Automation and High Throughput Technologies
Complex tumor microenvironments lead to intra- and inter-patient heterogeneity across multiple cancer types, which prevent consistent clinical treatment outcomes. Micro-physiological and cellular changes impact tumor cell survival and disease progression significantly. Therefore, it is critical to understand how tumor cells behave and respond to drugs under different environmental conditions. We have established 3D organoid cultures from colon cancer patient tissues. Organoids were treated with known clinical cancer drugs to examine how they respond to various treatments. To keep intact organoid structures, we performed whole mount immunostaining with tissue clearing techniques. Ki-67 (Cell proliferation), Cleaved-caspase 3 (Apoptosis), E-cadherin (Cell junction) and F-actin (Cytoskeleton) changes were examined during drug treatments. Cleaved-caspase 3 positive cell numbers were significantly increased after drug treatments but the degree of cell death differed depending on each drug type and dose. F-actin was enriched at the border between the outer surface of cells and the inner core in control organoids but this uniform pattern of actin cytoskeleton was lost in Staurosporine- and Irinotecan-treated organoids. To complement our fixed imaging workflow, we also performed live cell imaging on patient-derived organoids labeled using either Lentiviral-H2B transduction (Stable) or vital dyes (Transient). Dynamic imaging of H2B-GFP organoids illustrated cell division events, which were used to quantify cell proliferation rates in individual organoid. Cell proliferation was significantly reduced with Oxaliplatin treatment suggesting that this drug affects organoid growth by inhibiting cell divisions. DRAQ7 (Dead cells) staining was increased rapidly in entire organoids with Staurosporine and Irionotecan caused cell death at the outer surface of the organoids. Interestingly, we observed organoid contraction and shrinkage with Irinotecan and 5-Fluorouracil (5-FU) treatments highlighting the advantages of live cell imaging with regards to depicting dynamic drug- and concentration-dependent responses. Given tumor-stromal cell interactions are key microenvironmental factors influencing drug response, we also isolated cancer-associated fibroblasts (CAFs) from patient tissues to co-culture with patient-matched tumor organoids. H2B-GFP labeled organoids cultures was seeded on top of the fibroblasts to make physical contact between the two different cell types. By using high-resolution 3D imaging, we examined the impact of CAFs on the drug response of patient-derived organoids. 3D/4D organoid imaging is a powerful method to study the tumor microenvironment and screen preclinical drug compounds using patient tissues. We believe that a high-throughput automated imaging system coupled with a bio-repository of patient-derived organoids will expedite the identification of effective anti-cancer treatment options for individual patient as well as our understanding of biological questions regarding the complex tumor microenvironment.