Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Pribyl, Lee J.; Coughlin, Kathleen; Sueblinvong, Thanasak; Shields, Kristin; Iizuka, Yoshie; Downs, Levi S.; Ghebre, Rahel; Bazzaro, Martina

doi:10.3791/51581

Cited by 10 publications

(7 citation statements)

References 8 publications

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“…Our panel was optimized for single-cell suspensions obtained from primary tumor samples, and the six dissociation methods were selected to cover the spectrum of regular dissociation protocols applied to ovarian tumors [13,46]. In our study, we found that the method of tissue dissociation affected the marker expression of a few subgroups within the three major cell subsets.…”

Section: Discussionmentioning

confidence: 90%

Phenotypic Characterization by Mass Cytometry of the Microenvironment in Ovarian Cancer and Impact of Tumor Dissociation Methods

Anandan

Thomsen

Gullaksen

et al. 2021

Cancers

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Improved molecular dissection of the tumor microenvironment (TME) holds promise for treating high-grade serous ovarian cancer (HGSOC), a gynecological malignancy with high mortality. Reliable disease-related biomarkers are scarce, but single-cell mapping of the TME could identify patient-specific prognostic differences. To avoid technical variation effects, however, tissue dissociation effects on single cells must be considered. We present a novel Cytometry by Time-of-Flight antibody panel for single-cell suspensions to identify individual TME profiles of HGSOC patients and evaluate the effects of dissociation methods on results. The panel was developed utilizing cell lines, healthy donor blood, and stem cells and was applied to HGSOC tissues dissociated by six methods. Data were analyzed using Cytobank and X-shift and illustrated by t-distributed stochastic neighbor embedding plots, heatmaps, and stacked bar and error plots. The panel distinguishes the main cellular subsets and subpopulations, enabling characterization of individual TME profiles. The dissociation method affected some immune (n = 1), stromal (n = 2), and tumor (n = 3) subsets, while functional marker expressions remained comparable. In conclusion, the panel can identify subsets of the HGSOC TME and can be used for in-depth profiling. This panel represents a promising profiling tool for HGSOC when tissue handling is considered.

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Section: Discussionmentioning

confidence: 90%

Phenotypic Characterization by Mass Cytometry of the Microenvironment in Ovarian Cancer and Impact of Tumor Dissociation Methods

Anandan

Thomsen

Gullaksen

et al. 2021

Cancers

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“…Ovarian Surface Epithelial (OSE) cells and primary ovarian cancer cells were isolated from ovarian specimens excised from patients undergoing oophorectomy for benign conditions and cultured as we have previously described (17, 19, 20). …”

Section: Methodsmentioning

confidence: 99%

Small-Molecule RA-9 Inhibits Proteasome-Associated DUBs and Ovarian Cancer In Vitro and In Vivo via Exacerbating Unfolded Protein Responses

Coughlin

Anchoori

Iizuka

et al. 2014

Clinical Cancer Research

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Purpose Ovarian cancer is the deadliest of the gynecological malignancies. Carcinogenic progression is accompanied by up-regulation of ubiquitin-dependent protein degradation machinery as a mechanism to compensate with elevated endogenous proteotoxic stress. Recent studies support the notion that deubiquitinating enzymes (DUBs) are essential factors in proteolytic degradation and that their aberrant activity is linked to cancer progression and chemoresistance. Thus, DUBs are an attractive therapeutic target for ovarian cancer. Experimental Design The potency and selectivity of RA-9 inhibitor for proteasome-associated DUBs was determined in ovarian cancer cell lines and primary cells. The anticancer activity of RA-9 and its mechanism of action was evaluated in multiple cancer cell lines in vitro and in vivo in immunodeficient mice bearing an intra-peritoneal ES-2 xenograft model of human ovarian cancer. Results Here we report the characterization of RA-9 as a small-molecule inhibitor of proteasome-associated DUBs. Treatment with RA-9 selectively induces onset of apoptosis, in ovarian cancer cell lines and primary cultures derived from donors. Loss of cell viability following RA-9 exposure is associated with an Unfolded Protein Response (UPR) as mechanism to compensate for unsustainable levels of proteotoxic stress. In vivo treatment with RA-9 retards tumor growth, increases overall survival and was well tolerated by the host. Conclusions Our preclinical studies support further evaluation of RA-9 as an ovarian cancer therapeutic.

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“…Primary ovarian cancer cells were isolated from solid specimens using the modified method described by Pribyl et al . 77 . The tissue pieces were digested with 150 U/mL collagenase (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C and then transferred onto a nylon cell strainer (100 µm; VWR International, Radnor, PA) to separate EOC cells.…”

Section: Methodsmentioning

confidence: 99%

Detection and genotyping of CMV and HPV in tumors and fallopian tubes from epithelial ovarian cancer patients

Paradowska

Jabłońska

Studzińska

et al. 2019

Sci Rep

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Viral and bacterial infections are detected in epithelial ovarian cancer (EOC) tissues. Since the fallopian tubes are often affected by pelvic inflammatory disease (PID) and the majority of serous EOCs appear to originate from dysplastic lesions in the distal tube, it is relevant to consider the potential role that infectious agents may play in ovarian carcinogenesis. We sought to analyze the prevalence of human papillomavirus (HPV) and cytomegalovirus (CMV) in EOC tissue and fallopian tube specimens obtained at tumor resection. Ovarian cancer and fallopian tube tissue samples obtained from patients with EOC were analyzed by both qualitative and quantitative PCR to detect and quantify viral DNA. The presence of CMV and HPV DNA was detected in 70% and 74% cancerous ovarian tissues, respectively, and was significantly higher in EOC than in benign tumor cases (P ≤ 0.01). CMV or HPV infection was observed also in the fallopian tube samples. Infection with HPV16 was determined in 70% of EOC cases. Almost two thirds of EOC patients demonstrated coinfection with CMV and HPV in the pathological samples. The results revealed that the presence of CMV and HPV in EOC samples is common. CMV and HPV infections can be potential risks for EOC development.

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Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Cited by 10 publications

References 8 publications

Phenotypic Characterization by Mass Cytometry of the Microenvironment in Ovarian Cancer and Impact of Tumor Dissociation Methods

Phenotypic Characterization by Mass Cytometry of the Microenvironment in Ovarian Cancer and Impact of Tumor Dissociation Methods

Small-Molecule RA-9 Inhibits Proteasome-Associated DUBs and Ovarian Cancer In Vitro and In Vivo via Exacerbating Unfolded Protein Responses

Detection and genotyping of CMV and HPV in tumors and fallopian tubes from epithelial ovarian cancer patients

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