Single-cell sequencing unveils key contributions of immune cell populations in cancer-associated adipose wasting

Cancer-associated cachexia (CAC) is a multifactorial syndrome characterized by depletion of skeletal muscle and adipose tissue, with reduced food intake, elevated energy expenditure, excess catabolism, and systemic inflammation. Although CAC can occur with or without fat mass loss, multiple lines of evidence suggest that fat loss may precede and functionally drive muscle loss. White adipose tissue (WAT) is composed of adipocytes, adipose stem and progenitor cells (ASPCs), endothelial and smooth muscle cells, and diverse immune cells that together shape the adipose microenvironment and influence metabolic disease. Depot-specific features—particularly differences between subcutaneous (SAT) and visceral (VAT) depots—are well recognized. Prior single-cell transcriptomic studies have illuminated adipose cellular complexity mainly in obesity, with limited work in human tissues and none, to date, focused on CAC. This study aims to generate a high-resolution single-cell atlas of human SAT and VAT from gastric cancer patients with and without cachexia to identify depot- and disease-specific cellular changes—especially among progenitors and immune cells—that may drive adipose inflammation and catabolism in CAC.

Evidence that adipose alterations can precede or drive muscle wasting in CAC includes: (1) Genetic inhibition of WAT lipolysis via ablation of PNP42 or LIPE in cachexia animal models ameliorated myocyte apoptosis and proteasomal degradation, preserving adipose and gastrocnemius muscle mass. (2) Tumor-derived parathyroid hormone-related protein (PTHrP) from Lewis lung carcinoma induces browning of white adipocytes, increasing energy expenditure; anti-PTHrP treatment inhibited adipose browning and prevented skeletal muscle loss in cachectic animals. (3) Clinical observations indicate WAT alterations may precede muscle wasting in some patients. Additionally, prior single-cell studies in adipose tissue (mostly in obesity) revealed complex progenitor states and immune cell phenotypes, as well as depot-specific differences between SAT and VAT, but comprehensive single-cell profiling in human CAC had been lacking.

- Cohort: SAT and VAT samples from 8 gastric cancer patients (4 non-cachexia, 4 cachexia), with cachexia defined by >5% body weight loss and systemic inflammation. - Single-cell RNA sequencing: Stromal vascular fraction (SVF) isolated from adipose depots; 10x Genomics-based scRNA-seq performed. A total of 42,119 cells were captured; 33,856 single cells passed quality control and were analyzed. - Computational analysis: Unsupervised clustering and UMAP visualization identified major cell populations. Marker genes were used for annotation, including CD34/PDGFRA/THY1 for adipose progenitor cells (APCs), CD3/CD26 for T cell subsets, GNLY/NCAM for NK cells, IL3RA/CLEC4C for plasmacytoid dendritic cells (pDCs), PECAM1/CDH5 for endothelial cells, and PLN/CASQ2/ACTA2 for smooth muscle cells. Comparisons were made between depots (SAT vs VAT) and disease status (cachexia vs non-cachexia). - Quantification: Cell type proportions across conditions were compared; statistical tests included unpaired Student’s t-tests as reported. - Additional functional studies (as reported in results/abstract): In vitro assays tested pro-catabolic effects of activated CD8+ T cells on adipocytes. In vivo macrophage depletion in a CAC animal model assessed effects on adipose catabolism and cachexia severity.

- Single-cell atlas: From 33,856 high-quality SVF cells, nine major cell populations were identified across SAT and VAT, including adipose progenitors, macrophage/monocyte, T cell subsets, NK cells, pDCs, endothelial cells, and smooth muscle cells. - Depot differences: VAT exhibited substantially higher immune cell infiltration than SAT (23.3%–52.1% in VAT vs 11.0%–20.6% in SAT). SAT contained more blood vessel cells (2.0%–18.3% in SAT vs 0.0%–0.17% in VAT), consistent with greater vascularization in SAT. - Disease vs non-disease: Overall distributions of major cell types did not differ significantly between cachexia and non-cachexia within the same depot, but VAT of CAC patients showed higher immune cell fractions in SVF. - Progenitor and immune cell states in CAC: Clear pro-inflammatory transitions were observed in adipose progenitors, macrophages, and CD8+ T cells in CAC tissues, with remodeled intercellular communication (interactome) among these populations, suggesting synergistic promotion of inflammation. - CD8+ T cells: Activated CD8+ T cells specifically contributed to increased IFNG expression in adipose tissue from cachexia patients and exhibited significant pro-catabolic effects on adipocytes in vitro. In VAT, CD8+ T cells acquired a pro-inflammatory state with activated cytolytic effector pathways in CAC. - Macrophages: Macrophage depletion in a CAC animal model significantly rescued adipose catabolism and alleviated cachexia, implicating macrophages as causal contributors to adipose wasting. - Clinical differences (Table 1): Cachexia patients had lower BMI (21.9 ± 2.1 vs 25.6 ± 1.9; P=0.03), lower lymphocyte counts (1.40 ± 0.05 vs 1.64 ± 0.04 ×10^7/μL; P<0.01), lower albumin (36.6 ± 3.9 vs 41.7 ± 4.5 g/L; P<0.01), and lower prealbumin (0.18 ± 0.01 vs 0.21 ± 0.01 g/L; P<0.01). Pro-inflammatory cytokines were elevated: IL-6 (9.8 ± 1.3 vs 4.2 ± 0.8 pg/mL; P<0.01) and TNF-α (11.2 ± 1.8 vs 5.9 ± 1.3 pg/mL; P<0.01). More advanced TNM stage was observed in cachexia (III+IV: 3 vs 0; P=0.03).

This study addresses how depot- and disease-specific cellular alterations in adipose tissue contribute to CAC. Single-cell profiling reveals that in CAC, adipose progenitors, macrophages, and CD8+ T cells shift toward pro-inflammatory states and exhibit enhanced intercellular signaling, indicating a coordinated inflammatory microenvironment that promotes adipose catabolism. VAT’s higher immune infiltration and the emergence of cytolytic, IFNG-producing CD8+ T cells in CAC suggest that visceral fat may be particularly susceptible to immune-mediated wasting. Functional evidence supports causality: activated CD8+ T cells drive adipocyte catabolism in vitro, and macrophage depletion mitigates adipose catabolism and cachexia in vivo. These findings link immune activation with adipose wasting, providing mechanistic insights into how adipose inflammation may drive or exacerbate systemic features of CAC and potentially precede muscle loss.

The work provides a comprehensive single-cell atlas of human SAT and VAT in gastric cancer patients with and without cachexia, uncovering depot- and disease-specific cellular programs. Key contributions include identification of pro-inflammatory transitions in adipose progenitors, macrophages, and CD8+ T cells; heightened immune infiltration in VAT; and functional evidence that CD8+ T cells and macrophages causally promote adipose catabolism in CAC. These insights highlight immune–stromal crosstalk as a driver of adipose wasting and suggest therapeutic avenues targeting macrophages, IFNG-producing CD8+ T cells, or their intercellular signaling to mitigate cachexia. Future work could validate these signatures in larger cohorts, map longitudinal trajectories during CAC progression, and dissect molecular mediators of the altered interactome to guide targeted immunomodulatory interventions.