Erika Morera, PhD (Postodc) University of Oslo. Advisors: Vessela Kristensen & Pascal Duijf Problem or question being addressed: Breast cancer (BC) is the most prevalent cancer type in women worldwide [1]. BC classification depends on the overexpression of estrogen receptor (ER), progesterone receptor (PR), and the amplification of HER2 [2]. This molecular classification is routinely used in clinics for prognostic purposes and to choose specific treatment modalities. However, a high percentage of breast cancer (BC) tumors are still difficult to treat. Therefore, there is an urgent need to provide better or new treatment options for these patients. Immune cells in the tumor microenvironment have an important role in tumor progression and affect BC therapy response. Immune cells can recognize and inhibit tumor growth [3] and, in breast cancer, high immune cell infiltration has been associated with better prognosis [4, 5] and increased response to chemotherapy [6]. However, immune cells can also have harmful effects on the breast gland, promoting BC progression by releasing secreted molecules that induce inflammation and angiogenesis, which increase the survival and dissemination of BC cells [7, 8]. BCs with high genomic instability can lead not only to mutations but also to copy number alterations (CNAs). Previous studies from our group and others showed that specific CNAs are strongly associated with differences in immune cell infiltration (unpublished data). However, it is unknown how breast tumor cells and immune cells communicate and which specific immune cells and signaling molecules are involved in this process. Therefore, it is essential to study the immune cell populations in BC in the context of CNAs in the tumor cells, as well as how the heterotypic interactions of BC cells and immune cells occur and can be used to improve treatments for BC patients. Rationale for your approach: We would like to understand the mechanisms involved in the immune cell infiltration in BC and how cell-cell communication leads to different immune infiltration depending on different CNAs in BC cells. Currently, there are no good experimental models to study how immune cells infiltrate tumors, therefore it is important to develop new models. For this project, in collaboration with other researchers in Brisbane (QUT), a new model using innovative 3D hydrogels will be optimized to efficiently investigate immune cell infiltration in breast tumors. Advanced bioinformatics analyses will be applied as well in order to identify the cell subpopulations involved in the immune infiltration and the pathways that are relevant for the biological processes implicated. Details of suggested approach: Primary tumor cells derived from BC patients and BC cell lines with different CNAs will be cocultured with immune cells using a unique 3D hydrogel system developed in Brisbane [9]. This model mimics more closely in vivo conditions than the generally used basal membrane extracts (BME) systems and allows to study of changes in cellular composition and signaling. Using scRNA-seq and other omics analyses, cell co-cultures will be studied and subpopulations will be identified. The cell types found at the tumor site will be defined quantitatively and qualitatively. Genes involved in different pathways and molecules participating in the differences in immune cell infiltration will also be identified. How it will affect the broader field: We will study the heterotypic interactions between BC cells with different levels of CNA and immune cells. We shall explore the biological meaning of the processes and the cell subpopulations involved. With these experimental approaches, we want to classify BCs depending on their chromosomal alterations and identify alterations in the genetic material that can be associated with different types of immune cells. Our classification can be beneficial for immunotherapies used to treat BCs. Additionally, this project can enhance personalized treatment and improve the efficiency of treatments for BC patients. Altogether, unraveling the cell-cell communication mechanisms between tumor cells and immune cells in BC will be clinically applicable and improve treatment decisions. References: 1. Sung, H., et al., Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin, 2021. 71(3): p. 209-249. 2. Sørlie, T., et al., Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proceedings of the National Academy of Sciences, 2001. 98(19): p. 10869-10874. 3. Burnet, F.M., The concept of immunological surveillance. Prog Exp Tumor Res, 1970. 13: p. 1-27. 4. Ali, H.R., et al., Association between CD8+ T-cell infiltration and breast cancer survival in 12,439 patients. Ann Oncol, 2014. 25(8): p. 1536-43. 5. Mahmoud, S.M., et al., Tumor-infiltrating CD8+ lymphocytes predict clinical outcome in breast cancer. J Clin Oncol, 2011. 29(15): p. 1949-55. 6. Pruneri, G., A. Vingiani, and C. Denkert, Tumor infiltrating lymphocytes in early breast cancer. Breast, 2018. 37: p. 207-214. 7. Gordon, S. and F.O. Martinez, Alternative Activation of Macrophages: Mechanism and Functions. Immunity, 2010. 32(5): p. 593-604. 8. Carmeliet, P., Angiogenesis in life, disease and medicine. Nature, 2005. 438: p. 932. 9. Bray, L.J., et al., Three-Dimensional In Vitro Hydro- and Cryogel-Based Cell-Culture Models for the Study of Breast-Cancer Metastasis to Bone. Cancers (Basel), 2018. 10(9).