Donald P. Taylor

The Dormancy Dilemma: Quiescence versus Balanced Proliferation

Metastatic dissemination with subsequent clinical outgrowth leads to the greatest part of morbidity and mortality from most solid tumors. Even more daunting is that many of these metastatic deposits silently lie undetected, recurring years to decades after primary tumor extirpation by surgery or radiation (termed metastatic dormancy). As primary tumors are frequently curable, a critical focus now turns to preventing the lethal emergence from metastatic dormancy. Current carcinoma treatments include adjuvant therapy intended to kill the cryptic metastatic tumor cells. Because such standard therapies mainly kill cycling cells, this approach carries an implicit assumption that metastatic cells are in the mitogenic cycle. Thus, the pivotal question arises as to whether clinically occult micrometastases survive in a state of balanced proliferation and death, or whether these cells undergo at least long periods of quiescence marked by cell-cycle arrest. The treatment implications are thus obvious--if the carcinoma cells are cycling then therapies should target cycling cells, whereas if cells are quiescent then therapies should either maintain dormancy or be toxic to dormant cells. Because this distinction is paramount to rational therapeutic development and administration, we investigated whether quiescence or balanced proliferation is the most likely etiology underlying metastatic dormancy. We recently published a computer simulation study that determined that balanced proliferation is not the likely driving force and that quiescence most likely participates in metastatic dormancy. As such, a greater emphasis on developing diagnostics and therapeutics for quiescent carcinomas is needed.

Spontaneous dormancy of metastatic breast cancer cells in an all human liver microphysiologic system.

Background:Metastatic outgrowth in breast cancer can occur years after a seeming cure. Existing model systems of dormancy are limited as they do not recapitulate human metastatic dormancy without exogenous manipulations and are unable to query early events of micrometastases.Methods:Here, we describe a human ex vivo hepatic microphysiologic system. The system is established with fresh human hepatocytes and non-parenchymal cells (NPCs) creating a microenvironment into which breast cancer cells (MCF7 and MDA-MB-231) are added.Results:The hepatic tissue maintains function through 15 days as verified by liver-specific protein production and drug metabolism assays. The NPCs form an integral part of the hepatic niche, demonstrated within the system through their participation in differential signalling cascades and cancer cell outcomes. Breast cancer cells intercalate into the hepatic niche without interfering with hepatocyte function. Examination of cancer cells demonstrated that a significant subset enter a quiescent state of dormancy as shown by lack of cell cycling (EdU− or Ki67−). The presence of NPCs altered the cancer cell fraction entering quiescence, and lead to differential cytokine profiles in the microenvironment effluent.Conclusions:These findings establish the liver microphysiologic system as a relevant model for the study of breast cancer metastases and entry into dormancy.

A Microphysiological System Model of Therapy for Liver Micrometastases

Metastasis accounts for almost 90% of cancer-associated mortality. The effectiveness of cancer therapeutics is limited by the protective microenvironment of the metastatic niche and consequently these disseminated tumors remain incurable. Metastatic disease progression continues to be poorly understood due to the lack of appropriate model systems. To address this gap in understanding, we propose an all-human microphysiological system that facilitates the investigation of cancer behavior in the liver metastatic niche. This existing LiverChip is a 3D-system modeling the hepatic niche; it incorporates a full complement of human parenchymal and non-parenchymal cells and effectively recapitulates micrometastases. Moreover, this system allows real-time monitoring of micrometastasis and assessment of human-specific signaling. It is being utilized to further our understanding of the efficacy of chemotherapeutics by examining the activity of established and novel agents on micrometastases under conditions replicating diurnal variations in hormones, nutrients and mild inflammatory states using programmable microdispensers. These inputs affect the cues that govern tumor cell responses. Three critical signaling groups are targeted: the glucose/insulin responses, the stress hormone cortisol and the gut microbiome in relation to inflammatory cues. Currently, the system sustains functioning hepatocytes for a minimum of 15 days; confirmed by monitoring hepatic function (urea, α-1-antitrypsin, fibrinogen, and cytochrome P450) and injury (AST and ALT). Breast cancer cell lines effectively integrate into the hepatic niche without detectable disruption to tissue, and preliminary evidence suggests growth attenuation amongst a subpopulation of breast cancer cells. xMAP technology combined with systems biology modeling are also employed to evaluate cellular crosstalk and illustrate communication networks in the early microenvironment of micrometastases. This model is anticipated to identify new therapeutic strategies for metastasis by elucidating the paracrine effects between the hepatic and metastatic cells, while concurrently evaluating agent efficacy for metastasis, metabolism and tolerability.

All-human microphysical model of metastasis therapy

The vast majority of cancer mortalities result from distant metastases. The metastatic microenvironment provides unique protection to ectopic tumors as the primary tumors often respond to specific agents. 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. A real-time, all-human model of metastatic seeding and cancer cells that recapitulate metastatic growth and can be probed in real time by a variety of measures and challenges would provide a critical window into the pathophysiology of metastasis and pharmacology of metastatic tumor resistance. To achieve this we are advancing our microscale bioreactor that incorporates human hepatocytes, human nonparenchymal liver cells, and human breast cancer cells to mimic the hepatic niche in three dimensions with functional tissue. This bioreactor is instrumented with oxygen sensors, micropumps capable of generating diurnally varying profiles of nutrients and hormones, while enabling real-time sampling. Since the liver is a major metastatic site for a wide variety of carcinomas and other tumors, this bioreactor uniquely allows us to more accurately recreate the human metastatic microenvironment and probe the paracrine effects between the liver parenchyma and metastatic cells. Further, as the liver is the principal site of xenobiotic metabolism, this reactor will help us investigate the chemotherapeutic response within a metabolically challenged liver microenvironment. This model is anticipated to yield markers of metastatic behavior and pharmacologic metabolism that will enable better clinical monitoring, and will guide the design of clinical studies to understand drug efficacy and safety in cancer therapeutics. This highly instrumented bioreactor format, hosting a growing tumor within a microenvironment and monitoring its responses, is readily transferable to other organs, giving this work impact beyond the liver.

Modeling Boundary Conditions for Balanced Proliferation in Metastatic Latency

Purpose: Nearly half of cancer metastases become clinically evident five or more years after primary tumor treatment; thus, metastatic cells survived without emerging for extended periods. This dormancy has been explained by at least two countervailing scenarios: cellular quiescence and balanced proliferation; these entail dichotomous mechanistic etiologies. To examine the boundary parameters for balanced proliferation, we conducted in silico modeling. Experimental Design: To illuminate the balanced proliferation hypothesis, we explored the specific boundary probabilities under which proliferating micrometastases would remain dormant. A two-state Markov chain Monte Carlo model simulated micrometastatic proliferation and death according to stochastic survival probabilities. We varied these probabilities across 100 simulated patients each with 1,000 metastatic deposits and documented whether the micrometastases exceeded one million cells, died out, or remained dormant (survived 1,218 generations). Results: The simulations revealed a narrow survival probability window (49.7–50.8%) that allowed for dormancy across a range of starting cell numbers, and even then for only a small fraction of micrometastases. The majority of micrometastases died out quickly even at survival probabilities that led to rapid emergence of a subset of micrometastases. Within dormant metastases, cell populations depended sensitively on small survival probability increments. Conclusions: Metastatic dormancy as explained solely by balanced proliferation is bounded by very tight survival probabilities. Considering the far larger survival variability thought to attend fluxing microenvironments, it is more probable that these micrometastatic nodules undergo at least periods of quiescence rather than exclusively being controlled by balanced proliferation. Clin Cancer Res; 19(5); 1063–70. ©2013 AACR.

Hepatic nonparenchymal cells drive metastatic breast cancer outgrowth and partial epithelial to mesenchymal transition.

Nearly half of breast carcinoma metastases will become clinically evident five or more years after primary tumor ablation. This implies that metastatic cancer cells survived over an extended timeframe without emerging as detectable nodules. The liver is a common metastatic destination, whose parenchymal hepatocytes have been shown to impart a less invasive, dormant phenotype on metastatic cancer cells. We investigated whether hepatic nonparenchymal cells (NPCs) contributed to metastatic breast cancer cell outgrowth and a mesenchymal phenotypic shift indicative of emergence. Co-culture experiments of primary human hepatocytes, NPCs or endothelial cell lines (TMNK-1 or HMEC-1) and breast cancer cell lines (MCF-7 or MDA-MB-231) were conducted. Exposure of carcinoma cells to NPC-conditioned medium isolated soluble factors contributing to outgrowth. To elucidate outgrowth mechanism, epidermal growth factor receptor (EGFR) inhibition co-culture experiments were performed. Flow cytometry analyses and immunofluorescence staining were conducted to quantify breast cancer cell outgrowth and phenotype, respectively. Outgrowth of the MDA-MB-231 cells within primary NPC co-cultures was substantially greater than in hepatocyte-only or hepatocyte+NPC co-cultures. MCF-7 cells co-cultured with human NPCs as well as with the endothelial NPC subtypes grew out significantly more than controls. MCF-7 cells underwent a mesenchymal shift as indicated by spindle morphology, membrane clearance of E-cadherin, and p38 nuclear translocation when in HMEC-1 co-culture. HMEC-1-conditioned medium induced similar results suggesting that secretory factors are responsible for this transition while blocking EGFR blunted the MCF-7 outgrowth. We conclude that NPCs in the metastatic hepatic niche secrete factors that can induce a partial mesenchymal shift in epithelial breast cancer cells thus initiating outgrowth, and that this is in part mediated by EGFR activation. These data suggest that changes in the parenchymal cell and NPC ratios (or activation status) in the liver metastatic microenvironment may contribute to emergence from metastatic dormancy.

Improved Transplanted Stem Cell Survival in a Polymer Gel Supplemented with Tenascin C Accelerates Healing and Reduces Scarring of Murine Skin Wounds

Mesenchymal stem cells (MSCs) remain of great interest in regenerative medicine because of their ability to home to sites of injury, differentiate into a variety of relevant lineages, and modulate inflammation and angiogenesis through paracrine activity. Many studies have found that despite the promise of MSC therapy, cell survival upon implant is highly limited and greatly reduces the therapeutic utility of MSCs. The matrikine tenascin C, a protein expressed often at the edges of a healing wound, contains unique EGF-like repeats that are able to bind EGFR at low affinities and induce downstream prosurvival signaling without inducing receptor internalization. In this study, we utilized tenascin C in a collagen/GAG-based polymer (TPolymer) that has been shown to be beneficial for skin wound healing, incorporating human MSCs into the polymer prior to application to mouse punch biopsy wound beds. We found that the TPolymer was able to promote MSC survival for 21 days in vivo, leading to associated improvements in wound healing such as dermal maturation and collagen content. This was most marked in a model of hypertrophic scarring, in which the scar formation was limited. This approach also reduced the inflammatory response in the wound bed, limiting CD3e+ cell invasion by approximately 50% in the early wound-healing process, while increasing the numbers of endothelial cells during the first week of wound healing as well. Ultimately, this matrikine-based approach to improving MSC survival may be of great use across a variety of cell therapies utilizing matrices as delivery vehicles for cells.

An Integrated Biomedical Knowledge Extraction and Analysis Platform

High content screening (HCS) requires time-consuming and often complex iterative information retrieval and assessment approaches to optimally conduct drug discovery programs and biomedical research. Pre- and post-HCS experimentation both require the retrieval of information from public as well as proprietary literature in addition to structured information assets such as compound libraries and projects databases. Unfortunately, this information is typically scattered across a plethora of proprietary bioinformatics tools and databases and public domain sources. Consequently, single search requests must be presented to each information repository, forcing the results to be manually integrated for a meaningful result set. Furthermore, these bioinformatics tools and data repositories are becoming increasingly complex to use; typically they fail to allow for more natural query interfaces. Vivisimo has developed an enterprise software platform to bridge disparate silos of information. The platform automatically categorizes search results into descriptive folders without the use of taxonomies to drive the categorization. A new approach to information retrieval for HCS experimentation is proposed.

Abstract 5240: Breast cancer in the metastatic niche: A role for stress-induced dormancy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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

Abstract P1-07-01: Modeling breast cancer dormancy and re-emergence

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.