Unveiling functional heterogeneity in breast cancer multicellular tumor spheroids through single-cell RNA-seq

Abstract

Heterogeneity is an intrinsic characteristic of cancer. Even in isogenic tumors, cell populations exhibit differential cellular programs that overall supply malignancy and decrease treatment efficiency. In this study, we investigated the functional relationship among cell subtypes and how this interdependency can promote tumor development in a cancer cell line. To do so, we performed single-cell RNA-seq of MCF7 Multicellular Tumor Spheroids as a tumor model. Analysis of single-cell transcriptomes at twotime points of the spheroid growth, allowed us to dissect their functional relationship. As a result, three major robust cellular clusters, with a non-redundant complementary composition, were found. Meanwhile, one cluster promotes proliferation, others mainly activate mechanisms to invade other tissues and serve as a reservoir population conserved over time. our results provide evidence to see cancer as a systemic unit that has cell populations with task stratification with the ultimate goal of preserving the hallmarks in tumors. Cancer studies have established that tumors are complex and heterogeneous systems. These properties are grounded on genetic variations and diverse microenvironmental conditions that induce sizable differences in gene expression profiles, surface biomarkers, metabolism, growth rates, morphology, metastatic potential and response to therapy at a single cell level 1,2. From a clinical point of view, intratumoral (inside tumors) and intertumoral (between tumors) heterogeneity are critical factors that influence diagnosis outcomes and treatments in patients 1,3. Given their relevance, the understanding of tumor heterogeneity has emerged as a fundamental aim to improve treatment efficiency 4. To portray cancer intratumoral heterogeneity in human tissues, tumor microenvironment and their cellular population have been depicted in genome atlases for different cancer types 5-7. Notwithstanding the relevance of these atlases, big challenges into experimental designs come across to survey heterogeneity in human biopsies. For instance, proper cell dissociation methods must be implemented to reduce the risk of altering the transcriptional landscape 8. Additionally, it is hard to trace tumor dynamics due to invasive procedures and the inherent risk to patients 9. Interestingly, to overcome previous limitations, xenograft and organoid models have been used to emulate the temporal and three-dimensional organization of complex cell populations 10. On the other hand, intratumoral heterogeneity is an intricate property that influences even isogenic models providing complementary prosurvival functional roles, called functional heterogeneity 11,12. Functional heterogeneity is hard to be evaluated in the previously described models because of tumor complex interactions 9. To get the functional heterogeneity basics and design optimal treatments to overcome cancer, in vitro studies are the

MicroRNAs Regulate Metabolic Phenotypes During Multicellular Tumor Spheroids Progression

“…Together, these results suggest that the Q-MCTS can selectively enhance an invasive phenotype. As a whole, these observations mirror an orchestrated response to integrate the cancer phenotype, and it is in agreement with the fact that different tumor clonal subpopulations can diversify their task to maintain their growth and malignancy (14).…”

“…A major impediment in cancer treatment is the drug resistance due to the heterogeneous subpopulations of cells within the tumor with different cell-cycle phases (13,14). The cell cycle is the mechanism associated with proliferation, cellular division and DNA replication.…”

Recent Advances in Integrative Multi-Omics Research in Breast and Ovarian Cancer

“…Finally, merging single cell proteomics with other omics analysis has enable investigators to capture tumor-immune interactions in breast tumors [ 212 ]. Collectively, single-cell omics underscored intra- and inter-tumoral heterogeneity, identified subpopulation-specific vulnerabilities and emphasized the importance of addressing these vulnerabilities with combinatorial targeted therapeutic options [ 117 , 214 , 220 , 229 , 230 , 231 , 232 , 233 ].…”

In vitro Approaches to Model Breast Tumor Complexity

“…Growing BC cells in 3D has also revealed a more realistic drug response [21,37], cell proliferation and morphology [38], and better representation of tumor heterotypic phenotype and TME [39,40]. For example, single-cell RNA sequencing of breast cancer spheroids have uncovered cell clusters with specific functions (e.g., proliferation, invasion) that provide evidence of the heterotypic nature and complexity of breast tumors [41]. Figure 1 below depicts the main in vitro 2D and 3D culture modalities along with the most predominant co-culture models (discussed in the next subsection) to study cell crosstalk.…”