxification), which is a significant factor in determining efficacy and limiting toxicities of cancer therapies. The MPS incorporates hepatocytes and nonparenchymal cells (NPC) isolated from fresh human liver resections. BrCa cells (RFP+) are seeded on day 3 and afforded time to intercalate into the hepatic tissue until treatment with chemotherapy on day 7 for 72h. Surviving BrCa cells are stimulated on day 13 with LPS/EGF and cultured through day 15. Proliferation is monitored by RFP quantification, Ki67 staining and EdU incorporation. Physiological function of the hepatic tissue is monitored throughout the experiment by protein catabolism (urea), active metabolism (glucose, CYP P450) and injury markers (AST, ALT, A1AT, fibrinogen). Luminex assays (55 analytes) were used to provide insights into the communication networks in the hepatic metastatic milieu during different stages of dormancy and progression, and identify potential metastatic biomarkers via computational approaches. The MPS maintains the physiologic function of the hepatic niche through 15 days and BrCa cells effectively integrate into the established niche. Spontaneous dormancy is observed amongst a subpopulation of BrCa cells, indicated by the absence of Ki67 staining and EdU incorporation after 12 days of culture. Further, we demonstrate that the BrCa cells surviving chemotherapy (doxorubicin) are non-proliferating (Ki67-/EdU-). Notably, ‘re-awakening’ of the surviving non-proliferating cancer cells is observed in the presence of physiological inflammatory stressors (LPS/EGF). Luminex analyses of the milieu effluent identified signaling molecules from NPC influenced the metastatic cell fraction entering dormancy. This MPS provides unprecedented insights into the tumor biology of dormant micrometastases. We demonstrate the recreation of spontaneous, rather than engineered, BrCa dormancy in an all-human ex vivo hepatic MPS. Mimicking the dormancy and outgrowth observed in patients, we found that dormant breast cancer cells that are resistant to chemotherapy can be stimulated to re-emerge following an inflammatory insult. Ultimately, this MPS provides an accessible tool to identify new therapeutic strategies for metastasis during initial seeding, dormancy and re-emergence, while concurrently evaluating agent efficacy for metastasis, metabolism and dose-limiting toxicity. Citation Format: Amanda M Clark, Sarah E Wheeler, Donald P Taylor, Carissa L Young, Venkateswaran C Pillai, Donna B Stolz, Raman Venkataramanan, Douglas A Lauffenburger, Linda G Griffith, Alan Wells. Modeling breast cancer dormancy and re-emergence [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-01.

Treatment of triple negative breast cancer (TNBC) is particularly difficult due to lack of molecular targets. The vast majority of breast cancer patient mortality is a result of emergence of disseminated cancer cells from a so-called dormancy-like state allowing subsequent aggressive growth at the metastatic sites. Actin assembly and important regulators of actin assembly play a key role in promoting the dormancy to proliferation switch of extravasated breast cancer cells. MRTF (myocardin-related transcription factor - an important family of transcription cofactors for SRF)/SRF transcriptional axis plays a major role in transcriptional regulation of many major molecular components of actin cytoskeletal system. We hypothesize that MRTF plays a critical role in post-extravasation survival and outgrowth of TNBC cells. To test this hypothesis, we first performed matrigel-on-top assay (3D culture experiments which successfully predicts the post-extravasation pulmonary metastatic outgrowth competency of breast cancer cells) and found that loss-of-function (LOF) of MRTF, either by knockdown or small molecule inhibitor CCG-1423 (and its analog CCG-203971), significantly retards the outgrowth of isolated TNBC cells. In this model, LOF of MRTF also dramatically suppresses the progression of established outgrowth (an in vitro mimic of micro-to-macrometastasis progression) of TNBC cells. Translating these findings in vivo, in intracardiac-injection model of experimental metastasis, administration of MRTF inhibitor reduces the metastatic burden of TNBC cells in immunocompetent mice. These experimental data are consistent with clinical findings demonstrating significantly shorter progression-free survival with MRTF upregulation in breast cancer patients. To obtain further insight into the mechanism behind MRTF’s regulation of metastatic outgrowth, we performed RNAseq from TNBC grown in 3D culture treated with CCG and identified a number of differentially expressed genes related to cell proliferation/survival and invasion. In addition to these findings, we also found that LOF of MRTF causes a dramatic reduction of intracellular content of actin-binding protein Profilin-1 (Pfn1), an important regulator of actin cytoskeletal dynamics. Using a tetracycline-inducible Pfn1 knockdown system, we further demonstrated that acute Pfn1 depletion alone is sufficient to cause a major reduction of metastatic colonization of TNBC cells in vivo. Collectively, these findings suggest that MRTF-Pfn1 is an important signaling axis for metastatic outgrowth of TNBC cells. Citation Format: David M. Gau, Souvik Chakraborty, David Boone, Alan Wells, Partha Roy. MRTF|Profilin is an important signaling axis for metastatic outgrowth of triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-043.

Metastasis in breast cancer patients heralds mortality, as disseminated disease is generally chemoresistant. After tumor cells reach the ectopic tissue, they undergo an epithelial reversion to enter a period of quiescence, termed dormancy, which may last for decades before outgrowing again as mesenchymal/dedifferentiated masses. Thus, long-term, relatively non-toxic interventions that prevent metastatic outgrowth are needed to treat this mortal stage of tumor progression. Epidemiological analyses have suggested that statin usage, for cardiovascular indications, is correlated with a reduction in clinically-evident metastatic (though not in incidence of primary) breast cancer. The goal of this study is to demonstrate this is due to statins suppressing breast cancer cell proliferation and keeping the micrometastases in the dormant state. We have found that atorvastatin and simvastatin limit the growth of some cancer cell lines, but not others. The sensitive lines were marked by lacking surface E-cadherin, the hallmark of the mesenchymal phenotype. When E-cadherin is downregulated on epithelial tumor cells, the cells become growth inhibited by the statins. Furthermore, this is a direct effect, as we now have shown that hydrophilic statins are relatively ineffective compared to the membrane permeant lipophilic statins as tumor cells generally lack the transporters that enable these drugs to gain access to the cells. To determine whether the statins target the emergent metastatic tumor cells, we are using an all human microphysiological system (MPS) of the most common site for metastases, the liver. Briefly, a micro-hepatic tissue is established by seeding primary human liver cells in a porous scaffold subject to a physiological flow. RFP-labeled breast cancer cells are seeded into these microtissues and examined weeks later. Liver function and health are monitored by clinical chemistry assays performed on supernatant samples. We have previously shown that this system robustly reproduces tumor dormancy. Initial studies suggest that statins suppress the emergence of dormant tumor cells when challenged by stressors that lead to outgrowth. Additionally, atorvastatin suppresses proliferation of mesenchymal but not epithelial breast cancer cells in intrasplenic and mammary fat pad injection models for breast cancer metastasis to the liver and lung respectively. As 26% of adults currently take a statin for other medical conditions, these studies may suggest the best statin to use in the context of maintaining breast cancer dormancy long-term and delaying or avoiding the morbid emergence. Citation Format: Beckwitt CH, Clark AM, Warita K, Oltvai ZN, Wells A. Adjuvant statin therapy efficacy is dictated by tumor dormancy and statin lipophilicity in ex vivo and in vivo models of metastatic breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-20.

Background: Distant metastasis is the major cause of breast cancer (BrCa)-related mortality with half of the disseminated disease emerging clinically after 5 or more years of seeming “cure” of the primary tumor. The lack of relevant accessible model systems for metastasis has hindered our understanding of the molecular basis of metastasis and the development of therapies against these lethal outgrowths. Furthermore, standard culture techniques employ rigid surfaces that are associated with driving even non-transformed cells towards aggressive phenotypes. To address this gap, we previously developed an innovative all-human 3D ex vivo hepatic microphysiological system (MPS) that faithfully reproduces human liver physiology. This MPS also enables spontaneous dormancy in subpopulations of, but not all, BrCa cells. In this study, we query this subpopulation effect by examining matrix stiffness on the occurrence of spontaneous tumor dormancy; we compared a conventional polystyrene material (~3 GPa) to a PEG-based hydrogel biomaterial (~100 kPa) that more closely approaches the malleable physiologic consistency of the liver (~600 Pa). We hypothesized that providing a more physiological environment would promote BrCa dormancy and provide increased protection from chemotherapy as observed in patients, enabling the study of this elusive but critical stage in cancer progression. Methods: A hepatic all-human MPS was retrofitted with hydrogel scaffolding in place of the standard polystyrene. Hydrogel scaffolds were formed by micromolding with polydimethylsiloxane (PDMS) and UV-initiated free radical polymerization. Prior to polymerization, a SynKRGD peptide sequence was conjugated to PEGDa via Michael-type addition to aid in hepatocyte interaction with the hydrogel. The MPS incorporated de-identified excess primary human hepatocytes and non-parenchymal cells (NPCs) isolated from therapeutic patient liver resections. MDA-MB-231 BrCa cells (RFP labeled) were seeded on day 3 and allowed time to intercalate into the hepatic tissue prior to treatment with chemotherapies on day 7 for 72 h. Proliferation was determined by RFP quantification, Ki67 staining and EdU incorporation. Hepatic tissue function was monitored by protein catabolism (urea), metabolism (glucose, CYP P450) and injury markers (AST, ALT). Multiplex bead-based immunoassays of 77 analytes were used to assess physiologic tissue health, cancer markers and inflammatory signals as well as to discern signaling networks within the metastatic niche. Results: The use of hydrogels in this system further fostered spontaneous BrCa quiescent dormancy, as indicated by a lower presence of BrCa cells and decreased proliferation. Hepatic tissue in hydrogel scaffolds secreted lower levels of pro-inflammatory analytes and NPCs were more responsive to inflammatory stimuli. As a result, high dose chemotherapy that targets proliferating cells was less effective against BrCa cells within tissues supported by hydrogel scaffolds. Interestingly, proliferation (emergence) of BrCa cells was observed following treatment with the lower doses of chemotherapy. The lower doses may be damaging the hepatic tissue, causing cellular stress and activation of the more responsive NPCs, which in turn secrete signals that promote the outgrowth and emergence of BrCa cells. Conclusion: These data suggest that using hydrogel scaffolds to more closely align the physiologic and mechanical properties of the macro-environment imparts a healthier hepatic microenvironment. This allowed for interesting insights into the effect of different chemotherapeutic doses on dormant BrCa cells, and highlights this all-human hepatic model of metastasis fitted with hydrogel scaffolds as a critical tool in the assessment and development of therapeutic strategies to target dormant metastatic BrCa. Citation Format: Amanda M. Clark, Sarah E. Wheeler, Carissa L. Young, Jaclyn Shepard Neiman, Venkateswaran Pillai, Linda Stockdale, Donna Stolz, Douglas A. Lauffenburger, Raman Venkataramanan, Linda G. Griffith, Alan Wells. Mechanical properties of matrices strongly influence spontaneous tumor dormancy and responses to chemotherapy in a 3D model of metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A01.

Breast cancer is the second most commonly diagnosed cancer among American women. The American Cancer Society has estimated over 230,000 new cases of breast cancer, and over 40,000 deaths for the year 2015. Unfortunately, metastasis is the major contributor for the significant number of these deaths. The Epithelial-Mesenchymal Transition (EMT) is promoted by the Master Regulator known as Kaiso, a transcriptional repressor that is a member of the BTB/POZ family of zinc finger transcription factors. We have previously demonstrated that Epidermal Growth Factor Receptor (EGFR) signaling shuttles Kaiso into the nucleus, although, its mechanism is still not understood. EGFR signaling activates multiple downstream signaling cascades including the MAP Kinase cascade, which has a significant influence on proliferation, cell adhesion, gene expression, and cell survival. Mesenchymal cells demonstrate high levels of MAPK activity and disruption or inhibition of the cascade has proven to be a method for decreasing tumor growth. Therefore we hypothesize that subcellular localization and expression of Kaiso is modulated by EGFR induced MAPK activity. Using the TCGA Breast cancer dataset, we observed that Kaiso was altered in about 11% of patients, compared to 15% of E-cad expression and 13% of MAPK3 of 959 patients/cases of Breast cancers. MAPK3 (eRK1); MAPK6, MAPK7, MAPK9 AND MAPK14 (p38) seems to have trends in mutual exclusivity or co-occurrence with Kaiso (ZBTB33). To determine if MAPK3 is responsible for increased Kaiso expression, MCF-7 cells, which express low levels of Kaiso in the cytoplasm, were treated with 10ng/ml of EGF in the presence or absence of MAPK inhibitor, 20μM PD98093. EGF stimulation caused robust increase in Kaiso protein expression, while PD98093 inhibited EGF induced expression. Similarly, in Kaiso overexpressing, MDA-MB-231, blocking EGFR kinase activity with 500nM PD153035 resulted in an opposite effect as EGF stimulation in MCF-7 cells. Furthermore, we observed a significant decrease in p-ERK expression, re-expression of E-cadherin, and low EGFR expression levels in sh-Kaiso MDA-MB-231 cells compared to sh-Scr cells. The decreased p-ERK expression in sh-Kaiso MDA-MB-231 cells was also associated with decreased expression of mesenchymal markers ZEB1, ZEB2, Twist and SNAIL at both the mRNA and protein levels compared to the scrambled control. Similarly, we observed significant decrease in the somatic cell reprogramming factors Klf4, Oct4, Nanog expression in sh-Kaiso MDA-MB-231 as well, which coincide with a reversal of EMT. To our knowledge, this is the first study to investigate the molecular mechanism and/or functional consequences of increased Kaiso expression in breast cancer, and highlights the multiple roles that Kaiso plays in promoting aggressive disease. Citation Format: Shweta Tripathi, Balasubramanyam Karanam, Bo Ma, Alan Wells, Clayton Yates. Targeting subcellular localization of Kaiso to limit dissemination through MAPK signaling. [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 1680.

This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR11) of the Conference Proceedings. Citation Format: Amanda M. Clark, Sarah E. Wheeler, Carissa L. Young, Venkateswaran C. Pillai, Donna B. Stolz, Douglas A. Lauffenburger, Raman Venkataramanan, Linda G. Griffith, Alan Wells. Elucidating candidate biomarkers of breast cancer progression and dormancy using a 3D model of metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A41.

Purpose: Breast cancer is the most common cancer in women. The lung is one of the most common sites of breast cancer metastasis. While metastatic seeding is often accompanied by a dormancy-promoting mesenchymal to epithelial reverting transitions (MErT), we aimed to determine whether lung epithelial cells can impart this phenotype on aggressive breast cancer cells. Methods: Co-culture experiments of normal lung epithelial cell lines (SAEC, NHBE or BEAS-2B) and breast cancer cell lines (MCF-7 or MDA-MB-231) were conducted. Flow cytometry analysis, immunofluorescence staining for E-cadherin or Ki-67 and senescence associated beta-galactosidase assays assessed breast cancer cell outgrowth and phenotype. Results: Co-culture of the breast cancer cells had different effects on breast cancer cells. The epithelial MCF-7 cells were increased in number but still clustered even if in a slight more mesenchymal spindle morphology. On the other hand, the mesenchymal MDA-MB-231 cells survived but did not progressively grow out in co-culture. These aggressive carcinoma cells underwent an epithelial shift as indicated by cuboidal morphology and increased E-cadherin. Disruption of E-cadherin express in MDA-MB-231 using shRNA prevented this phenotypic reversion in coculture. In addition, we had noted enlarged MDA-MB-231 cells in the co-cultures with SAEC and NHBE but not BEAS-2B cells as suggestion of senescence. A subset of these cells stained with the senescence associated marker β-galactosidase (SA-β-gal). Lung cells limited cancer cell growth kinetics as noted by both (1) some of the cells becoming larger and positive for senescence markers/negative for proliferation marker Ki-67, and (2) Ki-67 positive cells significantly decreasing in MDA-MB-231 and MCF-7 cells after co-culture. Conclusions: Our data indicate that normal lung epithelial cells can drive an epithelial phenotype and suppress the growth kinetics of breast cancer cells by changing their phenotypes.Similar to the hepatocytes, these normal cells may promote the survival, seeding and even dormancy of aggressive mesenchymal carcinoma cells, with phenotypes similar to MDA-MB-231 cells, but also provide progressive signaling when stressed accounting for the spindle morphology and slightly increased growth rates of the epithelial carcinoma cells, such as those MCF-7 represents. In this latter manner, insults the small airway may provoke an outgrowth from dormant breast cancer cells. Citation Format: Masashi Furukawa, Alan Wells, Sarah Wheeler, Amanda M. Clark. Lung epithelial cells induce both phenotype alteration and senescence in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1426. doi:10.1158/1538-7445.AM2015-1426

Lymph node metastasis and local invasion of head and neck squamous cell carcinoma (HNSCC) is associated with a poor prognosis. However, little is known about the factors governing tumor cell invasion in HNSCC. Phospholipase Cgamma-1 (PLCgamma-1) contributes to tumor cell invasion in experimental systems when activated by the epidermal growth factor receptor (EGFR). We hypothesized that EGFR overexpression in HNSCC mediates invasion via PLCgamma-1. On EGFR ligand stimulation, phosphorylation of PLCgamma-1 increased in all of the HNSCC cell lines tested (4 of 4). In the presence of EGFR-specific tyrosine kinase inhibitor (PD153035) or an anti-EGFR antibody (C225), PLCgamma-1 activation was abrogated indicating that PLCgamma-1 was downstream of EGFR. Blocking cellular PLC with an inhibitor (U73122) reduced inositol phosphate turnover in all of the HNSCC cell lines examined, and treatment with the PLC inhibitor or antisense oligonucleotides targeting PLCgamma-1 significantly reduced in vitro invasiveness of HNSCC cell lines through Matrigel. To determine the clinical relevance of these findings, we compared levels of PLCgamma-1 in tumor and paired normal tissue from 33 patients with HNSCC. PLCgamma-1 levels were significantly higher (P0.0001) in the tumors compared with the normal mucosa of HNSCC patients. Levels of activated PLCgamma-1 were analyzed in 20 patients. Tumors expressed higher levels of phosphorylated PLCgamma-1 compared with normal adjacent mucosa (P = 0.05). Thus, PLCgamma-1 may mediate invasion and metastasis downstream of EGFR in HNSCC.

Mortality and morbidity of prostate cancer result from extracapsular invasion and metastasis. This tumor progression depends on active cell motility. Previous studies have shown that calpain-regulated rear detachment enabling forward locomotion is required for cell migration initiated by growth factor and adhesion receptors. Therefore, we asked whether calpain would be a target for limiting tumor progression, using as our model the PA DU-145 human prostate carcinoma cell line and a highly invasive subline, wild-type DU-145, derived from it. In vitro, the calpain-specific inhibitor CI-I (ALLN) and the preferential-but-less-specific inhibitor leupeptin decreased transmigration of both cell lines across a Matrigel barrier. These calpain inhibitors limited epidermal growth factor-induced motility but did not alter the growth rate of the tumor cells, as expected. Antisense down-regulation of the growth factor-activated calpain-2 (m-calpain) isoform also reduced transmigration and cell motility. These in vitro findings were then buttressed by in vivo studies, in which i.p. DU-145 tumor xenografts were treated with leupeptin. Tumor invasion into the diaphragm was reduced by leupeptin treatment for both the PA and wild-type DU-145 cells (from 1.7 to 0.78 for the parental line and 2.3 to 1.2 for the invasive derivative, respectively). Tumor cells of both types engineered to express calpain-2 antisense constructs also demonstrated a similar 50% reduced invasiveness in vivo. Finally, we found by gene expression survey of 53 human prostate tumors and 23 normal prostates that calpain was not up-regulated in relationship to invasiveness or metastatic activity, consistent with expectation from the biological role of this effector. Taken together, these results strongly suggest that epigenetic activation of calpain plays an important role in the invasion of human prostate cancer and that it can be targeted to reduce tumor progression.

Most cancer mortality results from distant metastases. The metastatic microenvironment protects ectopic tumors, these nodules are often resistant to agents that eradicate the primary mass. Although significant interventional progress has been made on primary tumors, the lack of relevant accessible model in vitro systems in which to study metastases has plagued metastatic therapeutic development – particularly among micrometastases. One third of women diagnosed with breast cancer (BC) will have metastatic disease which often presents years after a seeming cure from the primary malignancy. An in silico model of micrometastases strongly suggests that these disseminated cells are quiescent, or ‘dormant’, for long periods of time. Current models fail to recapitulate metastatic dormancy, in vivo due to issues of spontaneous metastases and rodent lifespan and in vitro due to the nascent state of organotypic organs or microphysiological systems (MPS). We hypothesize that even the most developed MPS do not allow tumors to attain dormancy due to continued stress signaling from stiff matrices and an artificial microenvironment. We use an innovative all human three dimensional liver MPS to faithfully reproduce human physiology and pathology. In the initial iteration, the liver cells are isolated from therapeutic partial hepatectomies, but as this source may be limiting, we are examining induced pluripotent stem cells (iPSC). Currently these iPSC-derived hepatocyte-like cells demonstrate cyp p450 activity and production of fibrinogen and urea through 15 days in our MPS, albeit at levels below fresh human hepatocytes; optimization protocols are underway. In the first phase of this work we optimized the flow rate and seeding of hepatocytes with non-parenchymal cells (NPCs) from fresh human liver resections. We found that higher flow rates produced poorer tissue formation and increased stress fibers/actin filaments. We maintained functioning hepatocytes in the MPS through 15 days. Hepatocyte function and injury was measured by urea, lactate, AST, ALT, A1AT, fibrinogen and cyp p450 assays. NPCs survived through the 15 day endpoint with immunofluorescent microscopy visualizing leukocytes, endothelial cells and macrophages. The proliferative MDA MB 231 BC cell line showed preliminary evidence of growth attenuation after 12 days of culture in a subpopulation of cells in our MPS. Luminex cancer panel studies are underway with systems biology modeling to describe a communication network in the early microenvironment of micrometastases. In parallel we are piloting hydrogel scaffolds that support tissue formation but provide a more physiologic rheology; stiff supporting materials yield an inflammatory phenotype in the NPC which forces even well-differentiated BC cells towards a mesenchymal phenotype. We found that hydrogels support hepatocytes through 15 days and incorporate cancer cells. Micropumps are also being developed by Draper Laboratories to allow for physiologic diurnal variations of hormones and nutrients to liver tissues to accurately assess dormancy and chemotherapy response. The completion of these studies will provide insights into the tumor biology of dormant micrometastases and an accessible tool for testing of therapeutics against metastatic BC in a metabolically competent system. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-08.

Prostate cancer (PCa) is the leading cause of cancer and the second leading cause of cancer death among men in the US, with tumor metastasis being the main reason behind high rates of mortality and morbidity. How these tumors escape the molecular signals that prevent epithelial cell migration from the prostate is being deciphered. A key motility ‘stop’ signaling network is via chemokines that bind to the common receptor CXCR3. CXCR3 is a G protein coupled receptor whose splicing is differentially regulated in prostate cancer. We have found over-expression of the CXCR3A splice isoform in prostate cancers and cell lines whereas normal prostate epithelium expresses by and large only the CXCR3B isoform, representing a possible switch in the expression between the CXCR3 splice variants. As these variants link differentially to pro- and anti-invasion signaling pathways, this may explain the relentless progression of the prostate carcinoma cells. The switch is present at both the RNA and protein level which suggests that the altering factor is at the transcriptional control level. In a series of PCa lines, the CXCR3 gene was scanned for insertions; deletions or substitutions that introduce a splice silencer; enhancer or a premature stop codon. Surprisingly, the gene was free from mutations except for a polymorphism in the intron in one of the cancer cell lines. A mutation is a permanent change whereas an epigenetic change is a reversible change that confers the cell with the ability to wield control over the expression of the isoforms at a given time. This suggests presence of a possible epigenetic change and explains the absence of mutations. To confirm the aforementioned, the CXCR3 epigenome was analyzed to find the intronic region differentially methylated at four different nucleotides while the upstream putative promoter was not. Strikingly, two of the differentially methylated sites were flanking splice sites while two of were adjoining the NK-2 transcription repressor binding site. A bichromatic CXCR3 reporter minigene has been designed to study the cell type specific expression pattern of the variants and the effect of each differentially methylated CpG nucleotide on splicing. Upon transfection of the wildtype minigene, it was found that the cancer cells express both the isoforms and as expected higher levels of CXCR3A when compared to normal prostate epithelial cells. The switch of isoforms was observed as: one subpopulation of cells that express both the isoforms; another that express solely one of the isoforms: either CXCR3A or CXCR3B. The mutant with guanines at the differentially methylated sites would validate the epigenomic link to splicing. The use of this reporter will be presented to dissect the plasticity of this epigenetic change during dissemination in experimental models of prostate cancer. Citation Format: Devi Sharanya Sampath Kumar, Alan Wells. CXCR3 epigenome switches splice variants in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2973. doi:10.1158/1538-7445.AM2013-2973

Metastasis of prostate cancer (PCa) almost always leads inexorably to death, but little is known about how cancer cells seed ectopic sites and survive in these inhospitable sites. Our lab has previously shown that carcinoma cells undergo a phenotypic change driven by the metastatic microenvironment, to attain an, at least partial mesenchymal (like)-to-epithelial reverting transition (MET/MErT). This phenotypic plasticity, marked by E-cadherin re-expression, plays a key role in metastatic breast and prostate cancer cell survival in vitro and is though to occur after seeding in the ectopic sites and subsequent treatment with chemotherapeutics. In this study we are seeking the pathway(s) involved in the mesenchymal to epithelial transition and subsequent cell survival. By using hepatocytes co-cultured with prostate cancer cells (DU145 or PC3) we found MET PCa cells displayed more resistance to chemotherapeutics, cell death inducing cytokines, or a combination of both. Moreover, E-cadherin knockdown in PCa cells abolished this survival advantage conferred by hepatocytes. This indicated the key role of E-cadherin in hepatocyted driven PCa cells survival. We focused next on the feedback pathways activated by E-cadherin homotypic binding, the MAP kinases. Our data showed that p38 MAPK regulated E-cadherin expression during DU145 cells MET driven by hepatocytes. By using immunofluorescence, the level of phosphor-p38 was much lower in DU145 cells co-cultured with hepatocytes, and the p38 was only detected in the nucleus, which suggested that hepatocytes suppressed p38 MAPK activity in DU145 cells. When co-cultured with hepatocytes, p38α knockdown DU145 displayed much higher levels of E-cadherin, especially in the rim of cells. Additionally, both p38 inhibitor SB202580 and p38α siRNA transfected cells expressed higher E-cadherin than control in DU145. Taken together, hepatocytes regulate PCa cells to undergo MET via suppressing p38 MAPK activity in the cancer cells. Citation Format: Bo Ma, Alan Wells. The mechanism of mesenchymal-to-epithelial transition driven by hepatocytes in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2820. doi:10.1158/1538-7445.AM2013-2820

Nearly half of breast cancer metastases will become clinically evident 5 or more years after the cancer was seemingly ablated. This implies that metastatic cancer cells survived over this extended timeframe without emerging as detectable nodules. Breast cancer metastatic dormancy has yet to be universally defined, and is not well understood as a potential means by which clinically evident metastases emerge after being undetected for years (or decades) from the primary tumor diagnosis. Various mechanistic and microenvironmental factors may work independently, collaboratively (or both) in order to allow a dormant metastatic breast cancer cell (or small cluster) to survive long term. At least one tumor dormancy hallmark is likely required: that of E-cadherin re-expression in order to form heterotypic connections between the carcinoma cells and the liver parenchyma. This re-expression is known as the mesenchymal-to-epithelial-reverting-transition (MErT). This re-expression provides not only survival signals to the breast cancer cells but may also establish cell polarity and protection from autocrine signaling (or extracellular factors) that otherwise may prompt the breast cancer cells to initiate metastatic outgrowth. The purpose of this study was to determine whether non-parenchymal liver cells (NPC) contribute to metastatic competency, initiate mesenchymal to epithelial reverting transition (MErT), and trigger metastatic dormancy. We co-cultured human or rat hepatic isolations with highly metastatic human immortalized breast cancer cells (MDA-MB-231). The hepatocytes, non-parenchymal hepatic supernatant, and liver sinusoidal endothelial cell enriched populations were independently cultured with the breast cancer cells in standard monolayer cultures. Because MDA-MB-231 cells do not survive in the hepatocyte culture media alone, we were able to assess the unique properties imparted by the experimental cultures. Confocal immunofluorescent imaging for proliferation and epithelial markers revealed a mesenchymal phenotypic transition only in the hepatocyte/breast cancer cultures. Interestingly the NPC/breast cancer co-cultures promoted cancer cell proliferation (but lower than with hepatocytes included) but did not impart carcinoma epithelial reversion. The liver sinusoidal endothelial cell (LSEC)-enriched co-cultures exhibited much lower breast cancer cell proliferation hinting toward a possible dormancy, though E-cadherin reversion was not detected. These data suggest that perturbations of the parenchymal and non-parenchymal cell ratios in the liver metastatic microenvironment may contribute to metastatic dormancy initiation or stability. Citation Format: Donald P. Taylor, Alan Wells. Liver nonparenchymal cells drive metastatic breast cancer survival but fail to initiate mesenchymal to epithelial reversion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C93.

In malignant melanoma, the most dangerous skin cancer, Tenascin-C (TNC) is overexpressed, especially at the invasive fronts of invasive melanomas. We found that the migration and invasiveness of a set of melanoma cell lines correlates with levels of TNC expression suggesting, surprisingly, that the melanoma cells themselves provide much of this matrix component. Adding TNC to matrices enhanced individual cell migration in cells that did not express much TNC, whereas it did not enhance the rapid motility of the cells expressing high levels. As TNC is a multidomain protein, the signaling pathways that promote this motogenic invasiveness are not obvious. Herein, we examined the potential role of the EGF-like repeats of TNC that have been shown to preferentially activate motogenic-signaling cascades. To analyze the effects of EGFL signaling on melanoma migration and invasion, we overexpressed TNC fragment containing the assembly and EGFL domains of TNC (TNCEGFL). Interestingly, this led to impaired cell migration in in vitro wound healing assays and live cell tracking experiments. This was likely due to a shift in the adhesion-contractility balance during cell migration. TNCEGFL expressing cells presented a spindle-shaped morphology and formed cords in culture. Expression of TNCEGFL delayed melanoma cell attachment and spreading, as determined in cell adhesion and inverted centrifugation assays. This anti-adhesive phenotype was concomitant with increased MLC2 phosphorylation. Inhibition of ROCK activity, which drives transcellular contractility, restored adhesion of TNCEGFL expressing cells and improved migration in 2D. In distinction to the findings in 2D, TNCEGFL expressing cells had higher invasion potential in Matrigel invasion assays, with cells expressing high TNC having rounded “ameboid” morphology, underlying the importance of morphological shifts, and possibly ROCK signaling, in 3D movement through barrier matrices during invasion and dissemination. These results imply that TNCEGFL repeats are involved in melanoma invasion both by directly influencing cell migration and indirectly changing the tumor microenvironment composition, possibly allowing transition from mesenchymal to ameboid migrational mode. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 485. doi:1538-7445.AM2012-485

Breast cancer dormancy in the metastatic site allows tumors to avoid clinical detection and evade treatment leading to recurrence after years or decades, with devastating mortality rates. How the aggressive carcinoma cells that initially underwent a cancer-associated Epithelial to Mesenchymal Transition (EMT) from the primary tumor to disseminate and maintain a long-term dormant state in an ectopic site is unknown. We have found that normal parenchymal cells of certain metastatic organs cause a partial reversion of the EMT to a more epithelial phenotype (Mesenchymal to Epithelial Reversion Transition, or MErT). Compared to carcinoma cells in the primary breast tumor, MErT is characterized by increased E-cadherin expression, MAPK and AKT signaling, and chemoresistance. While this phenotype should reflect low proliferation or quiescence, the MErT cells in vitro have heretofore maintained a highly proliferative state. Herein we demonstrate that a pre-stressed hepatic microenvironment allows for EMT cells to increase their MErT phenotype and to become dormant. Our research strategy is to modulate the initial liver stress through biochemical challenges applied to primary rat hepatocytes co-cultured with hepatic resident cells and a human immortalized breast cancer cell line (MDA-MB-231). Biochemical challenges (e.g. TGFβ, TNFα, IL-6, EGF, HGF, and DAMPS) are applied in varying concentrations and incubation periods in order to pre-stress the hepatic culture. In order to maintain hepatic metabolic function we implement a novel 3D perfused micro-well bioreactor. As a secondary aim we explore whether E-cadherin expression is required for metastatic seeding through splenetic injections in immune compromised mice. We aim to uncover novel targets that are not strictly against the cancer cells, but rather, against shared molecular pathways across cancer, parenchymal, and non-parenchymal cells within the hepatic niche. Sustaining and perhaps enhancing the ectopic site resilience may yield therapies directed toward maintaining dormant carcinomas so that secondary EMTs will be blocked and cancer recurrence greatly reduced. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5240. doi:1538-7445.AM2012-5240

Prostate cancer is the second leading cause of cancer death among men, with the metastasis of these tumors being the main reason for the high rates of mortality and morbidity. This tumor progression requires epithelial cell dedifferentiation, migration, invasion and growth in an ectopic site. CXCR3 is a G-protein coupled receptor whose splicing was found to be differentially regulated in prostate cancer. We have found over-expression of the CXCR3A splice isoform in prostate cancers and cell lines whereas normal prostate epithelium expresses by and large only the CXCR3B isoform, representing a possible switch in the expression between the CXCR3 splice variants. As these variants link differentially to pro- and anti-invasion signaling pathways, this may explain the relentless progression of the prostate carcinoma cells. The switch is present at both the RNA and the protein level which suggests that the altering factor is at the trancriptional control level. We hypothesized that a mutation in the gene or an epigenetic modification of the genome could possibly be regulating the splicing event and hence the switch in the expression. CXCR3 when scanned for mutations was found to be intact except for a polymorphism in the intronic region which coincided with the normal prostate cells. On the epigenetic regulation side, the CpG dinucleotides in the regulatory regions, the one intron and 5′ upstream region, susceptible to methylation changes were analysed for methylation differences. The upstream sequence does not show a differential methylation pattern whereas the intronic region shows differential methylation between the cancer and normal prostate cells at 3 different nucleotides. A possible explanation is that the methylation in the introns alters the speed of the transcription machinery allowing more time for a particular splice variant to be transcribed more than the other. A bichromatic reporter minigene has been designed for each of the differentially methylated CpG nucleotides, to study its effects on the splicing regulation of the CXCR3 gene. Further studies include expressing the minigene and documenting the splicing differences. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4016. doi:1538-7445.AM2012-4016

Epithelial to mesenchymal transition (EMT) is an oft-studied mechanism for the initiation of metastasis. We have recently shown that once cancer cells disseminate to a secondary organ, a mesenchymal to epithelial reverting transition (MErT) can occur, which we postulate is to enable metastatic colonization. Despite a wealth of in vitro and in vivo studies, evidence of MErT in human specimens is rare and difficult to document because clinically detectable metastases are typically past the micrometastatic stage at which this transition is most likely evident. We obtained specimens of primary and metastatic tumors from breast and prostate cancer patients and evaluated expression of various epithelial and mesenchymal markers by immunohistochemistry. Breast cancer metastases exhibited increased expression of membranous E-cadherin and β-catenin compared to primary tumors, consistent with EMT at the primary site and MErT at the metastatic site. Expression of epithelial markers connexins 26 and 43 was also increased, particularly in brain metastases. Despite the upregulation of epithelial markers in metastases, expression of mesenchymal markers vimentin and FSP1 was mostly unchanged or only slightly decreased. Furthermore, the switch in expression of E-cadherin to N-cadherin that is sometimes detected in EMT, was not found by our immunostaining. N-cadherin detection did not reveal a consistent pattern of expression in primary or metastastic tumors. We observed similar results in prostate cancer metastases; not only was E-cadherin expression increased but also expression inversely correlated with size of the metastasis, suggesting that a second EMT may occur in the ectopic site for tumor growth or to seed further metastases. In summary, we have observed an increased expression of epithelial markers and persistence of mesenchymal markers consistent with a partial MErT that may enable a second EMT at the metastatic site. Our results suggest that cancer cells continue to display phenotypic plasticity beyond the EMT that initiates metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3357. doi:10.1158/1538-7445.AM2011-3357

In melanoma, the most aggressive type of skin cancer, invasion through the extracellular matrix (ECM) is a harbinger of dissemination and mortality. Highly upregulated in both primary invasive and metastatic melanoma is tenascin C (TNC), a hexameric ECM protein with a multidomain structure composed of the globular N-terminal domain, 14.5 epidermal growth factor (EGF)-like repeats, fibronectin-III like repeats and the C-terminal globular fibrinogen-like domain. Our group previously showed that the EGF-like repeats of TNC are able to bind the EGF receptor (EGFR) with μM affinity and preferentially activate the motogenic signaling cascades. Both normal melanocytes and melanoma cells migrate faster on surfaces coated with TNC. Therefore, we hypothesize that the increased levels of TNC in melanomas promote tumor cell migration and invasion heralding dissemination through enhanced EGFR signaling. We expressed EGF-like repeats of TNC (TNC-EGFL) in melanoma cell lines already expressing endogenous TNC and observed change in the cell morphology, and more importantly a prominent anti-adhesive phenotype. TNC-EGFL expressing cells attached with a four-hour lag compared to parental cell lines and detached to a greater extent in inverted centrifugation adhesion assays. This was not due to decreased viability of TNC-EGFL expressing cells as determined by dye exclusion assay. As this phenotype could not be reverted by adding anti-EGFR antibody, we speculate that EGFR signaling might be intracellular. In two-dimensional migration assays, TNC-EGFL expressing cells closed a gap more slowly, possibly due to inability to properly attach during migration, emphasizing the need of concerted action of different TNC domains in regulating migration. On the other hand, quantitative PCR array analysis of a panel of ECM and adhesion molecules revealed changes in matrix metalloproteinase (MMP) expression, specifically MMP-2, MMP-9 and MMP-14, and increase in TGF β1 expression, proteins associated with melanoma invasiveness and vasculogenic mimicry. These results imply that TNC-EGFL repeats are involved in melanoma invasion both by directly influencing cell migration and indirectly by altering the tumor microenvironment and may have implications for development of anti tumor therapies directed at the extracellular matrix. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 421. doi:10.1158/1538-7445.AM2011-421

Numerous studies have shown that loss of E-cadherin through an epithelial to mesenchymal transition (EMT) contributes to tumor invasiveness. However, these studies focus only on the role of E-cadherin in initial dissemination. Interestingly, histopathology of metastases often appear more differentiated than the primary tumor, and several studies have shown that metastases often express E-cadherin despite loss from the primary tumor. We hypothesize that breast cancer cells re-express E-cadherin through a mesenchymal to epithelial transition (MET) to enable colonization within the liver, a common site of breast cancer metastases. We have previously shown that coculture of E-cadherin-negative MDA-MB-231 breast cancer cells with hepatocytes results in re-expression of E-cadherin, which leads to both ligation between cancer cells and hepatocytes as well as activation of the Erk and Akt signaling pathways. To explore the role of E-cadherin in hepatic colonization, we stably transfected full-length E-cadherin and E-cadherin shRNA constructs into MDA-MB-231 (231) cells. Wild-type 231 and single cell clones of 231-Ecad and 231-shEcad cells were seeded onto a hepatocyte monolayer and cultured for 5 days. Cancer cells were RFP-labeled and the number of cancer cells in each coculture was counted daily. Expression of E-cadherin affected the initial attachment of cancer cells to the hepatocyte monolayer, as twice as many 231-Ecad cells were counted 1 day after seeding compared to their E-cadherin-negative counterparts. In addition, wild-type 231 and 231-shEcad cells initially exhibited similar growth curves but began to diverge after 3 days of coculture, with 231 cells expanding twice as fast as 231-shEcad cells. The divergence coincides with a time course of E-cadherin re-expression. After 5 days of coculture, E-cadherin expression was observed by immunofluoresence in 231 and 231-Ecad cells but not in 231-shEcad cells as expected. Immunostaining also revealed that despite E-cadherin re-expression, vimentin expression persisted in 231 cells, suggesting that E-cadherin re-expression may not result in a full MET. Because E-cadherin-based adhesions have been shown to protect against cell death, all three cell lines were treated with staurosporine in the absence of hepatocytes and assayed for cell survival with calcein AM staining. 231-Ecad cells were more chemoresistant compared to 231 cells, and addition of an E-cadherin antibody abrogated this protection. Future studies will evaluate whether E-cadherin re-expression has a chemoprotective role in hepatocyte coculture and in vivo animal models. The role of E-cadherin down-regulation as a key initiator of metastasis has been clearly established. However, our work suggests that phenotypic plasticity of the metastatic cancer cell through E-cadherin re-expression may also be important for colonization and survival at distant sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2336.