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 metastases has hindered our understanding of the molecular basis of metastasis, the development of therapies, and the elucidation of biomarkers to detect these lethal outgrowths in real time. To address this gap, we developed an innovative all-human 3D ex vivo hepatic microphysiological system (MPS), which faithfully reproduces human physiology and effectively recreates spontaneous quiescence in a population of BrCa cells. Thus facilitating the investigation of BrCa behavior in a micrometastatic niche.
The MPS incorporated primary human hepatocytes and non-parenchymal cells (NPC) isolated from patient liver resections. BrCa cells (RFP+) were seeded on day 3 and allowed time to intercalate into the hepatic tissue before treatment with chemotherapies (such as doxorubicin) on day 7 for 72h. Surviving BrCa cells were stimulated on day 13 with physiological inflammatory stressors (LPS/EGF) and cultured through day 15. Proliferation was determined by RFP quantification, Ki67 staining and EdU incorporation. Physiological functioning of hepatic tissue was monitored at multiple time points by protein catabolism (urea), active nutrient metabolism (glucose, CYP P450) and injury markers (AST, ALT). Luminex assays (57 analytes) were used to identify potential metastatic BrCa biomarkers in the hepatic milieu during different stages, both dormancy and progression, and determine communication networks within the micrometastatic niche.
Spontaneous dormancy was observed amongst a subpopulation of BrCa cells (Ki67-/EdU-) after 12 days of culture. Following chemotherapy, the surviving BrCa cells were non-proliferating, quiescent (Ki67-/EdU-). Furthermore, re-emergence of the surviving non-proliferating cancer cells was then observed in the presence of physiological inflammatory stressors. Preliminary high-content analyses of secreted cytokines and growth factors identified candidate biomarkers of metastatic BrCa in hepatic tissue. Elevations in 11 soluble signaling factors in effluent from untreated proliferating BrCa were detected, of which actively growing cells were specifically associated with HGF, MCP-1, MIP-1β, RANTES (> 2-fold) compared to hepatic tissue alone and dormant BrCa effluent. Candidate biomarkers of dormancy were also identified when dormant cultures were compared to both hepatic tissue alone and proliferating BrCa cultures, 5 soluble signaling factors (HB-EGF, IL-18, VEGF-A, IL-10, IL-12p70). Further analyses found altered soluble signaling factors due to the presence of NPCs during attenuation and progression of BrCa, indicating NPCs within the hepatic microenvironment are implicated in regulating the dormant micrometastatic niche.
This MPS provides unprecedented insights into the tumor cell biology of growing and dormant micrometastases. First, it mimics the dormancy and outgrowth observed in patients - i.e. dormant breast cancer cells resistant to chemotherapy could be stimulated to re-emerge following an inflammatory insult. Second, this system provides an accessible tool enabling the identification of candidate biomarkers of BrCa progression and dormancy within the micrometastatic niche; significantly, the latter aspect of dormant biomarkers is exceedingly novel. Ultimately, these collective innovative capabilities are instrumental in the development of therapeutic strategies to target dormant metastatic BrCa.
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 PR11.