Characterising a 3D culture model of triple-negative breast cancer through systems-based molecular phenotyping

Abstract

Triple-negative breast cancer (TNBC) presents a worse prognosis than other subtypes, characterised by frequent distant recurrence, shorter recurrence-free period and limited therapeutic options. Developing effective oncology therapeutics proves particularly challenging, as indicated by the low success rate observed in oncology clinical trials. The use of 3D cell culture models is a potential approach to advance preclinical therapeutic
assessment by more closely representing the in vivo tumour microenvironment than conventional 2D cell culture models. Furthermore, the ultra-low adherent (ULA) 3D culture system has shown compatibility with ultra-high throughput drug screening assays and provides distinct hit detection compared to 2D culture drug screens.

In this study, we performed LC-MS/MS proteomic characterisation of TNBC cell lines cultured in the ULA culture system, providing insights into the cellular processes and signalling pathways in comparison to the 2D culture model. The pathway analysis revealed a significant increase in antioxidant expression and proteins associated with drug detoxification in the ULA culture system. Additionally, the HCC1143, HCC1806 and HCC1937 cell lines formed dense spheroids, while MDA-MB-231 cells displayed a loose aggregate morphology in the ULA culture system. The spheroid-forming cell lines exhibited a more pronounced elevation of glycolytic enzymes, as well as HIF-1 and AMPK signalling proteins, compared to MDA-MB-231 cells in the ULA culture system. This suggests that the morphological characteristics observed in the ULA culture system may be associated with the modulation of these pathways.

For the MDA-MB-231 cell line, transcriptomic profiling of the ULA and 2D culture models was performed to complement the proteomic dataset. Revealing diminished expression of genes and proteins associated with cell cycle, DNA replication and ECM-receptor interactions. Additionally, we performed a comparison of the MDA-MB-231 gene expression with The Cancer Genome Atlas TNBC patient data to assess the potential for clinical translation. The results indicated a good correlation between the patient data and both culture models, with the ULA culture system exhibiting a slightly closer correlation than the 2D culture system.

Compared to the 2D culture model, the HCC1143 ULA culture model exhibited reduced sensitivity to the chemotherapeutic agents, doxorubicin and paclitaxel. Proteomic assessment of chemotherapy treated HCC1143 spheroids revealed an increased expression of proteins associated with the oxidative stress response under doxorubicin treatment and elevated cell cycle proteins induced by paclitaxel treatment. This not only identified previously established therapeutic targets, validating the 3D culture model, but also revealed novel observations that warrant further investigation.

The findings of this study contribute to the understanding of the TNBC in vitro 3D culture model and its application in 3D culture drug screens. The adoption of 3D culture models in early drug screening is expected to improve the selection of therapeutics in the preclinical phase, ultimately supporting the development of more effective therapeutic options for TNBC.

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Identifiers

ORCID for Luis Coy: orcid.org/0000-0001-6466-6408

ORCID for Paul Skipp: orcid.org/0000-0002-2995-2959

ORCID for Andy Shapanis: orcid.org/0000-0003-4147-6956

ORCID for Charlie Birts: orcid.org/0000-0002-0368-8766

ORCID for Max Crispin: orcid.org/0000-0002-1072-2694

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