Mitochondria oscillate along a morphological continuum from fragmented individual units to hyperfused tubular networks. Their position at the junction of catabolic and anabolic metabolism couples this morphological plasticity, called mitochondrial dynamics, to larger cellular metabolic programs, which in turn implicate mitochondria in a number of disease states. In many cancers, fragmented mitochondria engage the cell with the biosynthetic capacity of aerobic glycolysis in service of proliferation and progression. Chemo-resistant cancers, however, favor remodeling dynamics that yield fused mitochondrial assemblies utilizing oxidative phosphorylation (OXPHOS) through the electron transport chain (ETC). In this study, expression of Mitofusin-2 (MFN-2), a GTPase protein mediator of mitochondrial fusion, was found to closely correlate to Jurkat leukemia cell survival post doxorubicin (DxR) assault. Moreover, this was accompanied by dramatically increased expression of OXPHOS respiratory complexes and ATP Synthase, as well as a commensurate escalation of state III respiration and respiratory control ratio (RCR). Importantly, CRISPR knockout of MFN-2 resulted in a considerable decrease of doxorubicin (DxR) median lethal dose compared to a treated wildtype control, suggesting an important role of mitochondrial fusion in chemotherapy sensitivity and acute resistance.

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Achieving high data quality in single-cell RNA-seq (scRNA seq) experiments has always been a significant challenge stemming from minute signal that can be detected in individual cells. Droplet-based scRNA-seq additionally suffers from ambient contamination, comprising nucleic acid materials released by dead cells into the loading buffer and co-encapsulated with real cells, which further washes out real biological signals. Here, we developed quantitative, ambient contamination-based metrics and an associated software package that can both evaluate current datasets and guide new experimental optimizations. We performed a series of experimental optimizations using the inDrops platform to address the mechanical and microfluidic cell encapsulation aspect of an scRNA-seq experiment, with a focus on minimizing ambient contamination. We report improvements that can be achieved via cell fixation, microfluidic loading, microfluidic dilution, and nuclei versus cell preparation; many of these parameters are inaccessible on commercial platforms. We provide insights into previously obscured factors that can affect scRNA-seq data quality and suggest mitigation strategies that can guide future experiments.

Our HTAN (Human Tumor Atlas Network) study presented in Chen et al. documented single-cell RNA-sequencing (scRNA-seq) data on 128 human colonic specimens for mapping out distinct paths for polyp to cancer transformations. What we have not detailed is our optimizations on the droplet-based scRNA-seq approach that generate high quality data from challenging tissues such as the colonic epithelium. In this work, we provide a systematic dissection of tissue processing and microfluidic encapsulation factors that affect data quality generated on colonic epithelial cells using an open droplet-based scRNA-seq platform (inDrops). We optimized our process based on these factors to significantly decrease dying cell bodies and ambient RNA from being co-encapsulated with true cells, which led to the development of the current scRNA-seq protocol used for human specimens. We have also developed pre-filtering data metrics to score data quality stemming from degree of ambient contamination. We envision that these experimental optimizations reduce the amount of introduced artifacts, leading to more rigorous biological interpretations. Citation Format: Deronisha L. Arceneaux, Zhengyi Chen, Joey Simmons, Bob Chen, Cody Heiser, Yanwen Xu. The effects of tissue processing and microfluidic factors on single-cell RNA-sequencing data quality in colorectal tissues [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B031.