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.

Figure 1:. ɣ-H2AX foci in PDOs before and after irradiation.

Representative images of DAPI and ɣ-H2AX foci in PDOs before and after irradiation at 10x with 63x insets. Scale bars: 10 μm.

Figure 2:. RAD51 foci and geminin in PDOs before and after irradiation.

Representative images of DAPI, geminin, RAD51, and co-staining of geminin/RAD51 foci PDOs before and after irradiation at 10x with 63x insets. Scale bars: 10 μm.

Figure 3:. RPA and 53BP1 in PDOs before and after irradiation.

Representative images of (A) DAPI, RPA; (B) geminin, 53BP1, and co-staining of geminin/53BP1 foci at 10x with 63x insets. Scale bars: 10 μm.

Figure 4:. The JCountPro software quantification workflow.

(A) Under the object analysis, the blue channel is selected to identify the blue objects, nuclei, then it is automatically optimized by selecting the auto segmentation (red arrow). (B) Under auto split, the object size (nuclei) is adapted to the size of the image and magnification. The identify object button is selected to test the parameters (1), and the identify objects in all images (red arrow) button is selected to identify objects (nuclei) for each image. (C) Under the foci analysis tab, the images are inputted and the foci counting parameters are set: first, the color of the foci is selected under the focus channel, green; the top hat index is set to 12; the H dome settings are set to have a dome height percentage of 30 and a threshold percentage of 28; the shape and size of the foci are optimized to the image size with the manual parameters of maximum focus size pixels at 60 and minimum roundness x 100 at 96; finally, the noise filter is applied. The settings are tested by applying the top hat, H dome, and foci count (1–3). To quantify the ɣ-H2AX foci per cell, press start (red arrow). (D) For the RAD51 foci, the settings are applied as illustrated for ɣ-H2AX foci, except the focus channel which is changed to red; however, to identify nuclei that are stained with geminin, the second channel is selected for green, and analysis is changed to intensity. The parameters are tested by applying the top hat, H dome, and foci count (1–3), then pressing start (red arrow) to quantify all RAD51 foci and evaluating the intensity of green per cell in each image. Scale bar: 10 μm.