Purpose: The tumor microenvironment (TME) consists of a heterogenous cellular milieu that can influence cancer cell behavior. Its characteristics have an impact on treatments such as immunotherapy. These features can be revealed with single-cell RNA sequencing (scRNA-seq). We hypothesized that scRNA-seq analysis of gastric cancer together with paired normal tissue and peripheral blood mononuclear cells (PBMC) would identify critical elements of cellular deregulation not apparent with other approaches.Experimental Design: scRNA-seq was conducted on seven patients with gastric cancer and one patient with intestinal metaplasia. We sequenced 56,167 cells comprising gastric cancer (32,407 cells), paired normal tissue (18,657 cells), and PBMCs (5,103 cells). Protein expression was validated by multiplex immunofluorescence.Results: Tumor epithelium had copy number alterations, a distinct gene expression program from normal, with intratumor heterogeneity. Gastric cancer TME was significantly enriched for stromal cells, macrophages, dendritic cells (DC), and Tregs. TMEexclusive stromal cells expressed distinct extracellular matrix components than normal. Macrophages were transcriptionally heterogenous and did not conform to a binary M1/M2 paradigm. Tumor DCs had a unique gene expression program compared to PBMC DCs. TME-specific cytotoxic T cells were exhausted with two heterogenous subsets. Helper, cytotoxic T, Treg, and NK cells expressed multiple immune checkpoint or co-stimulatory molecules. Receptor-ligand analysis revealed TME-exclusive intercellular communication.Conclusions: Single-cell gene expression studies revealed widespread reprogramming across multiple cellular elements in the gastric cancer TME. Cellular remodeling was delineated by changes in cell numbers, transcriptional states, and intercellular interactions. This characterization facilitates understanding of tumor biology and enables identification of novel targets including for immunotherapy.

It has long been hypothesized that abnormalities in lipid biology contribute to degenerative brain diseases. Consistent with this, emerging epidemiologic evidence links lipid alterations with Parkinson disease (PD), and disruption of lipid metabolism has been found to predispose to α-synuclein toxicity. We therefore investigated whether Parkin, an E3 ubiquitin ligase found to be defective in patients with early onset PD, regulates systemic lipid metabolism. We perturbed lipid levels by exposing Parkin +/+ and Parkin -/-mice to a high-fat and -cholesterol diet (HFD). Parkin -/-mice resisted weight gain, steatohepatitis, and insulin resistance. Introduction PARK2 mutations associate with an autosomal recessive juvenileform of Parkinson disease (PD) (1). However, the role of Parkin in the development of PD in mice is surprisingly modest, as the genetic deletion of Parkin (2, 3) and the combined KO of Parkin with additional "candidate Parkinson susceptibility genes" PINK1 and DJ-1 do not substantially replicate the human condition (4). These unexpected findings suggest that the biological function of Parkin may not primarily modulate neurodegeneration but rather that PARK2 mutations increase biological susceptibility to stressors that manifest with substantia nigra neurodegeneration. Thus, the characterization of the functional actions of Parkin could be instrumental in delineating its role in the pathophysiology underpinning the development of PD.Parkin encodes an E3 ubiquitin protein ligase and contains 465 amino acids with multiple distinct domains, including a ubiquitinlike domain, a unique Parkin-specific domain, 2 RING domains, and an in-between-RING domain (5). The structure of this protein with its multiple binding domains is most likely central to the myriad of functions prescribed to Parkin (6). Additionally, the subcellular location of Parkin appears to be dynamic, with a predominant cytosolic localization with redistribution to the nucleus (7) and to the outer mitochondrial membrane (8, 9). Parkin exhibits mono- and multiubiquitination functions (10), and studies show that classical as well as nonclassical ubiquitin linkages facilitate proteosome-dependent and independent Parkin effects (11)(12)(13)(14).

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