# A single-cell derived spheroid approach to dissect intratumoural heterogeneity in colorectal cancer: cell lines show changes in proteomes and therapeutic response to 5-FU

## TL;DR

This study uses single-cell spheroids and proteomics to explore tumor diversity in colorectal cancer and how it affects drug response.

## Contribution

A new experimental framework combining single-cell spheroids and proteomics to study intratumoural heterogeneity and drug response in CRC.

## Key findings

- Spheroids showed significant heterogeneity in morphology, growth, viability, and proteomic signatures.
- ITH-associated proteins and pathway variations were identified, including tumour suppressor and oncogenic signaling.
- Spheroids exhibited differential sensitivity to 5-fluorouracil, highlighting heterogeneous therapeutic responses.

## Abstract

Colorectal cancer (CRC) stands as a significant contributor to cancer-related mortality. Owing to its prognostic and therapeutic implications, intratumoural heterogeneity (ITH) presents a considerable challenge. We have developed an experimental framework integrating single-cell derived spheroids with proteomic profiling to facilitate a molecular, proteomic, and therapeutic characterization of intratumoural heterogeneity during CRC progression.

Single cells from the commercially available colorectal cancer cell lines SW480 (primary colorectal adenocarcinoma) and SW620 (locoregional lymph node metastasis of the same donor) were isolated using fluorescence-activated cell sorting (FACS) and subsequently cultured forming spheroids. This platform allowed controlled interrogation of clonal diversity through proliferation and viability assays, alongside deep proteomic characterization using label-free liquid chromatography–mass spectrometry (LC-MS) with data-independent acquisition. To evaluate its utility for therapeutic testing, chemotherapy response was measured after 72 h of incubation with 5-fluorouracil (5-FU).

The single-cell derived spheroid system demonstrated significant heterogeneity, as evidenced by variations in morphology, growth dynamics, viability, and proteomic signatures. Protein profiling identified ITH-associated proteins (WDR5, CKB, IPO11, ATP6V1F, DCXR and PCCB) and underscored pathway variations including tumour suppressor and proto-oncogenic signalling, vascularization and metabolic regulation. Furthermore, individual spheroids exhibited differential sensitivities to 5-fluorouracil, demonstrating the platform’s capacity to resolve heterogeneous therapeutic responses.

Our study establishes a robust and scalable method that integrates single-cell spheroids with proteomics to model and quantify ITH in CRC. By capturing clinically relevant diversity across morphology, viability, proteomic profiles and drug response, this approach provides a foundation for translating spheroid- and proteomics-based assays into personalized therapeutic testing.

The online version contains supplementary material available at 10.1007/s00432-025-06418-0.