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

Tania Velletri,Massimiliano Pagani,Raoul J P Bonnal,Pietro Lo Riso,Giuseppe Testa,Stefano Piccolo,Nicoletta Colombo,Domenica Cilli,Alejandro López-Tobón,Carlo Emanuele Villa,Luca Marelli,Michela Lupia,Marco De Simone,Saverio Minucci,Raffaele Luongo,Bianca Barzaghi,Ugo Cavallaro

doi:10.1038/s41418-021-00878-w

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.

Highlights

High Grade Serous Ovarian cancer (HGSOC) constitutes a major unmet need in oncology as one of the most lethal gynecological cancers, due to the failure of surgery and chemotherapy at eradicating the disease, the ensuing nearly invariable recurrence and very limited therapeutical progress over the past decades [1, 2]
We found that in such non-adherent conditions fically, the single cell-derived metastatic ovarian cancer spheroids no sMOCS are generated from single cells plated in medium alone platform here presented is endowed with several (Fig. 2b), while only the supplementation with cell-free patients’
We found that, across patients, ascitic fluid supplementation is strictly required to enable the growth of individual tumor cells into sMOCS and their propagation, pointing to the functional relevance of components shared among metastatic ascites samples

Summary

INTRODUCTION

High Grade Serous Ovarian cancer (HGSOC) constitutes a major unmet need in oncology as one of the most lethal gynecological cancers, due to the failure of surgery and chemotherapy at eradicating the disease, the ensuing nearly invariable recurrence and very limited therapeutical progress over the past decades [1, 2]. Cells from primary 2D heterogeneity of the original tumor, our approach is intended to cultures at the first in vitro passage were suspended in ovarian rapidly and reliably enrich for the core subpopulation of cancer stem cells medium with or without ascitic fluid (12.5%) and metastatic HGSOC propagating CICs, making such homoge- plated into 96 wells ultra-low attachment plates at the density of neously selected cell populations experimentally tractable. We used of the spheroid in a single well To this end, 2D primary cell cultures of patients with HGSOC composition of HGSOC ascites and trace its propagation in both (Fig. 3a) were labeled with PKH27 and sorted by flow cytometry. 2D primary cell cultures of patients with HGSOC composition of HGSOC ascites and trace its propagation in both (Fig. 3a) were labeled with PKH27 and sorted by flow cytometry This method establishes the feasibility of enriching with 12.5% of patients’ ascitic fluid. Again in the same conditions for a short-term propagation through passage 2 and passage 3 (P2 and P3, respectively)

RESULTS

DISCUSSION

Findings

MATERIALS AND METHODS