3d Is Not Enough: Recapitulating The Tumor Microenvironment Is Necessary To Elicit Increased Proliferation Of Triple Negative Breast Cancer Cells In Vitro
Xue Dong, MD, PhD1, Sabrina Shih, BA2, Carly Askinas, BS3, Nicholas Vernice, BA1, Jason Spector, MD1.
1Weill Cornell Medical College, New York, NY, USA, 2Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA, 3Tulane University School of Medicine, New Orleans, LA, USA.
Purposes: Breast cancer (BC) is the most common cancer worldwide in women. Although many therapeutics have been successful in treating tumors in labs, only a small fraction of those agents prove efficacious in patients. Thus, there is an urgent need for high fidelity in vitro test platforms which closely resemble the local tumor microenvironment in order to develop more effective breast cancer therapeutics for clinical application. Herein we describe a tissue-engineered 3D biomimetic platform (BMP), derived from patient specific breast tissues that contains all components of the breast tumor microenvironment (glandular epithelial organoids, adipocytes, stromal vascular fraction (SVF)) within which are embedded engineered vascular channels. Tumor spheres may then be placed precisely at predetermined distances between the vessels allowing for detailed studies of the tumor/vessel interactions. Methods: Polydimethylsiloxane (PDMS) molds were created using custom designed 3D-printed Poly lactic acid (PLA) molds. 3D-printed PLA stamps and 22G catheters were used for the fabrication of tumor spheres and putative vascular channels within the same Z coordinate, with the spheres separated by 3.5mm from each other and 1.5mm from either channel. The BMP was fabricated using adipocytes and other patient-derived tissue components mixed within neutralized 1% (w/v) Type I collagen to form the main structure in PDMS molds; red-fluorescent MDA-MB-231 cells mixed with 1% collagen at 40,000 cells/1.6uL were added into the wells that were pre-formed with PLA stamps in the BMP bulk to create the tumor spheres. Twenty-four hours after plating, red-fluorescent smooth muscle cells (SMC) and green-fluorescent endothelial cells (EC) were seeded sequentially within the channels at 5 million cells/mL. Control constructs were made by generating vascular structures and tumor spheres within a collagen-only matrix. Constructs were cultured for 7, 14 and 21 days, and analyzed with H&E and immunofluorescent staining. Results Patent vascular channels lined with SMC and EC were visualized within the channels the day after seeding. Fluorescent images showed SMC sprouts had formed from pre-formed channels over 21 days in culture. Concurrently, the tumor spheres were noted to increase in size and individual cells were seen invading into surrounding collagen matrix over time. Successful fabrication of biomimetic platform containing patient-derived tissue components was proved by different staining methods. Immunofluorescent staining of Ki67 revealed an increased tumor cell proliferation in biomimetic platform on Day 7, 14 and 21 when compared to tumors cultured in 3D collagen-only group. Conclusion 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 spheres that closely resemble early tumors. The increased tumor proliferation noted within the biomimetic platform underscores the importance of recapitulating the TME in ex vivo investigation and the mechanism underlying this observation is currently being interrogated. This advanced tissue engineered platform represents a highly potent tool that holds significant promise for diagnostic and therapeutic applications in breast cancer study.
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