A tumor deconstruction platform identifies definitive end points in the evaluation of drug responses

Abstract

Tumor heterogeneity and the presence of drug-sensitive and refractory populations within the same tumor are almost never assessed in the drug discovery pipeline. Such incomplete assessment of drugs arising from spatial and temporal tumor cell heterogeneity reflects on their failure in the clinic and considerable wasted costs in the drug discovery pipeline. Here we report the derivation of a flow cytometry-based tumor deconstruction platform for resolution of at least 18 discrete tumor cell fractions. This is achieved through concurrent identification, quantification and analysis of components of cancer stem cell hierarchies, genetically instable clones and differentially cycling populations within a tumor. We also demonstrate such resolution of the tumor cytotype to be a potential value addition in drug screening through definitive cell target identification. Additionally, this real-time definition of intra-tumor heterogeneity provides a convenient, incisive and analytical tool for predicting drug efficacies through profiling perturbations within discrete tumor cell subsets in response to different drugs and candidates. Consequently, possible applications in informed therapeutic monitoring and drug repositioning in personalized cancer therapy would complement rational design of new candidates besides achieving a re-evaluation of existing drugs to derive non-obvious combinations that hold better chances of achieving remission.

Evaluation of Epigenetic Drug Targeting of Heterogenous Tumor Cell Fractions Using Potential Biomarkers of Response in Ovarian Cancer

“…Several studies demonstrating stabilization of expression patterns in response to epigenetic drug treatments remain limited due to lack of complete evaluation/prediction of long-term clinical responses (46,47). Importantly, intratumoral heterogeneity and minimal residual disease (MRD) are almost never considered in such studies and can become major deterrents in achieving drug efficacy (27,28,48). This realization led us to critically dissect out drug responses not only in terms of reversal of putative epigenetic biomarker expression patterns, but also evaluate functional associations vis-a-vis specific targeting of discrete tumor cell fractions as resolved through differential regenerative potential and genetic instability.…”

“…These consolidated efforts identified a subset of epigenetic biomarkers associated with ovarian cancer transformation and progression. We further evaluated the suitability of these biomarkers in predicting in vivo tumor responses to four epigenetic drugs through resolution of discrete cellular fractions within xenografts (27,28). Together, our findings present a novel and comprehensive approach for the derivation of potentially predictive biomarkers that provide a read-out for evaluating epigenetic drug efficacy and tumor responses for the development of next-generation drugs in serous ovarian adenocarcinoma.…”

“…Toward a mechanistic understanding of drug cytotoxicity, we delineated the specific cellular targets of each drug vis-a-vis discrete cellular subsets resolved through flow cytometry based on the cancer stem cell (CSC) hierarchy, genetically unstable populations (aneuploidy) within xenografts and differential cell cycling as described earlier (28). Briefly, label-chase of vital membrane dyes (PKH26/PKH67) resolves the CSC hierarchy as three cell subsets with a decreasing order of regenerative potential, viz., PKH hi cells (quiescent CSCs), PKH lo (progenitors), and PKH neg (host and differentiated tumor cells; Supplementary Fig.…”

“…For analysis of heterogeneous populations, unlabeled tumor cells were used as controls for gating total dye quenching events, while freshly labeled cells were used as positive controls. Propidium iodide (PI), Hoechst-PyroninY staining was carried out as described (28).…”

Functional Balance between TCF21-Slug defines phenotypic plasticity and sub-classes in high-grade serous ovarian cancer

“…Suspension cultures for HGSC cell lines were generated as described by earlier studies 56 . Briefly, 5 × 10 4 cells/well were seeded in ultra-low attachment plate (Corning) in serum free medium and incubated at 37 °C for 14 days.…”