A Vascularized, Three-dimensional Biomimetic Platform for Patient-Specific, ex-vivo studies of Breast Cancer Invasion and Metastasis.
Daniel Lara, BS1, Karel-Bart Celie, BA2, Yoshiko Toyoda, BA1, Matthew Wright, BA2, Justin Buro, BA3, Arash Samadi, BS1, Sofya Oshchepkova, BA1, Kristy Brown, PhD1, Jason Spector, MD1.
1Weill Cornell Medicine, New York, NY, USA, 2Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA, 3George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
Introduction:Breast cancer (BC) is the most common non-skin cancer in females, affecting 12.5% of women throughout their lifetime. Partly due to a lack of models that accurately mimic the tumor microenvironment of individual patients, many preclinical successes fail translation into the clinic. Current two-dimensional models fail to replicate the cellular behaviors and interactions that occur in vivo, and newer three-dimensional systems, though promising, all lack vasculature which is crucial to understanding the mechanisms of tumor cell invasion and metastasis. We have engineered an advanced three-dimensional biomimetic platform derived from patient specific tissues that contains all components of the breast tumor microenvironment (glandular organoids, adipocytes, stromal vascular fraction (SVF)) surrounding engineered vascular structures that can be precisely positioned at predetermined distances from BC spheroids (BCS) allowing for highly novel ex-vivo investigations of the interactions between human tumor cells and blood vessels. Methods: Polydimethylsiloxane (PDMS) wells were created using 3D-printed molds that include stages for localization of BCS, and putative vascular channels (VC). Type I collagen was neutralized at 0.3% and 0.6% w/v. Red-fluorescent MDA-MB-231 cells were mixed with 0.6% collagen at a density of 40,000 cells/uL; 1uL of the collagen/cancer cell mix was plated on stages of the PDMS molds and allowed to nucleate. A biomimetic platform made with BODIPY stained adipocytes and all other patient-derived tissue components mixed within both concentrations of collagen was plated in the pre-designed well, surrounding the BCS and VC. Twenty-four hours after plating, fluorescently labeled endothelial cells (EC) and smooth muscle cells (SMC) were seeded within the channel at a concentration of 5 million cells/mL. Control constructs were made by generating vascular structures and BCS within a collagen-only matrix, and by creating full biomimetic platforms with vascular channels in the absence of cancer cells. Constructs were cultured for 7 days, formalin-fixed, counterstained with DAPI, and analyzed with confocal microscopy. Results After 7 days in culture, confocal microscopy revealed successful fabrication of biomimetic platforms with a type I collagen (different concentrations) extracellular matrix containing patient-derived adipocytes, SVF and breast duct organoids. Patent VC lined with fluorescently labeled SMC and EC were visualized within the platform, with vascular walls located within 1mm of the red fluorescent, triple-negative MDA-MB-231 cancer foci. Invasion of BC cells into the surrounding tissue was identified by the presence of red fluorescent cells within the biomimetic platform in constructs containing type I collagen at both 0.3% and 0.6% w/v. Decreased vascular integrity was observed in constructs containing BCS when compared to those without BC cells. Conclusion With the aid of three-dimensional printing technology, we have successfully engineered an advanced, patient-specific, biomimetic platform of the breast cancer microenvironment that not only replicates patient tissue characteristics, but also includes vascular structures and cancer foci that closely resemble early tumors. Observed BC invasion into the surrounding microenvironment, and the platform's ability to mimic patient specific tissue with extremely high fidelity, make this platform a highly versatile and powerful tool that holds significant promise for diagnostic and therapeutic applications in the study of breast cancer.
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