Quantifying Autophagy by Flow Cytometry Revealed a Link to Leukemic Tumor Growth

Flow cytometry is a powerful technology for investigating many aspects of cell biology and for isolating cells of interest. Flow cytometry utilizes highly focused, extremely bright beams of light (usually from lasers) to directly reveal aspects of cells (e.g. size and granularity) by the way light is scattered, or indirectly by introducing fluorescent probes to cell compartments (e.g. through DNA binding dyes that stain nuclei or fluorescently-labeled antibodies that specifically detect cellular proteins). The power of flow cytometry derives from the fact that it quantitatively analyzes individual cells, thus permitting the identification of subpopulations within a sample. The power of single cell analysis is compounded by the ability to measure multiple parameters simultaneously on each individual cell, to do this very fast (in excess of 20,000 cells/second), and to isolate/purify/sort desired subpopulations (up to 4 simultaneously). One of the many uses of flow cytometry today is the analysis of autophagy by measuring autophagic flux and quantifying the content of autophagic vesicles in cells. Autophagy is a catabolic delivery pathway for excess or damaged cytoplasmic constituents to the lysosomes where macromolecules are broken down and their components freed for anabolic activities. Upon induction following metabolic stress, autophagy maintains mitochondrial health and metabolic pathways being induced following metabolic stress under the control of the mTOR complex 1 (mTORC1). Under favorable conditions, activated mTORC1 signals for cell growth, promotion of translation, cell cycle progression, and glycolysis while inhibiting autophagy. To maximize cell mass during proliferation, suppression of self-catabolism may be vital for growth activities and indeed, it was found that induction of autophagy prolongs cell survival at the cost of cell size and growth.

Activation of the Akt/mTOR pathway is a common feature of cancers, including leukemias and is required for proliferation in acute myeloid leukemia (AML) models. Knockout of autophagy genes in mice is associated with hyper-proliferation in some tissues and eventual tumor development. Previous studies indicated that mice without the autophagy gene Atg7 in the hematopoietic system develop pre-leukemic myeloproliferation. However, it remains unclear how Atg7 promotes cell proliferation and whether this is an Atg7-specific function. With the literature demonstrating both tumor-promoting and -inhibiting roles for autophagy in leukemia, its involvement in the biology of cancer cells is still controversial. It is, however, well accepted that transformation events leading to AML may occur at the stem or progenitor cell stage. Hematopoietic stem cells (HSCs) strike a fine balance between quiescence, self-renewal, and differentiation. When this balance is perturbed, the consequences may include biased differentiation and/or hematopoietic malignancies. In steady-state hematopoiesis, the majority of HSCs are quiescent. Quiescent cells are particularly hardy and able to survive long periods of metabolic stress. HSCs downregulate protein synthesis and activate pathways that sustain them during periods of non-division. Therefore, autophagy may be required for maintenance of the long-lived HSC, as their slow turnover prevents the dilution of damaged macromolecules to daughter cells, similar to a post-mitotic neuron or cardiomyocyte. Moreover, autophagy controls mitochondrial quality.

Using Enzo’s CYTO-ID® Autophagy detection kit and flow cytometry analysis, Dr. Watson and colleagues from John Radcliffe Hospital Oxford found that autophagy levels were highest in the most immature human and mouse hematopoietic stem and progenitor cells (HSPCs). They also demonstrated that loss of Atg5 results in an identical HSPC phenotype as loss of Atg7, confirming a general role for autophagy in HSPC regulation. Compared to more committed/mature hematopoietic cells, healthy human and mouse HSCs displayed enhanced basal autophagic flux, limiting mitochondrial damage and reactive oxygen species in this long-lived population. Moreover, human AML blasts typically only displaying heterozygous Atg deletions readily showed reduced expression of autophagy genes and displayed decreased autophagic flux with accumulation of unhealthy mitochondria. Also, heterozygous loss of autophagy in an MLL-ENL model of AML led to increased proliferation in vitro, a glycolytic shift, and more aggressive leukemias in vivo. Taken together these data are compatible with autophagy limiting leukemic transformation. With autophagy gene losses also identified in multiple other malignancies, these findings point to low autophagy providing a general advantage for tumor growth.

From our complex CELLESTIAL® portfolio of fluorescent probes and assay kits for cellular analysis to our large portfolio of antibodies validated in flow cytometry, Enzo Life Sciences provides a complete set of flow cytometry tools, some of which are described below.

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