Imaging Hypoxia in TNBC 3D Microtissues

3D microtissues are now well-established as an improved in vitro recapitulation of tumor and the likely response or resistance to therapeutic interventions.  To that end, a lab at the University of Saskatchewan, Saskatoon, has developed a model micro-tissue platform for triple-negative breast cancer cell lines (TNBC) and having the feature that these microtissues are free-floating in a collagen-based hydrogel as a defined extracellular matrix and to perhaps better establish and in vitro tumor microenvironment.

In one measure of the changes over time during growth of microtissues they were monitored longitudinally for emergence of hypoxic regions from day 7 to 21.  This was achieved non-destructively using HypoxiTRAK™, simply added to culture medium and providing a direct fluorescence readout throughout the time-course.  As expected, these microtissues did show increases in hypoxic microenvironments over the measured period.

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Reference:

Daneshvar BN, Bosso M, Greene R, Dzikowski T, Johannson M, Gagnon A, Chamberlain MD.  A novel in vitro 3D cancer model based on modular tissue engineering approach. bioRxiv preprint, March 1 2025.  DOI:/10.1101/2025.02.25.64023

Screening Concept Aims to Aid Diagnosis for VUS

Fascinating new research headed by a team in Utrecht has applied the strategies now aiding drug discovery to inform and direct clinical investigations of variants of unknown significance (VUS) that are a product of the genetic revolution in medical diagnosis.

With the emergence of VUS that result from their detection by DNA sequencing but which may nonetheless confound a diagnosis or, for example, genetic counselling it has been, thus far, impractical and uneconomical to consider the enormous battalion of tests that might be required to provide a definitive diagnosis, with no certainty of an effective outcome.

They authors of this new work demonstrate that by creating a screening approach they can provide a morphological- and organelle-based feature set for cluster analysis to better direct the clinical investigations for patients with VUS.

They chose the Imagestream imaging flow cytometer to meet statistical sampling demands and to collect image information, from both the assay-designed and inherent image parameters.

Imaging Flow Cytometry and DRAQ5 have long been combined as a solid foundation for assays (500+ co-citations) and this work is no different.  The authors chose DRAQ5 as the DNA counterstain here and it shows its many capabilities: for the fixed cell assays, for the live cell assays, to confirm cytoplasmic-to-nucleus translocations, to show nuclear morphology (and changes thereof) and nuclear staining intensity (i.e. DNA content as an indicator of altered proliferation) compared to controls.

Patient -derived fibroblasts were chosen due to the accessibility of samples via skin biopsies and also given that fibroblasts have a large cytoplasm.

They chose six assays that reflected changes in important organelles and pathways that would allow tractability to downstream investigations. From these they were able to correlate the VUS of well-known genes with abberancies in the relevant positive controls while in those cases where there was a gene of uncertain significance, differences in one or more of the assay readouts gave direction for onwards investigation. 

What also transpired was that a broader, untargeted approach combining all the ca. 300 features that could be extracted from each assay, including those of the nuclear and brightfield images, proved able to separate samples into defined clusters, despite the susceptibility identified being non-tractable directly from any of the 6 assays - akin to phenotypic screening "hits" in drug discovery, and perhaps specifically the "cell painting" assay. These diagnostic "hits" were then confirmed by orthogonal analysis, for example increased intensity of a nucleus correlating to de novo DNA synthesis in the S phase of the cell cycle and therefore indicating different cell proliferation compared to healthy controls, shown by simple DNA histograms.  These correlated to a common genetic variant.

This work may signal a route to a screening-type strategy as a powerful yet relatively low-cost intermediate gateway to aid the conclusion of a detailed diagnosis with affected pathways that can assist clinicians and patients uncover the landscape of the VUS in question.

Reference:

Muffels et al. Imaging flow cytometry‑based cellular screening elucidates pathophysiology in individuals with Variants of Uncertain Significance.  Genome Medicine (2025) 17:12

DOI:10.1186/s13073-025-01433-9

Powerful Spheroid Imaging Methodology using DRAQ5

This new work builds on earlier studies to determine a best methodology to clear and image tumour spheroids (Nürnberg et al. 2020) previously reviewed here.  This showed the performance advantage of a low-cost glycerol-based RI correction and nucleus counterstaining with DRAQ5.  The authors, from the laboratory of Rüdiger Rudolph (Mannheim Univ. of Applied Sciences, DE), described a full workflow from tumor spheroid seeding through treatment, and onto whole mount clearing, staining, immunofluorescence imaging and image analysis.

A significant part of this work was the deployment of a deep-learning-based segmentation tool which could give single cell information within the context of the complete 3D spheroid whole mount rather than the limited information available from a cryosection or a 2D optical section of a spheroid.  One fascinating finding was that it was possible to differentiate the tumor and stromal (fibroblast) cells on the basis of nuclear morphology alone which further simplifies the preparative steps and aids the interrogation of the interactions between two cell types in co-culture.

Pancreatic cell line mono-cultures and co-cultures with fibroblasts were exposed to different cytostatic treatments and show features consistent with previous findings on these drugs and the presence or absence of the co-cultured stromal cells.

The results show strong correlation between the whole mount procedure and cryosections.  Using their procedure, DRAQ5 maintained good penetration and staining of nuclei throughout the spheroids. Where can I buy DRAQ5?

The authors concede a limitation of the study in that these results are on single pancreatic cancer cell line but they signal their intention to test their workflow on patient material with the ultimate goal of informing personalized medicine.

References:

Vitacolonna, Mario, et al. "A spheroid whole mount drug testing pipeline with machine-learning based image analysis identifies cell-type specific differences in drug efficacy on a single-cell level." BMC cancer 24.1 (2024): 1542

Clear sense - optical clearing of microtissues, tissue slices. BioStatus Blog, June 2021.