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.