Single-cell imaging to completely define the micro-metabolic state of the tumor

Many large pharmaceutical companies have been able to deploy SARS-CoV-2 vaccines in record time by re-equipping their experimental gene therapies with existing mRNAs to carry a new payload. While these cancer vaccines originally encoded a single RNA-molecule neoantigen concatamer that could be optimally presented on specific HLA receptors to enhance anti-tumor responses of CD8 + T cells, they can now provide Optimized versions of the advanced protein receptor binding domain to elicit the production of protective neutralizing antibodies.

But things did not end here. These same companies have also worked hard to reorient their drastically streamlined drug discovery pipelines to screen for small molecule inhibitors that could block SARS infection, life cycle or inflammatory sequelae in record time. . As the winners of vaccine battles and treatment challenges begin to emerge, an inspired industry is now reigniting the war on cancer with renewed vigor and purpose.

On Monday, we reported on new technologies, like Mission Bio’s Tapestri platform, that simultaneously assess protein biomarkers and genetic defects in individual cells throughout the cancer lifecycle. While this type of information is essential for adjusting therapies as new resistant clones appear in heterogeneous tumors, there is more to tell. The genetic profile of a cell indicates what it is able to do, i.e. what substrates he could potentially use and what substrates they absolutely need – but not so much on what the cells are Actually do at any time. In other words, it indicates how quickly they metabolize substrates – at the highest level, it would be either through glycolysis, aerobic glycolysis, or mitochondrial oxidative phosphorylation.

Imaging modalities such as positron emission tomography (PET 18F-FDG), computed tomography, and hyperpolarized magnetic resonance imaging (MRI) have been invaluable tools in distinguishing metabolic states in tumors, but they completely lack sufficient resolution to study tumor heterogeneity. cellular level. For this, researchers need cell culture and in vivo models where individually labeled cells can be imaged. In a recent article by Cell reports, researchers have developed a technique that does just that.

Glycolysis in tumors is frequently upregulated, leading to increased biosynthesis of metabolic intermediates necessary for cell proliferation. Cells can achieve this through upregulation of phosphatidylinositol 3-kinase (PI3K) signaling, either by acquisition of activating mutations, by inactivating mutations in negative regulatory phosphatase PTEN, or by increased receptor tyrosine kinase signaling. PI3K signaling works in part via AKT (protein kinase B) to enhance glycolytic flow, as well as via other mediators that ultimately control actin backbone and local membrane dynamics. In estrogen receptor positive (ER +) tumors, several PI3K inhibitors have now been approved as treatments, which could also be explored for more resistant triple-negative tumor subtypes.

The researchers used a combined approach of glucose fluorescence resonance energy transfer (FRET) imaging, metabolomics, detection of reactive oxygen species, and potential fluorescence imaging of the mitochondrial membrane (with TMRE tag) to define tumor heterogeneity at the single cell level. They also used flow cytometry to isolate high and low glucose cells for culture for more specific analysis. MCF-7 breast cancer cells were grown in different types of media to reflect different tumor environments, and then cultures were implanted into the mammary fat pad of mice. Implantation of tumor cell lines into the species and tissue of origin (called orthotopic models) can preserve normal immune interactions.

Alternatively, patient-derived xenografts (PDX) can also be implanted into immunosuppressed mouse models to assess human cancers within a defined setting. Traditionally, cancer cells have been implanted subcutaneously for practical study in mice. However, these tumors ulcerated prematurely, terminating the study prematurely. New test protocols like subQperior, in which cells are injected directly into the breast fat pad stroma, have emerged as workhorse tumor models for cancers of all kinds.

The researchers found that the tumors were interspersed with distinct regions of glucose-rich and low-glucose cells that maintained their condition for more than 10 hours. These metabolic states were found to be inherited to daughter cells during mitosis, and even neighboring cells could be radically different. The researchers suspected that the basis of the inherited metabolic state might be epigenetic regulation. By testing several inhibitors, they found that while PI3K inhibition destroyed many cells, better results were obtained when certain proteins containing “bromodomain” were simultaneously inhibited. Bromodomains are 110 amino acid motifs that recognize epigenetic acetylation marks on the N-terminal tails of histone proteins. When the lysine acetylation bromodomain “drives” are imbalanced, cells may be refractory to PI3K inhibition. At least 43 bromodomain proteins, many histone acetyltransferases, have been identified to date.

Researchers have observed that cancer cells in different states have different vulnerabilities. They found that glucose-rich cells are particularly dependent on extracellular pyruvate, but that this vulnerability is masked in the presence of low-glucose cancer cells or stromal fibroblasts. One of the regulators of pyruvate dehydrogenase turns out to be Akt kinase, suggesting additional ways to potentially treat these cancers. The combination of single-cell genetic multi-ohm predictors and protein markers with single-cell metabolics in the field should ultimately provide the comprehensive information needed to defeat any cancer.

© Science X Network 2021