Single cell-derived spheroids capture the self-renewing subpopulations of metastatic ovarian cancer

Abstract

High Grade Serous Ovarian cancer (HGSOC) is a major unmet need in oncology, due to its precocious dissemination and the lack of meaningful human models for the investigation of disease pathogenesis in a patient-specific manner. To overcome this roadblock, we present a new method to isolate and grow single cells directly from patients' metastatic ascites, establishing the conditions for propagating them as 3D cultures that we refer to as single cell-derived metastatic ovarian cancer spheroids (sMOCS). By single cell RNA sequencing (scRNAseq) we define the cellular composition of metastatic ascites and trace its propagation in 2D and 3D culture paradigms, finding that sMOCS retain and amplify key subpopulations from the original patients' samples and recapitulate features of the original metastasis that do not emerge from classical 2D culture, including retention of individual patients' specificities. By enabling the enrichment of uniquely informative cell subpopulations from HGSOC metastasis and the clonal interrogation of their diversity at the functional and molecular level, this method provides a powerful instrument for precision oncology in ovarian cancer.

Fig. 1. Generation of sMOCS from HGSOC ascites.

a Scheme illustrating the main steps of the method for generating single cell ovarian cancer spheroids (sMOCS) from HGSOC ascites. Ascitic fluid is centrifuged; the cell pellet is processed for the removal of red blood cells and dissociated as single cell suspension for monolayer culture of tumor cells (step 1); the remaining supernatant after the first centrifugation is processed in order to remove the residual fraction of cells and is used as supplement for growing and culturing sMOCS (step 2). Tumor cells from monolayer culture are dissociated, resuspended in specific culture media for growing single cell from HGSOC ascites and plated by limiting dilution at the density of 1 cell per well in a low-adhesion 96 well V bottom plate (step 3). sMOCS at first passage (P1) are observed after 8–12 days in culture as tridimensional structure of about 200–220 μm of diameter and then propagated through dissociation of a single spheroid in single cells. The single cells are resuspended in the described media and plated as previously indicated in order to obtain the next passages in culture (P2 and P3). The scheme illustrates that fresh ascites, monolayer culture of tumor cells, and sMOCS at different passages are then processed for scRNAseq to obtain their transcriptomic profile. b Bright field image analysis (x4magnification for 2D, scale bar 1000 μm; x40 magnification for sMOCS P1, scale bar 400 μm; x20 magnification for sMOCS P2 and P3, scale bar 200 μm).

Fig. 2. Metastatic ascitic fluid is required for cell proliferation and generation of sMOCS.

a Bright field images representative of the morphology of primary cultures derived from HGSOC ascites of different patients (x4 magnification for 2D monolayer culture of three different patients, scale bar 1000 μm). b Bright field image analysis (x4 magnification for patient 1, patient 2 and patient 3, scale bar 1000 mm) of a single cell from 3 different patients plated in STEM media only and the respective fluorescent signal for PKH67 + (green). c Bright field image analysis (x4 magnification for patient 1, scale bar 1000 μm; x20 magnification for patient 1, scale bar 200 μm) of a single cell from patient 1 cultured in STEM media supplemented with ascitic fluid and fluorescent signal for PKH67. d Time-lapse of a single well followed during cell division. Day 0: only a single cell is present in the well. Day 2: first mitotic division. Day 4: the small spheroid is formed. Day 5: the small spheroids divide in single cells. Day 6: all the cells derived from the original spheroid start to divide. Day 10: multiple monoclonal spheroids are grown.

Fig. 3. SMOCS forming efficiency increase during passages in vitro.

a Scheme illustrating the main steps of the culturing method to derive sMOCS with a dedicated timeline indicating the number of days required in culture for each stage. b Bright field image of representative images of sMOCS from three different patients at three different passages: P1, P2, and P3, (x20 magnification for sMOCS passage 1(P1) for patient 1, 2, and 3; for sMOCS P2 from patient 2 and 3; for sMOCS P3 from patient 3, scale bar 200 μm); (x40 magnification for sMOCS passage 2 from patient 2 and for sMOCS P3 from patient 1 and 2, scale bar 400 μm). c Scatter plot showing the sMOCS forming efficiency (sSFE) as percentage for each patient and for each passage. Primary cultures derived from HGSOC ascites were grown under non-adherent conditions in 96 well plate V bottom in presence of the media supplemented with ascitic fluid to test their ability to generate monoclonal spheroids. The experiment was performed on 4 independent samples. sSFE was calculated as the ratio between number of monoclonal derived spheroids and the number of cells seeded. d Graph showing single cell spheroid forming efficiency (sSFE) (mean + SEM) of sMOCS from different patients at passage 1, 2, and 3. P1 n = 4, P2 n = 4, P3 n = 4. Unpaired t test, *p < 0.05; **p < 0,01; ***p < 0.001.

Fig. 4. single cell RNAseq reveals enrichment of CICs in sMOCS.

a Number of cells for samples and patients analyzed after filtering and quality control. b UMAP of single cell transcriptomes from cells in (a), where each cell is represented by a point; each color tone identifies different conditions among the same patient, yellow tones: fresh ascites; turquoise tones: 2D culture; purple tones: sMOCS;. Top right: the magnification of the central area of the UMAP is enriched mainly for sMOCS cells but also for some fresh ascites and 2D cultured cells. c Diffusion map of single cell transcriptomes from cells in (a), where each cell is a point; yellow tones: fresh ascites; turquoise tones: 2D culture; purple tones: sMOCS. Bottom right: magnification of the region where all the samples from different patients and conditions converge, enriched in sMOCS cells. d–g Enrichment analysis of Cancer stem and EMT signature. Left: diffusion maps of all the conditions and patients with a color scale defined by the z score of the respective signature; right: frequency plot showing the variation in the distribution of cells as a function of the z score of the indicated signature. The higher the percentage of cells with high z score, the more enriched the sample is for the indicated signature.

Fig. 5. sMOCS capture relevant features of the fresh tissue while preserving hallmarks of HGSOC ascites that do not emerge from 2D culture.

a Pathway analysis on the groups of genes and 2D culture; (b) pathway analysis on the groups of genes that correlate among fresh ascites and monoclonal 3D system; (c) heatmap of differentially expressed genes between sMOCS and 2D culture; (d) pathway analysis performed on DEGs in (c): color scale define the predicted activation/deactivation of the pathway; orange identifies the pathways activated in sMOCS; (e) IPA causal network derived from DEGs in (c): color scale define the expression of downstream regulated genes and their associated function, the orange tone identifies the genes and functions activated in sMOCS.

Fig. 6. Drug treatment of sMOCS highlights inter- and intra-patient variability in response.

a, b Comparison of the response of sMOCS vs 2D cultures to drug treatment. sMOCS and 2D cultures were treated with carboplatin (untreated, 5 µM, 10 µM, 25 µM, 50 µM, 75 µM). Cell viability was measured with CellTiter-Glo^® 3D Cell Viability Assay (for sMOCS) and CellTiter-Glo^® Luminescent Cell Viability Assay (for 2D cultures). a Results obtained from three independent naive patient samples, shown as means + SD. P value was calculated by a two-tailed unpaired Student’s t test, and the difference among monoclonal-derived spheroids and 2D cultures resulted statistically significant: 0.0262. b Data obtained from cells derived from a patient sample after chemotherapy (post-chemotherapy), at relapse, shown as means + SD. P value was calculated as above: 0.0454. c, d Intrapatient heterogeneity of response to drug treatment using sMOCS. P2 sMOCS were dissociated and cells seeded at 500 cells/well, and then derived spheroids were treated with carboplatin (untreated, 50 µM, 100 µM, 150 µM, 200 µM) and measured for cell viability as described above. In (c), spheroids at P3 derived from six cancer initiating cells of patient 17AS27 were analysed, in (d) spheroids at P3 derived from five cancer initiating cells of patient 18AS18. P values were determined by a two-tailed unpaired Student’s t test. In (c), the following pairs showed a significant P value: #1 vs #2: 0.0152, #2 vs #4: 0.0304. Comparison of the response of sMOCS vs 2D cultures and intrapatient heterogeneity of response to drug treatment. a After 7 days, cell viability was measured.Response variability shown as percentage error between sMOCS/2D cells derived from 6 patients. b, c Spheroids were treated with carboplatin (untreated, 50 µM, 100 µM, 150 µM, 200 µM) and measured for cell viability. In (b), spheroids derived from six individual cell clones of patient 17As27 were analysed, in (c) spheroids derived from five individual cell clones of patient 18As18.