A Tissue-Engineered Organo-Typic Model of Breast Cancer Metastasis
Julia L. Jin, B.S.1, Jaime L. Bernstein, B.S.1, Matthew R. Zanotelli, M.S.2, Yoshiko Toyoda, B.A.1, Andrew Abadeer, M.Eng1, Sarah J. Karinja, B.A.1, Omer Kaymakcalan, M.D.1, Alexandra Lin, B.A.1, Peter Torzilli, PhD3, John P. Morgan, PhD1, Jason A. Spector, M.D.1.
1Weill Cornell Medical College, New York, NY, USA, 2Cornell University, Ithaca, NY, USA, 3Hospital for Special Surgery, New York, NY, USA.
BACKGROUND: Abnormal vasculature and extracellular matrix (ECM) alter tumor microenvironmental (TME) mechanical and metabolic properties, thereby increasing cancer cell invasive and metastatic potential, enabling immunosuppression and treatment resistance, and driving evolution and adaptive survival of cancer cell populations. Characteristic properties include hypoxia, low pH and high interstitial fluid pressure. In breast cancer, TME density is considered an independent risk and prognostic factor. However, breast cancer research has suffered from a lack of model systems that can recapitulate this complex 3D-microenvironment, in vitro, including the complex interplay between the many resident cell types and evolving ECM density. How cell fate regulation, tumor angiogenesis and breast cancer progression occur within an intact complex vascular network and TME remains undefined. Herein we present an organo-typic model of breast cancer, with the full complement of breast adipose, vascular and epithelial cells, functional epithelial ducts and perfused vascular networks within a biocompatible collagen construct. We tune the mechanical and metabolic properties of this system to study their influence on tumor progression and vascular remodeling.
METHODS: Breast adipose, vascular and epithelial cells were harvested from patient-derived breast tissue discarded from the clinic, and used to create vascularized and epithelialized breast tissue in Type-I collagen matrices, enzymatically stiffened with varying ribose solutions. Specifically, Pluronic F127 fibers, were sacrificed in collagen, creating multiple microchannels which were seeded separately with breast epithelial cells and with vascular cells, including human-aortic-smooth-muscle (HASMC) and human-umbilical-vein-endothelial (HUVEC) cells. Adipocytes and stromal cells were embedded in the bulk collagen along with breast-cancer-tumor-spheroids. Constructs were cultured with varying adipocyte concentrations and levels of endogenous oxygen. Scaffolds were imaged with confocal and multiphoton microscopy (MPM) to visualize cellular growth progression and spatial-arrangement. Collagen fibers were characterized quantitatively using confocal reflectance microscopy and image analysis.
RESULTS: Confocal reflectance image analysis showed no statistical change of fiber length or pore area in enzymatically altered collagen. Dosing collagen with 200mM of ribose increased biomechanical stiffness to appropriate physiologic tumor levels (4kPa). Microvessels in non-cancerous constructs exhibited contiguous cell-cell junctions indicating confluent, healthy endothelia with intact cytoskeletons and stable architecture. In cancer constructs, degradation of the vascular lining and aberrant cell organization was observed. Capillary-like neovessels ranging from 10-80μm in diameter invaded tumor spheroids. MPM revealed the intravasation of metastatic breast cancer cells within micro-neovessels. In comparison, less stiff hydrogels lacked significant development of neovessels within constructs, suggesting a correlation between tumor angiogenesis and ECM stiffness.
CONCLUSION: This novel model faithfully recapitulates in vivo human breast tissue, overcoming the limitations of previous 2D/3D culture models and effectively recapitulating tumor angiogenesis. In addition to allowing for significantly greater understanding of the complex interplay that occurs within the tumor microenvironment with the goal of determining the role of metabolism and ECM mechanics on breast cancer cell behavior, our biomimetic model can ultimately be used with tumor cells extracted from individual patients to develop patient specific ex vivo 3D culture precision medicine systems. These individual models will allow us to develop patient-specific targeted approaches to treating breast cancer.
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