Cancer is a rapidly evolving, multifactorial disease that accumulates numerous genetic and epigenetic alterations. This results in molecular and phenotypic heterogeneity within the tumor, the ...complexity of which is further amplified through specific interactions between cancer cells. We aimed to dissect the molecular mechanisms underlying the cooperation between different clones.
We produced clonal cell lines derived from the MDA-MB-231 breast cancer cell line, using the UbC-StarTrack system, which allowed tracking of multiple clones by color: GFP C3, mKO E10 and Sapphire D7. Characterization of these clones was performed by growth rate, cell metabolic activity, wound healing, invasion assays and genetic and epigenetic arrays. Tumorigenicity was tested by orthotopic and intravenous injections. Clonal cooperation was evaluated by medium complementation, co-culture and co-injection assays.
Characterization of these clones in vitro revealed clear genetic and epigenetic differences that affected growth rate, cell metabolic activity, morphology and cytokine expression among cell lines. In vivo, all clonal cell lines were able to form tumors; however, injection of an equal mix of the different clones led to tumors with very few mKO E10 cells. Additionally, the mKO E10 clonal cell line showed a significant inability to form lung metastases. These results confirm that even in stable cell lines heterogeneity is present. In vitro, the complementation of growth medium with medium or exosomes from parental or clonal cell lines increased the growth rate of the other clones. Complementation assays, co-growth and co-injection of mKO E10 and GFP C3 clonal cell lines increased the efficiency of invasion and migration.
These findings support a model where interplay between clones confers aggressiveness, and which may allow identification of the factors involved in cellular communication that could play a role in clonal cooperation and thus represent new targets for preventing tumor progression.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Introduction: Cancer is a rapidly evolving, multifactorial disease that accumulates numerous genetic and epigenetic alterations. This results in phenotypic and molecular heterogeneity within ...the tumor, the complexity of which is further amplified through specific interactions between cancer cells and the tumor microenvironment. In this context, cancer may be perceived as an “ecomolecular” disease that involves cooperation between several neoplastic clones and their interactions with other cell types and secreted factors present in the microenvironment. Cancer is therefore analogous to complex ecosystems such as microbial consortia. We are currently aiming at dissecting the molecular mechanisms underlying the cooperation between different clones.
Methods: We produced clonal cell lines derived from the MDA-MB-231 breast cancer cell line, using UbC-StarTrack system, which allows tracking of multiple clones by color. The characteristics of each clone were determined by measuring migration, proliferation and metabolic activity. In vivo analysis by orthotopic breast injection and intravascular tail vein injection was performed to assess the tumorigenic capacity of these clones. We studied potential collaborations between clones by determining the influence of secreted factors on growth rate of different clones by medium complementation with supernatant or exosomes from different clones. In vivo, we used zebrafish as a model system to study the migration of individually or co-injected clones.
Results: Characterization of these clones in vitro revealed clear differences in proliferation, cell metabolic activity and morphology among them. In vivo, all the individually injected clones were able to form tumors but the growth rates differed among them. Injection of an equal mix of clones led to the formation of tumors where some clones displayed a growth or survival advantage. In vitro the complementation of growth medium with medium from other clone increased the proliferation rate of the other clones. Co-injection of clones in zebrafish increased the efficiency of migration.
Conclusions: These results confirm that even in stable cell lines heterogeneity is present. Malignant properties were enhanced when some clones were combined or treated with exosomes or medium from other clones. These results clearly support our hypothesis that tumor clones cooperate in cancer progression and that this cooperation is mediated by secreted factors. Finally, we are performing expression arrays of mRNA, microRNas and lncRNAs and determining epigenetic state by methylation arrays, in order to identify potential factors that are differentially expressed among clones and are therefore bona fide candidates for clonal cooperation promoting factors. It is anticipated that this knowledge will facilitate the design of new and more effective therapeutic approaches that are directed to the tumor ecosystem as a sum of different clones.
Citation Format: Ana Martín-Pardillos, Angeles Valls-Chiva, Eva Bejar Serrano, Roberto Piñeiro Cid, Pablo Hurtado Blanco, Angel Días-Lagares, María Magdalena Castro, Juan Antonio Juan Antonio Cámara Serrano, Santiago Ramon y Cajal. Clonal cooperation in cancer progression: A new paradigm in cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2183.
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