# Metabolic profiling of therapy-induced senescent cancer cells via TPEF, MALDI-MS, and RNA-sequencing

## TL;DR

This study explores how cancer cells become senescent after treatment and identifies metabolic changes that could be targeted to prevent cancer recurrence.

## Contribution

The paper introduces a multi-modal approach combining TPEF, MALDI-MS, and RNA-sequencing to uncover metabolic traits of senescent cancer cells.

## Key findings

- Senescent cells show mitochondrial dysfunction and altered NAD(P)H/FAD distribution.
- Lipid remodeling involving cardiolipin precursors was consistently observed.
- Engulfing-senescent cells exhibit distinct gene expression related to lipid metabolism and communication.

## Abstract

Despite advances in cancer therapies, treatment failure from resistance and recurrence remains a major clinical challenge. Therapy-induced senescence (TIS), a state of stable cell cycle arrest with sustained metabolic activity, has emerged as a driver of inflammation, tumor persistence, and relapse. However, the heterogeneity of TIS complicates its detection and targeting. Here, we applied a multi-modal strategy to characterize metabolic alterations in senescent cancer cells induced by doxorubicin or γ-irradiation across three tumor cell lines: MCF7, HeLa, and TPC-1. Mitochondrial dysfunction was assessed using MitoTracker and JC-1 staining, while two-photon excitation fluorescence (TPEF) microscopy enabled label-free visualization of intracellular NAD(P)H and FAD distribution. Lipid remodeling was evaluated by MALDI mass spectrometry imaging, and RNA sequencing was performed on control, senescent, and engulfing-senescent MCF7 cells to identify differentially expressed genes and enriched pathways. Senescent cells displayed mitochondrial dysfunction, with altered NAD(P)H/FAD distribution and decreased membrane potential. TPEF confirmed redistribution of coenzymes, reflecting redox changes. Lipidomics revealed consistent remodeling, notably involving cardiolipin precursors. Transcriptomic profiling showed engulfing-senescent MCF7 cells possess a distinct signature marked by increased lipid metabolism, endocrine signaling, and cell–cell communication. Overall, our findings reveal conserved and cell type–specific metabolic traits of TIS, highlighting metabolic vulnerabilities for senolytic intervention.

The online version contains supplementary material available at 10.1038/s41598-025-32573-y.