Morphologic and molecular matching of patient-derived organoids to parent tumors
Thirty-eight patients with gynecological cancer were enrolled during 2020~2022. Tumors from 28 patients were successfully generated into organoids, including 15 with ovarian cancer, 11 with endometrial cancer, and 2 with cervical cancer. Among the ovarian cancer PDOs, four were high-grade serous carcinoma (HGSC), four were mucinous, three were clear cells, one was carcinosarcoma, and one was endometrioid (Table 1). The organoid culture success rate was 71.4%, with a higher success rate in mucinous and clear cell types than in HGSC.
Different pathologic types presented with different appearances (Figure 1A). HGSC organoids displayed papillary branching, with a glandular-like protruding contour. Endometrioid organoids showed a glandular pattern with smaller cells and more confluent surface. Mucinous cancer organoids showed vanished glandular architecture and a simple, non-stratified cell lining outside with mucin-like content. Clear cell cancer organoids had polyhedral, flattened cells with vacuolated cytoplasm.
As shown in Figure 1A, two HGSC organoids from different patients displayed different cell densities and arrangements, suggesting its interpatient heterogeneity. Interestingly, within a single patient, organoid sizes, cellular densities, and protruding contours were distinct; these morphologic differences thus also indicate intrapatient heterogeneity (Supplementary Figure S1).
With hematoxylin and eosin (H&E) staining, mucinous organoid cells were spheroid with the cell apical at inner side and basal at outer side. The organoid cells had a high nucleus/plasma ratio, and nuclear polymorphism. Few immune cells or fibroblasts were identified (Figure 1B). High-power field showed that organoid cells had vacuolated and secretions. We further assessed organoid biomolecular preservation with H&E and immunohistochemical staining of organoids, parent tumors, and Human Protein Atlas (proteinatlas.org) references.
The characteristics of organoids and parental tumors were compared. As shown in Figure 2, the organoids displayed a high nucleus/cytoplasm ratio, irregular nuclei, and prominent nucleoli, recapitulating all malignancy features. p53 immunostain showed null expression in HGSC PDOs (Figure 2A) and was matched to the parent tumors. In addition, the strong positive PAX8 and weak positive WT1 were compatible with parent tumors and the Human Protein Atlas (https://www.proteinatlas.org/). In primary mucinous ovarian cancer, the immunohistochemistry expression of PAX8 is usually low [15]. Mucinous carcinoma PDOs presented with diffuse CK7 positive, focal PAX8 positive, and CK20 negative, matching the parent tumors and reference data (Figure 2B). This cumulative evidence indicates the successful establishment of ovarian cancer-derived organoids, of which the morphology and molecular characteristics were consistent with parent tissues.
Heterogenous chemosensitivity profile of ovarian cancer organoids
We tested drug responses in 10 OV-PDOs using guideline-recommended chemotherapeutic agents, including paclitaxel, cisplatin, carboplatin, epirubicin, doxorubicin, gemcitabine, topotecan, and olaparib. The drug sensitivity analysis is presented in Figure 3 and estimated IC50 values are shown in Supplementary Figure S2. The areas under the dose–response curves for each drug were also calculated (Figure 4). Each PDO had a unique dose–response curve for each drug, indicating interpatient heterogeneity even within the same histology type. A lower area under the curve (AUC) indicates a more sensitive response, as demonstrated in Figure 4 (smaller circles indicate better drug choices).
The HGSC-4 OV-PDO was relatively sensitive to epirubicin and topotecan. The HGSC-6 OV-PDO was relatively sensitive to paclitaxel, gemcitabine, and topotecan. The HGSC-5 OV-PDO responded poorly to all drugs. For the clear cell ovarian cancer (CCC) OV-PDOs, three were more sensitive to topotecan and CCC-3 was more sensitive to gemcitabine. Between the two mucinous OV-PDOs, MC-5 was more sensitive to gemcitabine and topotecan. These results demonstrate an OV-PDOs-guided precision therapy approach.
Clinical relevance of organoid drug testing
The clinical courses of four patients, who included platinum-sensitive, resistant, and refractory profiles, were correlated with their OV-PDOs drug response results (Figure 5).
Patient HGSC-4 had stage IIIC platinum-sensitive HGSC and had undergone primary suboptimal debulking surgery (Figure 5A). After surgery, she received adjuvant chemotherapy with paclitaxel and carboplatin, based on guideline recommendations. The tumor markers decreased and the clinical image revealed partial tumor response after six cycles. Because of intolerable neurological toxicity, especially hand numbness, the regiment was changed to liposomal doxorubicin (Lipodox). After six cycles of Lipodox, she was tumor-free. There was no tumor recurrence during the following 18 months. In vitro drug testing revealed that this patient’s OV-PDOs were sensitive to taxanes, platinum, and anthracycline, which is compatible with her clinical course.
Patient EM-2 had platinum-resistant, stage IIB, dedifferentiated endometrioid ovarian cancer. She had a suboptimal debulking surgery following adjuvant chemotherapy, with six cycles of paclitaxel and carboplatin to achieve complete remission (Figure 5B). After four months, tumor recurrence at the presacral area was found. She had chemotherapy with three cycles of Lipodox, but the tumor progressed. She then underwent chemotherapy with gemcitabine and cisplatin, and a second, optimal debulking operation. There was no evidence of disease for 16 months (at the time of manuscript preparation). Her OV-PDOs, derived at the second debulking surgery, showed resistance to paclitaxel, cisplatin and Lipodox and relative sensitivity to gemcitabine and topotecan. Thus, topotecan may be a better drug choice in the event of future recurrence.
Patient HGSC-5 had platinum-refractory, stage IIIC HGSC. She received four cycles of neoadjuvant chemotherapy with paclitaxel and carboplatin, followed by optimal interval debulking (Figure 5C). Tumor progression developed after two further cycles of paclitaxel and carboplatin. Chemotherapy was then shifted to Lipodox. Tumor progression occurred again after three cycles of Lipodox, at which time gemcitabine was administered. The tumor still progressed after three cycles. The patient began palliative care and expired a few months later. Her OV-PDO drug tests revealed multiple drug resistances to these chemotherapeutic agents.
Patient CCC-2 had platinum-refractory, stage IIIC CCC. Metastatic para-aortic lymph node was found by the general surgeon, and she received three cycles of neoadjuvant treatment with bevacizumab, paclitaxel, and cisplatin at the gynecologic department (Figure 5D). Because of proteinuria and nephrotoxicity, she had paclitaxel and carboplatin for two more cycles. Imaging showed a growing para-aortic tumor; therefore, she underwent interval suboptimal debulking surgery. She received adjuvant chemotherapy with gemcitabine and carboplatin. However, she was found to have jaundice before the next chemotherapy cycles, and imaging revealed rapid tumor growth. Family counseling led to the decision to take palliative care for the rest of her life, and she expired two weeks later. Her OV-PDOs, derived at the interval debulking surgery, presented relative resistance to paclitaxel, carboplatin, and gemcitabine.