Poster S10 - In-Vivo dECM Breast Cancer Model Reveals Genetic Insights into Cancer Progression with Age

Introduction: Breast cancer is one of the most commonly diagnosed cancers globally. Despite the advancements made in its prognosis and treatment, it remains a prevalent and highly metastatic disease that continues to be a major barrier towards human health. One of the major risk factors of breast cancer is aging. In 2020, over 2.3 million new cases and 685,000 deaths from breast cancer occurred, with both numbers only expected to increase by 2040 due to population growth and aging as the sole factors. Breast cancer incidence increases drastically with age, and with the increase in longevity, it has become a significantly larger health problem. In recent breast cancer and aging studies, a specific interest is the extracellular matrix (ECM). The ECM is an intricate network of molecules that provide structural and biochemical support. Within the ECM exists a variety of proteins and components that regulate its structure, mechanics, and drug resistance, of which both cancer and age can drastically alter, causing a more tumor-permissive environment. Within breast cancer, the ECM has been a key focus in understanding various metastatic niches and downstream ECM-mediated signaling pathways. The Zorlutuna Lab has previously shown that the aged ECM predisposes both normal and cancerous cells to a more invasive phenotype and may harbor components that could lead to cancer initiation and/or progression. This study aims to serve as a more in-depth in-vivo follow up to continue to assess how age affects tumor growth and development along with the identification of potential biomarkers.

Materials and

Methods: Mouse 4th mammary glands were isolated from young (2-4 months) and aged (24-48 months) C57BL/6 mice. The dissected tissues were then decellularized and delipidized to generate decellularized matrices (dECM). The matrices were reseeded with MCF10DCIS.com and implanted into Rag1-/- mice (one young and one aged matrix per mouse). After 4 weeks, the mice were sacrificed and the tumors were excised to measure volume, and tumor samples were then processed for scRNA-sequencing to identify cell clusters in the aged and young environment. Cell clusters and relevant gene expressions were analyzed by 10x Genomics. Further, in-vivo experiments were performed to explore the genes of interest identified in the previous set of in-vivo experiments. MCF10DCIS.com cells were knockdown of genes of target or control (pLKo) via shRNA and validated through western blot. Aged dECM were then reseeded with validated cells and implanted into Rag1-/- mice to compare the tumor growth and transcriptome similar to the previous experiment setup. Cell clusters and relevant gene expressions were analyzed by 10x Genomics and compared with the first set of data.

Results, Conclusions, and Discussions: In the initial setup, MCF10DCIS.com cells seeded atop young and aged mouse dECM were implanted in-vivo for four weeks to assess tumor growth differences. The mice were then sacrificed, and the tumors were excised to have their volume evaluated along with transcriptome through scRNA-sequencing. The tumor size was found to be significantly increased in the aged matrices in comparison to the young matrices (p=0.0032). Additionally, scRNA-sequencing performed on the tumor cells identified cell clusters enriched specifically on aged matrices or younger matrices. Interesting results were found from this on the cancer phenotype and innate immune cells. The selected genes of targets were then knocked down on the MCF10DCIS.com cells to validate the selected genes of interest. Rag1-/- mice were implanted with a control and experimental recellularized dECM on either fat pad and were sacrificed after 6 weeks with the tumors excised for analysis. Tumor sizes were altered with the genetic knockdown.
In this study, we generated same-subject age-mimetic in-vivo models with aged and young dECM reseeded with tumor cells and implantation. scRNA-seq allowed us to identify cell clusters representative of tumors in aged and young microenvironment and select 3 genes of interest. Genetic knockdown of the 3 selected genes confirmed that these genes play a significant role in the inception of a more invasive phenotype of tumor cells observed in the aged tumor microenvironment.

Acknowledgements (Optional): This study is funded by NIH award number 5R01EB027660-02 and 1R01CA275423-01A1