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Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays.

ABSTRACT: Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate identification and localization of proteins, glycans, and small molecules. While this technique is well-established in two-dimensional (2D) cell culture, less is known about its use in three-dimensional (3D) cell models. Ovarian cancer organoids are 3D tumor models that recapitulate tumor cell clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Thus, they are superior to cell lines for the evaluation of drug sensitivity and functional biomarkers. Therefore, the ability to utilize immunofluorescence on primary ovarian cancer organoids is extremely beneficial in understanding the biology of this cancer. The current study describes the technique of immunofluorescence to detect DNA damage repair proteins in high-grade serous patient-derived ovarian cancer organoids (PDOs). After exposing the PDOs to ionizing radiation, immunofluorescence is performed on intact organoids to evaluate nuclear proteins as foci. Images are collected using z-stack imaging on confocal microscopy and analyzed using automated foci counting software. The described methods allow for the analysis of temporal and special recruitment of DNA damage repair proteins and colocalization of these proteins with cell-cycle markers.

SUBMITTER: van Biljon L

PROVIDER: S-EPMC10881227 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays.

van Biljon Lillian L Fashemi Bisiayo B Rodriguez Jeimmy J Graham Olivia O Compadre Amanda A Fuh Katherine K Khabele Dineo D Mullen Mary M

Journal of visualized experiments : JoVE 20230224 192

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate identification and localization of proteins, glycans, and small molecules. While this technique is well-established in two-dimensional (2D) cell culture, less is known about its use in three-dimensional (3D) cell models. Ovarian cancer organoids are 3D tumor models that recapitulate tumor cell clonal heterogeneity, the tumor microenvironment, ...[more]

PMID: 36912550

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Project description:BackgroundOvarian cancer is the most lethal gynecological cancer. As the primary treatment, chemotherapy has a response rate of only 60-70% in advanced stages, and even lower as a second-line treatment. Despite guideline recommendations, which drugs will be most effective remains unclear. Thus, a strategy to prioritize chemotherapy options is urgently needed. Cancer organoids have recently emerged as a method for in vitro drug testing. However, limited clinical correlations have been assessed with test results from cancer organoids, particularly in gynecological cancers. We therefore aimed to generate patient-derived organoids (PDOs) of ovarian cancer, to assess their drug sensitivities and correlations with patient clinical outcomes.MethodsPDOs were generated from fresh tumors obtained during surgical resection, which was then cultured under matrix gel and appropriate growth factors. Morphological and molecular characterization of PDOs were assessed by phase contrast microscopy and paraffin-embedded histopathology. Expressions of PAX8, TP53, WT1, CK7, and CK20 were tested by immunohistochemical staining and compared with parental tumor tissues and the human protein atlas database. PDOs were subjected to in vitro drug testing to determine drug sensitivity using Titer-Glo® 3D Cell Viability Assay. PDO viability was measured, and area under the curve calculated, to compare responses to various compounds. Correlations were calculated between selected patients' clinical outcomes and in vitro drug testing results.ResultsWe established 31 PDOs. Among them, 28 PDOs can be expanded, including 15, 11, and 2 from ovarian, endometrial, and cervical cancers, respectively. The PDOs preserved the histopathological profiles of their originating tumors. In vitro drug testing of 10 ovarian cancer PDOs revealed individual differential responses to recommended drugs, and interpersonal heterogeneity in drug sensitivity, even with the same histology type. Among four patients who were platinum sensitive, resistant, or refractory, PDO drug responses correlated well with their clinical courses.ConclusionIn vitro drug testing using ovarian cancer organoids is feasible and correlates well with patient clinical responses. These results may facilitate development of precision chemotherapy and personalized screening for repurposed or new drugs.

Project description:BackgroundHigh grade serous ovarian cancer (HGSOC) is among the deadliest human cancers and its prognosis remains extremely poor. Tumor heterogeneity and rapid acquisition of resistance to conventional chemotherapeutic approaches strongly contribute to poor outcome of patients. The clinical landscape of HGSOC has been radically transformed since the advent of targeted therapies in the last decade. Nevertheless, the lack of predictive biomarkers informing on the differential clinical benefit in select subgroups, and allowing patient-centric approaches, currently limits the efficacy of these novel therapies. Thus, rational selection of the best possible treatment for each patient represents a clinical priority in order to improve outcome, while limiting undesirable effects.Main bodyIn this review, we describe the state of the art and the unmet needs in HGSOC management, illustrate the treatment options that are available and the biomarkers that are currently employed to orient clinical decisions. We also describe the ongoing clinical trials that are testing new therapeutic approaches for HGSOC. Next, we introduce the organoid technology as a promising, expanding strategy to study cancer and to develop personalized therapeutic approaches. In particular, we discuss recent studies that have characterized the translational potential of Patient's Derived Organoids (PDOs) to inform on drug sensitivity of HGSOC patients.ConclusionsPDOs can predict the response of patients to treatments and may therefore guide therapeutic decisions. Although preliminary results appear encouraging, organoids still need to be generated and expanded efficiently to enable drug screening in a clinically meaningful time window. A new generation of clinical trials based on the organoid technology should guarantee tailored approaches to ovarian cancer management, as it is now clear that the one-size-fits-all approach cannot lead to efficient and meaningful therapeutic advancements.

Project description:Patient-derived tumor organoids (PDOs) hold immense potential for personalized drug sensitivity testing, but accurate efficacy determination is crucial for clinical translation. This study investigated factors influencing the accuracy and reproducibility of drug sensitivity measurements in PDOs, focusing on half-maximal-inhibitory-concentration (IC50) calculation methods, drug concentration numbers, and plate types. PDOs were established from six primary cancer tissues, including two cervical resections, one lung biopsy, one lung pleural effusion, one breast biopsy, and one gastric resection. They were subjected to drug sensitivity assays with 21 single/combined treatments, encompassing chemotherapy and targeted therapy drugs, with concentrations standardized in fold. Utilizing 6- and 12-concentration setups, IC50 derived from GraphPad-Dose-response-Inhibition (DRI), LC-logit, and LC-probit methods were compared. Relative changes (RCs) in IC50 and area-under-the dose-response-curve (AUC) between setups and the impact of plate type on cell viability measurements were assessed. In the 12-concentration setup, no significant IC50 differences were observed among the calculation methods. Notably, GraphPad-DRI and LC-logit exhibited minimal RCs between the 6- and 12-concentration setups (0.035 and -0.033, respectively), indicating accurate IC50 quantification even with fewer drug concentrations. AUC correlated strongly with GraphPad-DRI-derived IC50 (R = 0.858) and demonstrated lower variance between technical replicates. Furthermore, opaque-bottom plates yielded higher precision in cell viability measurements compared to transparent-bottom plates. This study provides valuable insights into optimizing drug sensitivity testing in PDOs. By demonstrating the robustness of specific IC50 calculation methods and the feasibility of using fewer drug concentrations, this study contributed to the standardization and reliability of PDO-based drug sensitivity assays.

Dataset Information

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays.

Publications

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays.

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