# Integrative SMR and single cell & spatial analysis reveals the spatial heterogeneity and prognostic value of CASP9-mediated apoptotic pathways in clear cell renal cell carcinoma

## TL;DR

This study shows that apoptosis, especially CASP9-driven pathways, plays a key role in the spatial organization and progression of clear cell kidney cancer.

## Contribution

The study introduces a novel integrative approach combining SMR, single-cell, and spatial analysis to reveal the spatial and prognostic role of CASP9 in ccRCC.

## Key findings

- Apoptosis-high tumor cells with elevated CASP9 cluster near macrophage-rich regions and show distinct spatial organization.
- A CASP9-associated five-gene signature accurately predicts patient survival in ccRCC.
- CASP9 is genetically linked to renal cancer risk and regulates tumor-macrophage interactions.

## Abstract

Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of renal cancer and remains a clinical challenge due to its frequent resistance to therapy and poor prognosis in advanced stages. Apoptosis, a fundamental tumor-suppressive mechanism, exhibits paradoxical roles in cancer, wherein apoptotic tumor cells can also contribute to immunosuppression and tumor progression. However, the spatial dynamics, transcriptional heterogeneity, and prognostic relevance of apoptosis-related gene programs in ccRCC remain poorly defined.

We performed an integrative analysis combining single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and summary-based Mendelian randomization (SMR) to dissect apoptosis-related malignant cell states in ccRCC. Cancer cells were stratified based on apoptosis gene signatures and CASP9 expression. Cell–cell communication was assessed using CellChat and spatial interaction networks were constructed using RCTD and mistyR. SMR was employed to link genetically regulated CASP9 expression with renal cancer risk. A CASP9-associated prognostic model was developed using LASSO Cox regression and DeepSurv on TCGA and E-MTAB-1980 cohorts.

We identified transcriptionally and spatially distinct apoptosis-high and apoptosis-low malignant cell subpopulations. Apoptosis-high tumor cells, characterized by elevated CASP9 expression, preferentially localized near macrophage-enriched stromal regions and exhibited stronger spatial clustering. Ligand–receptor modeling revealed directional signaling via the SPP1–CD44 axis between CASP9-high cancer cells and macrophages. SMR analysis provided genetic evidence supporting CASP9 as a causal gene for renal cancer. CASP9-high cells demonstrated distinct developmental trajectories and formed multicellular spatial modules with macrophages and cycling cells. A five-gene apoptosis-related signature derived from CASP9-stratified tumor cells robustly predicted patient survival across both training and validation cohorts. Low-risk patients exhibited enriched immune infiltration, increased immune checkpoint expression, and enhanced immune pathway activity.

Our study reveals that apoptosis, particularly CASP9-driven programs, defines a spatially organized, immunosuppressive malignant cell state in ccRCC. CASP9 acts as both a genetic driver and spatial regulator of tumor–macrophage interactions, contributing to disease progression. The CASP9-associated risk model demonstrates strong prognostic utility and highlights apoptosis as a promising therapeutic axis in ccRCC.

The online version contains supplementary material available at 10.1007/s12672-025-03798-0.