Engineered 3D Model of Cancer Stem Cell Enrichment and Chemoresistance

Abstract

Intraperitoneal dissemination of ovarian cancers is preceded by the development of chemoresistant tumors with malignant ascites. Despite the high levels of chemoresistance and relapse observed in ovarian cancers, there are no in vitro models to understand the development of chemoresistance in situ . Method: We describe a highly integrated approach to establish an in vitro model of chemoresistance and stemness in ovarian cancer, using the 3D hanging drop spheroid platform. The model was established by serially passaging non-adherent spheroids. At each passage, the effectiveness of the model was evaluated via measures of proliferation, response to treatment with cisplatin and a novel ALDH1A inhibitor. Concomitantly, the expression and tumor initiating capacity of cancer stem-like cells (CSCs) was analyzed. RNA-seq was used to establish gene signatures associated with the evolution of tumorigenicity, and chemoresistance. Lastly, a mathematical model was developed to predict the emergence of CSCs during serial passaging of ovarian cancer spheroids. Results: Our serial passage model demonstrated increased cellular proliferation, enriched CSCs, and emergence of a platinum resistant phenotype. In vivo tumor xenograft assays indicated that later passage spheroids were significantly more tumorigenic with higher CSCs, compared to early passage spheroids. RNA-seq revealed several gene signatures supporting the emergence of CSCs, chemoresistance, and malignant phenotypes, with links to poor clinical prognosis. Our mathematical model predicted the emergence of CSC populations within serially passaged spheroids, concurring with experimentally observed data. Conclusion: Our integrated approach illustrates the utility of the serial passage spheroid model for examining the emergence and development of chemoresistance in ovarian cancer in a controllable and reproducible format.

Expansion of Circulating Tumor Cells from Patients with Locally Advanced Pancreatic Cancer Enable Patient Derived Xenografts and Functional Studies for Personalized Medicine

“…Cells were removed from culture surfaces by gentle trituration and collected in conical tubes. Pancreatic CTC spheroids were generated on 384 well hanging drop array plates, following extensively established protocols [37][38][39][40]. Briefly, cell suspensions were adjusted such that 20 µL contained 100 pancreatic CTCs.…”

“…Cells were trypsinized, washed with ice-cold PBS, and fixed at a concentration of 2 × 10 6 cells/mL in ice-cold 70% ethanol. For γH2AX analysis, samples were incubated with a mouse anti-γH2AX-specific antibody (clone JBW301; Millipore, Burlington, MA, USA) overnight at 4 • C, followed by incubation with a fluorescein isothiocyanate-conjugated secondary antibody (Sigma-Aldrich), as previously described [37]. For quantification of γH2AX positivity, a gate was arbitrarily set on the control, untreated sample to define a region of positive staining for γH2AX of approximately 5%.…”

Immobilization rapidly selects for chemoresistant ovarian cancer cells with enhanced ability to enter dormancy

“…There is currently no rapid way to predict how responsive ovarian cancer patients will be to chemotherapy before treatment, as patient‐derived xenograft (PDX) avatar models take months to establish (Scott, Mackay, & Haluska, 2014). Recently, three‐dimensional (3D) in vitro models using patient tumor cells have shown promise as platforms for predicting patient drug response and require less time than PDX models (Raghavan et al, 2017; Rashidi et al, 2019); however, even these methods can take weeks. The ability to rapidly identify patients (within days) who would not respond to chemotherapy would enable clinicians to adjust monitoring strategies or suggest alternative therapies that are often only used once conventional treatment no longer works.…”

Novel Therapeutic Strategies for Ovarian Cancer Stem Cells

“…Signaling within the tumor microenvironment (tumor niche), including oxygenation, cell-to-cell contact and secreted factors, could induce differentiated tumor cells to re-acquire stem celllike properties (62). Additionally, radio-and chemotherapy treatments have been shown to enrich CSC subpopulations in residual tumors because of selective pressure on drug-resistant cells (65)(66)(67) and due to tumor cell plasticity (64). Even though the CSC state has high plasticity, it is of high clinical importance as a potential marker for clinical outcome and target for anticancer treatment (68,69).…”