Single-cell high-dimensional imaging mass cytometry: one step beyond in oncology.

Solid tumors have a dynamic ecosystem in which malignant and non-malignant (endothelial, stromal, and immune) cell types constantly interact. Importantly, the abundance, localization, and functional orientation of each cell component within the tumor microenvironment vary significantly over time and in response to treatment. Such intratumoral heterogeneity influences the tumor course and its sensitivity to treatments. Recently, high-dimensional imaging mass cytometry (IMC) has been developed to explore the tumor ecosystem at the single-cell level. In the last years, several studies demonstrated that IMC is a powerful tool to decipher the tumor complexity. In this review, we summarize the potential of this technology and how it may be useful for cancer research (from preclinical to clinical studies).

A 31-plex panel for high-dimensional single-cell analysis of murine preclinical models of solid tumors by imaging mass cytometry.

Currently, the study of resistance mechanisms and disease progression in cancer relies on the capacity to analyze tumors as a complex ecosystem of healthy and malignant cells. Therefore, one of the current challenges is to decipher the intra-tumor heterogeneity and especially the spatial distribution and interactions of the different cellular actors within the tumor. Preclinical mouse models are widely used to extend our understanding of the tumor microenvironment (TME). Such models are becoming more sophisticated and allow investigating questions that cannot be addressed in clinical studies. Indeed, besides studying the tumor cell interactions within their environment, mouse models allow evaluating the efficacy of new drugs and delivery approaches, treatment posology, and toxicity. Spatially resolved analyses of the intra-tumor heterogeneity require global approaches to identify and localize a large number of different cell types. For this purpose, imaging mass cytometry (IMC) is a major asset in the field of human immuno-oncology. However, the paucity of validated IMC panels to study TME in pre-clinical mouse models remains a critical obstacle to translational or basic research in oncology. Here, we validated a panel of 31 markers for studying at the single-cell level the TME and the immune landscape for discovering/characterizing cells with complex phenotypes and the interactions shaping the tumor ecosystem in mouse models.

An IDH1-vitamin C crosstalk drives human erythroid development by inhibiting pro-oxidant mitochondrial metabolism.

The metabolic changes controlling the stepwise differentiation of hematopoietic stem and progenitor cells (HSPCs) to mature erythrocytes are poorly understood. Here, we show that HSPC development to an erythroid-committed proerythroblast results in augmented glutaminolysis, generating alpha-ketoglutarate (αKG) and driving mitochondrial oxidative phosphorylation (OXPHOS). However, sequential late-stage erythropoiesis is dependent on decreasing αKG-driven OXPHOS, and we find that isocitrate dehydrogenase 1 (IDH1) plays a central role in this process. IDH1 downregulation augments mitochondrial oxidation of αKG and inhibits reticulocyte generation. Furthermore, IDH1 knockdown results in the generation of multinucleated erythroblasts, a morphological abnormality characteristic of myelodysplastic syndrome and congenital dyserythropoietic anemia. We identify vitamin C homeostasis as a critical regulator of ineffective erythropoiesis; oxidized ascorbate increases mitochondrial superoxide and significantly exacerbates the abnormal erythroblast phenotype of IDH1-downregulated progenitors, whereas vitamin C, scavenging reactive oxygen species (ROS) and reprogramming mitochondrial metabolism, rescues erythropoiesis. Thus, an IDH1-vitamin C crosstalk controls terminal steps of human erythroid differentiation.

The WNT/ß-catenin pathway is a transcriptional regulator of CYP2E1, CYP1A2, and aryl hydrocarbon receptor gene expression in primary human hepatocytes.

The wingless-type MMTV integration site family (WNT)/ß-catenin/adenomatous polyposis coli (CTNNB1/APC) pathway has been identified as a regulator of drug-metabolizing enzymes in the rodent liver. Conversely, little is known about the role of this pathway in drug metabolism regulation in human liver. Primary human hepatocytes (PHHs), which are the most physiologically relevant culture system to study drug metabolism in vitro, were used to investigate this issue. This study assessed the link between cytochrome P450 expression and WNT/ß-catenin pathway activity in PHHs by modulating its activity with recombinant mouse Wnt3a (the canonical activator), inhibitors of glycogen synthase kinase 3ß, and small-interfering RNA to invalidate CTNNB1 or its repressor APC, used separately or in combination. We found that the WNT/ß-catenin pathway can be activated in PHHs, as assessed by universal ß-catenin target gene expression, leucine-rich repeat containing G protein-coupled receptor 5. Moreover, WNT/ß-catenin pathway activation induces the expression of CYP2E1, CYP1A2, and aryl hydrocarbon receptor, but not of CYP3A4, hepatocyte nuclear factor-4α, or pregnane X receptor (PXR) in PHHs. Specifically, we show for the first time that CYP2E1 is transcriptionally regulated by the WNT/ß-catenin pathway. Moreover, CYP2E1 induction was accompanied by an increase in its metabolic activity, as indicated by the increased production of 6-OH-chlorzoxazone and by glutathione depletion after incubation with high doses of acetaminophen. In conclusion, the WNT/ß-catenin pathway is functional in PHHs, and its induction in PHHs represents a powerful tool to evaluate the hepatotoxicity of drugs that are metabolized by CYP2E1.