2.1 Culture and characterization of ovarian cancer organoids from patient-derived tumors

In an effort to closely emulate the intricate morphological characteristics and crucial cell–cell interactions present in original ovarian tumors, we devised a novel organoid culture protocol. This method circumvents the enzymatic dissociation of tumors into single cells, thereby preserving the structural integrity and cellular heterogeneity of the original tumor tissue. We totally established 7 organoids in this research, including OV004 (61 years old, HGSOC), OV014 (68 years old, HGSOC), OV015 (52 years old, HGSOC), OV016 (69 years old, HGSOC), OV076 (59 years old, right-sided fallopian tube mass), OV077 (96 years old, unknown) and OV078 (67 years old, unknown). Utilizing precision-cutting techniques, we sectioned patient-derived ovarian tumors into fragments approximately 1 mm in size. These fragments were sourced from various regions within the tumors to encompass a broad representation of the tumor's cellular composition (Figure 1A). Notably, our protocol deliberately abstained from removing red blood cells and tissue debris, a step we hypothesized would aid in maintaining the organoid's fidelity to the tumor's microenvironment.

Upon incubation at standard culture conditions, we observed the formation of three distinct ovarian cancer organoids. Each organoid exhibited successful culture establishment and, within approximately 2 weeks, demonstrated spherical growth, indicative of their proliferative capacity. Histological examination of the organoids revealed the preservation of key features characteristic of ovarian tumors, including the presence of intra-organoid vascular structures. Interestingly, despite our method not incorporating a red blood cell removal step, the vascular features within the ovarian cancer organoids were distinctly observable, with blood vessels persisting for up to 18 days in culture (Figure 1B). Immunohistochemical analysis further substantiated the presence of vascular structures, with CD31 expression delineating a tubular vascular pattern within the organoids (Figure 1B). Moreover, our culture conditions facilitated the preservation of diverse cell types, suggesting the retention of the cellular diversity within the organoids, especially CD4+ and CD8+ T cells (Figure 1C).

The ovarian cancer organoids’ capacity for subculture was also evaluated. Following a 28-day incubation period, the organoids were mechanically divided and redistributed across new culture vessels. Remarkably, these secondary organoids mirrored the initial spherical morphology observed in their predecessors, although they lacked vascular components, likely attributable to the absence of hematopoietic functionality (Figure 1D).

Our study demonstrates the successful establishment of ovarian cancer organoids that not only maintain growth ability but also show potential in preserving the native immune microenvironment and blood vessel. This advancement represents a significant step forward in the development of biologically relevant in vitro models for ovarian cancer, offering promising avenues for future therapeutic research and personalized medicine approaches.

2.2 Preservation of tumor molecular characteristics in ovarian cancer organoids

Addressing the inherent challenge of replicating the complex heterogeneity of ovarian tumors, our study focuses on the capacity of ovarian cancer organoids to mirror the diverse cellular makeup of the parent tumors accurately. Employing scRNA-seq at day 14 postformation, we identified distinct cell clusters within the ovarian cancer organoids. Cross-referencing with recent single-cell sequencing datasets from ovarian cancer tissues, the cellular composition of our organoids is largely similar to that of public data (Figure 2A and B). These included T cells (positive for CD3 and CD8), endothelial cells (positive for CD34 and FLT1), smooth muscle cells (DCN and ACTA2 positive), monocytes and macrophages (LYZ and CD163 positive), and B cells (CD79A positive). This evidence confirms the existence of equivalent cell populations within the ovarian cancer organoids, validating the capacity of our organoid model to faithfully recapitulate the cellular diversity inherent to ovarian tumors (Figure 2B).³³

Focusing on the organoids’ microenvironment, particularly the vascular and immune components, we analyzed the expression of key marker genes. Notably, the presence of vascular endothelial markers, such as FLT1, CD34, and KDR, underscores the organoids’ capability to maintain blood vessel-like structures. Simultaneously, the detection of T-cell markers, including CD3 and CD8, highlights the preservation of immune cell subsets within the ovarian cancer organoids. This cellular composition, closely mirroring that of the original ovarian tissues, emphasizes the ovarian cancer organoids’ utility as a robust model for studying ovarian cancer in a physiologically relevant context (Figure 2C and D).

To assess the genetic stability of ovarian cancer organoids over the culture period, whole exome sequencing (WES) was performed, revealing the preservation of key ovarian cancer-related mutations, including CSMD3, TP53, and GABRA6. Comparative analysis between the organoids and their tissue of origin demonstrated a high correlation, affirming that ovarian cancer organoids maintain the genetic landscape of the parent tumors throughout the culture process (Figure 2E and F).

Our findings underscore the ovarian cancer organoids’ remarkable ability to preserve the complex cellular and molecular landscape of ovarian tumors, offering a promising platform for in-depth studies on tumor biology, drug testing, and personalized medicine approaches in ovarian cancer.

2.3 Preservation of tumor pathological phenotypes in ovarian cancer organoids

The criticality of maintaining not only cellular types but also their inherent organizational architecture to emulate biological function is paramount in our study. Our assessment of the organoids' pathological features elucidates this cellular organization and the expression profiles of pivotal biomarkers. Utilizing hematoxylin and eosin (H&E) staining, we established a clear parallelism in the tissue architecture between the organoids and their clinical counterparts (Figure 3A), affirming the organoids' capability to conserve the structural integrity observed in vivo. Additionally, the nuclear-to-cytoplasmic (N/C) ratio, a metric indicative of malignancy, was closely studied. The comparative N/C ratios, 1.056 in tissue versus 0.901 in organoids for one patient pair and 0.524 versus 0.578 for another, demonstrate the organoids' proficiency in mirroring the cytological features pertinent to cancer assessment (Figure 3B).

In the clinical setting, Ki67 serves as a prognostic marker for ovarian cancer, reflecting cellular proliferation rates, while PAX8 identifies the gynecological origin of the malignancy, correlating with patient outcomes. Our meticulous immunohistochemical (IHC) evaluation demonstrated analogous expression and localization patterns for Ki67 and PAX8 across organoids and tissue samples (Figure 3C). Despite some quantitative differences—38.8–42.1% for Ki67 and 41.2–51.3% for PAX8 in organoids versus 70.5–72.2% and 56.5–60.1% in tissues, respectively (Figure 3D)—these variations provide insights into the microenvironmental adaptation within the organoid culture.

Collectively, these findings substantiate the organoids' utility in preserving and reflecting the pathological nuances of ovarian cancer, showcasing their potential as an instrumental model for the pathological study and therapeutic assessment of this disease. Through these organoid models, we can achieve a more nuanced understanding of ovarian cancer's pathology, facilitating advanced research and personalized treatment approaches.

2.4 Enhanced cisplatin sensitivity assessment through patient-derived ovarian cancer organoid models

Central to our investigation has been the application of patient-derived ovarian cancer organoids for evaluating cisplatin responsiveness, particularly in the context of patients previously treated with carboplatin and paclitaxel. To establish a rapid screening method for drug sensitivity, we assessed the impact of cisplatin on ovarian cancer organoids. Notably, after a 3-day exposure to 16.7 µM of cisplatin, the treated organoids exhibited a marked structural collapse compared to the untreated control group, suggesting significant sensitivity to cisplatin at this concentration (Figure 4A). Further elucidation of cisplatin's effect was obtained through flow cytometry analysis of cell apoptosis. This revealed a substantial increase in the fraction of early-stage apoptotic cells (PI-/Annexin V+) posttreatment, with the extent of early-stage apoptosis amplifying in response to higher cisplatin concentrations. Conversely, the proportion of cells in late-stage apoptosis remained unchanged, underscoring cisplatin's mechanism of action through the induction of early apoptosis (Figures 4B and C). Corroborating this, caspase-3 levels, indicative of early-stage apoptosis, were significantly elevated in cisplatin-treated cells (Figure 4D). This dose-dependent effect highlights the ineffectiveness of low cisplatin concentrations in suppressing ovarian cancer organoids’ activity, emphasizing the drug's cytotoxic efficacy at higher doses. Indeed, different concentrations of cisplatin have different inhibitory effects on organoids (Figure 4E). Immunocytochemistry (ICC) analysis further demonstrated a diminished proliferation capacity within the organoids following cisplatin treatment, as evidenced by a significant reduction in Ki67 expression. In contrast, PAX8 expression remained unaffected, indicating the specificity of cisplatin's action on proliferative markers (Figure 4F).

Collectively, our findings affirm that cisplatin significantly curtails the proliferative capabilities of ovarian cancer organoids, aligning with clinical observations and underscoring the utility of ovarian cancer organoids in devising personalized treatment plans. This study also reinforces the predictive value of organoids in fast-assessing drug sensitivity.

2.5 Deepened molecular understanding of cisplatin responsiveness through patient-derived ovarian cancer organoid models

In order to study the intricacies of cisplatin's impact on ovarian cancer at the molecular level, we conducted RNA-seq analysis on two sets of patient-derived ovarian cancer organoids subjected to cisplatin treatment. The analysis revealed significant alterations in the gene expression profiles under the influence of cisplatin, demonstrating the drug's pervasive action across various patient-derived models (Figure 5A). Among the top 1000 genes modulated by cisplatin treatment, there was a substantial overlap, with 339 genes upregulated and 346 genes downregulated in both sets of organoids (Figure 5B). This overlap highlights the consistent and broad-spectrum efficacy of cisplatin across different ovarian cancer backgrounds.

Further exploration of these changes through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses pinpointed specific clusters of genes that were differentially regulated by cisplatin treatment. Notably, there was an enhanced expression of genes associated with apoptosis in the cisplatin-treated organoids, in contrast to the control group, which showed elevated expression of genes involved in cell division (Figure 5C). This differential gene expression pattern aligns with our understanding that cisplatin exerts its anticancer effects primarily through the induction of apoptosis.

Particularly, the analysis shed light on the differential pathway activation between the control and cisplatin-treated groups (Figure 5D). Control organoids demonstrated heightened activity of genes related to cancer proteoglycans, indicating an active metabolic state conducive to cancer progression. Conversely, the cisplatin-treated organoids exhibited a marked upregulation of genes within the p53 signaling pathway, suggesting that cisplatin's mechanism of inhibiting cancer cell proliferation is mediated through apoptosis, triggered by the activation of the p53 pathway. This observation is in harmony with previous study,^{34, 35} underscoring the pivotal role of the p53 pathway in cisplatin-induced apoptosis mechanisms. Moreover, our RNA-seq analysis unveiled a notable reduction in the expression of genes crucial for cell proliferation and tumor genesis, such as Ki67 and BRCA1/2, following cisplatin administration. This finding substantiates the drug's efficacy in curtailing oncogenic processes within the organoids.

In summary, notwithstanding the interpatient heterogeneity, the organoid models consistently recapitulated the clinical efficacy of cisplatin, corroborating the notion that these organoids retain patient-specific biological features and substantiating their value in drug sensitivity testing. This convergence of clinical and organoid-based responses to cisplatin underscores the potential of organoid models in facilitating personalized oncology, particularly in predicting and understanding patient-specific responses to chemotherapy.

4.1 Patient information

Ovarian cancer tissues were collected from 7 individual patients (OV004(61 years old, HGSOC), OV014(68 years old, HGSOC), OV015(52 years old, HGSOC), OV016(69 years old, HGSOC), OV076(59 years old, Right-sided fallopian tube mass), OV077(96 years old, unknown) and OV078(67 years old, unknown)) with ovarian cancer at Obstetrics and Gynecology Hospital of Fudan University in 2022, after obtaining informed consent of the patient before surgery. OV004 was used for scRNA-seq. OV014, OV015, and OV016 were used for living microscopy observe and subsequent experiments. OV014 and OV015 were used for cisplatin treatment, ICC staining and RNA-seq. OV076, OV077, and OV078 were utilized for live microscopic observation. These patients underwent a laparotomy primary debulking surgery with optimal cytoreduction. Our study was approved by the Ethics Committee of the Obstetrics and Gynecology Hospital of Fudan University.

4.2 Organoids formation, culture, and subculture

Patient-derived OC tissues were washed 4−6 times with organoid washing buffer (LSTO00100201, Shanghai Lisheng Biotech, China) and then dissociated to about 1 mm diameter pieces with surgical scissors, no collagenase, trypsin or dispase needed. Tumor pieces were resuspended ovarian cancer organoid medium (LSTO00100501, Shanghai Lisheng Biotech, China) and transferred into new dishes. The ovarian organoids were maintained with at 37°C, 5% CO₂, and medium was semichanged per week. When the diameter of organoids reached 2 to 4 mm (about 4 weeks), the organoids were collected and mechanically dissociated, then subcultured as described above.

4.3 Whole exome sequencing

For each sample, 200 ng genomic DNA was sheared to 150−200 bp fragments to construct libraries. The whole exome was captured using AIExome® Human Exome Panel V3 with TargetSeq One® Hyb & Wash Kit v2.0 (iGeneTech Co., Ltd, Beijing, China) and sequenced on DNBSEQ-T7 with 150-bp reads.

4.4 Immunofluorescence and immunohistochemistry

Tissue or organoids were first fixed with 4% PFA (BL539A, Biosharp, China), then dehydrated with 10%, 20% and 30% sucrose solution sequentially. Embedding medium with samples (7.5% gelatin and 10% sucrose in 1× PBS) was cooled at 4°C for 10 min and quickly frozen on dry ice for 10 min, then stored at −80°C. Materials were sectioned using Leica CM 1950. On-plate adherent cells were fixed with 4% PFA for 20 min at room temperature. Permeabilization of cells and sections was performed with PBS+0.25% TritonX-100 for 18 min at room temperature, followed by blocking with 3% BSA.

For immunofluorescence, rabbit anti-Ki67 (1:1000; MA5-14520, ThermoFisher, England) and anti-PAX8 (1:1000; 10336-1-AP, Proteintech, China) were used as primary antibodies to stain proteins overnight at 4°C. Samples were then washed 5 times with 0.125% PBST and incubated with the CY3-anti-rabbit secondary antibody (1:1000) for 3 h at room temperature. Nuclei were stained by DAPI (1:1000).

For immunohistochemistry, mouse anti-CD31 (1:250; ab9498, Abcam, USA) were used as primary antibodies to stain CD31 positive cells. Samples were incubated overnight at 4°C then washed 5 times with 0.125% PBST and incubated with the HRP-anti-mouse secondary antibody (1:500; ab6789, Abcam, USA) for 3 h at room temperature. Recolor the slices using DAB substrate kit (ab64328, Abcam, USA).

4.5 RNA sequencing

For bulk RNA, RNA Isolation Kit (DP451, TIANGEN, China) was used to isolate cell mRNA. Four to six 2-mm-diameter organoids were collected as input for each group of organoids. Concentration of RNA was measured using Qubit4 fluorometer. Reverse transcription was performed with RT Kit (KR118, TIANGEN, China) and libraries were generated with RNA Library Prep Kit (E7530L, NEB, USA).

For single cell RNA, libraries were generated with 10× genomics protocol using 15,000 cells as input. Both libraries were sequenced on the Illumina Hiseq 4000 with PE-150 bp reads for subsequent analysis.

4.6 Bioinformatics analysis

For RNA-seq, the kallisto software was utilized to quantify transcript abundance, employing Fragments Per Kilobase of transcript per Million mapped reads to estimate RNA expression levels. Differentially expressed genes were identified using the Morpheus online platform. In the case of scRNA-seq, the raw data were processed using Cell Ranger, with subsequent analyses conducted via the Seurat package. For WES, the BWA-MEM algorithm facilitated alignment of raw sequence data to the reference human genome (hg38). Downstream analyses, including variant calling, were performed using the Genome Analysis Toolkit and Mutect2.

4.7 Cisplatin sensitivity test

Organoids were cultured in 6-well plates as mentioned above, and cisplatin was added into medium at 8/48 h after the initial time point at final concentrations of 4.175, 8.35, and 16.7 µM. At 168 h, the organoids were collected and sectioned as described above.

4.8 Microscopy observing

All bright-field images were taken using Leica Ti-1 microscope. Fluorescence images were taken using Leica K5 microscope.

4.9 Caspase3 activity assay

Organoids cultured with protocols described above were dissociated with 0.25% trypsin and then cultured in 96-well ultralow attachment plates at 10,000 to 20,000 cells per well. Cisplatin was added to concentration of 16.7 µM, and caspase3 activity assay was performed using Promega G8091 kit after 3 days treatment.

4.10 CTG assay

Organoids were cultured in 6-well plates as mentioned above, and cisplatin was added into medium at the beginning of culture. At 0, 12, 24, and 36 h respectively, same amount of organoids were collected and performed CTG assay with CellTiter-Glo Luminescent Cell Viability Assay Kit (G7572, Promega).

4.11 Cell cytometry

Organoids were dissociated with collagenase NB 4 in 0.25% trypsin, then collect 1 × 10⁶ cells in 1.5 mL EP tube and centrifuge at 250 × g for 3 min. Cells were suspended in 1× PBS and stained with PI (0.01 mg/mL) and annexin V (1:100) for 15 min. Cells were then centrifuged at 250 × g for 3 min and collected. The cell flow cytometry was performed with a BD FACS Verse kit (Franklin Lakes, NJ, USA) following standard procedures.

4.12 Statistical analysis

In ICC assay and qPCR, data analysis was performed using Prism software. p Value < 0.05 was considered statistically significant. All data are expressed as mean ± standard deviation.