Assaying 3D cell cultures in metastatic breast cancer

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(colon, kidney) using identical staining conditions, with addition of a subsequent formaldehyde fixation, which remain unpublished due to unconnected issues with the projects.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.

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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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Cancer Drug Screening in PDTOs - key parameters

A leading laboratory in 3D in vitro screening has recently published important findings in the establishment of patient-derived tumor organoids for therapeutic drug screening.

It is now widely acknowledged that 3D cell culture is better able to represent the in vivo environment than the historically-used 2D equivalent.  The initial steps into 3D have been based on spheroids, organoids and more recently, patient-derived tumor organoids (PDTOs) - which recapitulate features of the source tissue.  Thus, the latter represent improved physiological mimics and potentially a useful representation of the natural variation across both healthy control and patient material.  Being derived from patient material PDTOs may combine multiple cell types or generate these from undifferentiated tumor cells.  

Despite the advantages, these PDTOs present challenges in imaging and as a result of variability in size.  In addition, no determination has been made of the canonical features which discriminate drug effects on PDTOs.  However, the technological developments in imaging techniques and data analysis mean that it is possible to interrogate the complexity of PDTOs at the level of the whole structure and down to the granularity of the individual cells within.

In this context, the team led by Asst. Prof. Shannon Mumenthaler at USC designed a thorough examination of this. They generated PDTOs from cells isolated from colorectal cancer biopsies, cells which were transduced with H2B-GFP.  The resulting H2B-GFP⁺ PDTOs were cultured in the presence or absence of model anti-cancer drugs with different modes of action.  The PDTOs were monitored in a time lapse mode over 6/7 days in the presence of each drug and with the far-red fluorescing viability probe DRAQ7™.  A number of organoid-level morphological features and cell level features were monitored for their correlation given the a priori knowledge of the drugs tested.

In brief summary, this primary study identified that organoid volume, surface and live cell count  (GFP⁺ minus DRAQ7⁻ events) were the strongly correlated parameters amongst those tested.  Additionally, as organoids became very large the emergence of a necrotic core could be detected by DRAQ7™ staining.

The authors acknowledge that a more extensive study is needed but these findings indicate the key parameters were sentinel for drug responses, when tracked over time, as is possible with H2B-GFP expression and combined with DRAQ7™ as a real-time monitor of cell death.

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Reference & further reading

Kim, Seungil, et al. "Comparison of cell and Organoid-Level analysis of Patient-Derived 3D organoids to evaluate tumor cell growth dynamics and drug response." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25.7 (2020): 744-754

Real-time cell health monitoring in 2D/3D with DRAQ7™ - latest papers. BioStatus Blog June 22, 2018

Info & ordering on DRAQ7™