Nucleo-mitochondrial asymmetry profiles the proliferative engine and spatial niche reconstruction in clear cell renal cell carcinoma.

BACKGROUND

Tumor heterogeneity is the key driver of disease progression and therapeutic resistance in clear cell renal cell carcinoma (ccRCC). Within this landscape, mitochondrial (MT) heterogeneity has emerged as a critical but poorly understood feature. This study identified a specific manifestation of MT heterogeneity termed "nucleo-mitochondrial expression asymmetry (NMA)". It is characterized by a dysregulated burst of mitochondrial DNA (mtDNA)-encoded genes compared to the nuclear genome, marking a pivotal tipping point in tumor proliferation and spatial reconstruction.

METHODS

We employed an integrative multi-omics approach combining single-cell RNA sequencing (scRNA-seq), spatial transcriptomics (stRNA-seq), and mass spectrometry imaging (MSI)-based spatial metabolomics from the Tongji Renal Cell Carcinoma (TJ-RCC) cohort. To identify and characterize the profound NMA malignant subpopulations, we utilized Gaussian Mixture Model (GMM) clustering, CytoTRACE 2 for differentiation potential, and scFEA for metabolic flux inference. We implemented neighborhood and pseudo-spatiotemporal map (pSM) analyses to quantify spatial reconstruction. We validated these findings through mitochondrially encoded cytochrome c oxidase I (MT-CO1) immunohistochemistry (IHC) in an independent cohort of 53 patients.

RESULTS

We identified a unique malignant subpopulation (C0) defined by NMA, where nucleo-mitochondrial coordination significantly decreased to R = 0.30 compared to R = 0.50 in other clusters. C0 functioned as a proliferative engine, exhibiting the highest ribosomal activity, peak differentiation potential, and concentrated G2M/S-phase activity. Metabolic modeling and MSI revealed that C0-dominant regions act as metabolic hubs, correlating with total metabolic flux (R = 0.631) and the physical accumulation of tricarboxylic acid (TCA) cycle intermediates. Spatially, C0 abundance was highly predictive of global MT gene scores (R = 0.852). As ccRCC progressed, NMA-driven niches underwent a dramatic reconstruction: transitioning from an "immune-active core" in the early stages to stroma-shielded "metabolic islands" in the advanced stages. Furthermore, we observed a resurgence of NMA and the C0 subpopulation in metastatic lesions. Clinical validation confirmed that MT-CO1 protein levels-a histological proxy for NMA-positively correlated with the proliferation marker Ki67 (r = 0.702) and served as an independent prognostic factor for overall survival.

CONCLUSION

This study characterized NMA as a hallmark of ccRCC progression and spatial niche reconstruction, offering a novel, clinically actionable framework for metabolic risk stratification via MT-CO1.