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Triple negative breast tumors contain heterogeneous cancer cells expressing distinct KRAS-dependent collective and disseminative invasion programs

Oncogene volume 42, pages 737–747 (2023)Cite this article

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Abstract

Inter-patient and intra-tumoral heterogeneity complicate the identification of predictive biomarkers and effective treatments for basal triple negative breast cancer (b-TNBC). Invasion is the initiating event in metastasis and can occur by both collective and single-cell mechanisms. We cultured primary organoids from a b-TNBC genetically engineered mouse model in 3D collagen gels to characterize their invasive behavior. We observed that organoids from the same tumor presented different phenotypes that we classified as non-invasive, collective and disseminative. To identify molecular regulators driving these invasive phenotypes, we developed a workflow to isolate individual organoids from the collagen gels based on invasive morphology and perform RNA sequencing. We next tested the requirement of differentially regulated genes for invasion using shRNA knock-down. Strikingly, KRAS was required for both collective and disseminative phenotypes. We then performed a drug screen targeting signaling nodes upstream and downstream of KRAS. We found that inhibition of EGFR, MAPK/ERK, or PI3K/AKT signaling reduced invasion. Of these, ERK inhibition was striking for its ability to potently inhibit collective invasion and dissemination. We conclude that different cancer cells in the same b-TNBC tumor can express different metastatic molecular programs and identified KRAS and ERK as essential regulators of collective and single cell dissemination.

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Fig. 1: Organoids display heterogeneity in invasive behavior in a model of basal triple negative breast cancer.
Fig. 2: Specific invasive phenotype RNA sequencing reveals distinct molecular pathways.
Fig. 3: KRAS is required for both collective and disseminative invasion ex vivo and for metastasis formation in vivo.
Fig. 4: EGFR, MAPK, PI3K/AKT pathways are involved in C3(1)-TAg organoid invasion.

Data availability

The RNA-seq data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus [28] and are accessible through GEO Series accession number GSE215069 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE215069). Additional requests for information or data will be fulfilled by the corresponding author upon request.

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Acknowledgements

We thank all members of the Ewald Laboratory for critical discussions. AJE received support for this project through grants from: the Breast Cancer Research Foundation (BCRF-21-048), Twisted Pink Foundation, Hope Scarves, the Cindy Rosencrans Fund for Triple Negative Breast Cancer Research, the Jayne Koskinas Ted Giovanis (JKTG) Foundation, METAvivor Foundation, and the National Institutes of Health / National Cancer Institute (U01CA217846, U54CA268083, 3P30CA006973). JSB receives support from: the Jayne Koskinas Ted Giovanis (JKTG) Foundation and the National Institutes of Health / National Cancer Institute (U01CA217846).

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Authors and Affiliations

  1. Department of Cell Biology, Center for Cell Dynamics, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Elodie Henriet, Eloise M. Grasset, Matthew Dunworth, Meagan Haynes & Andrew J. Ewald

  2. Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, 21205, USA

    Hildur Knutsdottir, Joel S. Bader & Andrew J. Ewald

  3. Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Joel S. Bader & Andrew J. Ewald

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  1. Elodie Henriet
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Contributions

EH and AJE conceptualized the project and designed the experiments. EH performed most of the experiments. HK and JSB performed RNA-seq data analysis. EMG performed the in vivo experiments. MD contributed to qPCR data collection and analysis. MH was in charge of maintaining the mouse colony. EH and AJE wrote the manuscript with useful inputs from all authors.

Corresponding author

Correspondence to Andrew J. Ewald.

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Competing interests

AJE has unlicensed patents and patent applications related to the use of K14 as a prognostic biomarker, to the use of antibodies in cancer treatment, and to 3D culture protocols for tumor organoids. AJE’s spouse is an employee of Immunocore. JSB is a founder of Neochromosome and consultant for its parent company Opentrons Labworks Inc and serves on the SAB of Reflexion Pharmaceuticals. The other authors declare no competing interests.

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Henriet, E., Knutsdottir, H., Grasset, E.M. et al. Triple negative breast tumors contain heterogeneous cancer cells expressing distinct KRAS-dependent collective and disseminative invasion programs. Oncogene 42, 737–747 (2023). https://doi.org/10.1038/s41388-022-02586-2

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