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Purpose <p>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.</p> Methods <p>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).</p> Results <p>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.</p> Conclusion <p>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.</p>

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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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月24日

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  • Helene Sophia Radloff,
  • Michael Kohl,
  • Thorben Sauer,
  • Sonja Hartwig,
  • Sven Geisler,
  • Stefan Lehr,
  • Timo Gemoll

摘要

Purpose

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.

Methods

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).

Results

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.

Conclusion

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.