Research scientists and experimental medicine experts from Fraunhofer Institute for Toxicology and Experimental Medicine in Regensburg and affiliates have sought to unpick some of the confounding traits of cells that lead to the development of metastatic breast cancer in bone/bone marrow.
Studying the molecular mechanisms driving dissemination of breast cancer cells to the bone marrow and the pre-requisites for their survival, genomic changes and proliferation in the distant niche. Importantly they show that disseminated cancer cells (DCC) are initially driven to a dedifferentiated lineage, and maintained by IL-6 trans-signalling that supported the theory of involvement of a micro-environment. Later, for full development of metastatic potential the lineage must become IL-6 independent (and thereby evade micro-environment control), apparently by accumulation of mutations leading to autonomous activation of the PI3K pathway.
The authors suggest that this more detailed understanding of the progression of DCC may offer new opportunities for therapeutic intervention.
Along with the detailed single cell transcriptomics and genomics, assays to mimic the microniches in the bone marrow there was significant utilisation of 3D cell culture (in this case, mammospheres) to assess the clonal potential of different phenotypes and stimuli.
For the latter mammosphere numbers were counted automatically on the Perkin Elmer Opera Phenix platform by labeling the spheres with CyTRAK Orange™ - orange fluorescent cell permeant DNA and cytoplasm dye. Staining of spheres was achieved in a facile manner by the addition of the cell permeant probe at a final concentration of 10 µM for 1 hour, prior to analysis. Spheres were only counted when above 50 µm diameter.
This successful demonstration of the utility of CyTRAK Orange™ for this application, follows earlier anecdotal reports on other 3D microtissues (colon, kidney) using identical staining conditions, with addition of a subsequent formaldehyde fixation, which remain unpublished due to unconnected issues with the projects. CyTRAK Orange™, therefore may have broad utility for the end-point labeling of whole in vitro-derived microtissues and spheroids.
Reference:
Werner-Klein, M., Grujovic, A., Irlbeck, C. et al. Interleukin-6 trans-signaling is a candidate mechanism to drive progression of human DCCs during clinical latency. Nat Commun 11, 4977 (2020). https://doi.org/10.1038/s41467-020-18701-4
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