3D TUMOR MODELING TO IMPROVE BIOMARKER IDENTIFICATION AND CANCER OUTCOMES.

Purpose: Cancer-associated proteins annexina2 (ANXA2) and MIEN1 are overexpressed in various cancers including triple negative breast cancer, prostate, and colorectal cancers. Therefore, these proteins are being investigated as potential biomarkers and therapeutic targets. We will use a 3D tumor model to mimic in vivo tumor growth and evaluate changes in tumor characteristics after silencing ANXA2 and MIEN1 to understand their correlative/causal relation to tumor behavior. Methods: Ultra-low attachment 3D culture plates were used to culture tumor spheroids in breast cancer cells(MDA-MD-231) with ANXA2 knock-down using sh-RNA and colorectal cancer cells(HT29) with MIEN1 knock-out (KO) using CRISPR-Cas9. Changes in spheroid growth, migration and invasion, drug sensitivity, and immune cell killing will be evaluated. Boyden chambers with Matrigel inserts were used to evaluate migration and invasion. Spheroids were imaged using confocal microscopy for size estimation. Results: Optimal initial seeding density was 5000 cells and spheroids were grown for 7-days. Average tumor spheroid size was 1360±420µm with no differences in spheroid size with MIEN1 KO. 50% reduction in migration and invasion potential were observed after MIEN1 KO in colorectal cancer cells in a 96-hour time-course experiment. Experiments with ANXA2 knock-down are ongoing with spheroid sizes averaging 1700±260µm. Future experiments include evaluating changes in protein expression, drug sensitivity, immune cell killing as a result of knockout of MIEN1. Conclusion: We expect the results to identify functional role of cancer-associated proteins ANXA2 and MIEN1 in tumor spheroid growth, metastasis, drug sensitivity, and susceptibility to immune cell killing. Funding: Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number R01CA220273 (JKV).