219 - Lineage Tracing to Characterize Response to Radiation and Immunotherapy in Breast Cancer

Purpose/Objective(s):

Immunotherapy (IT) is less effective in estrogen-receptor (ER) positive (+) than ER negative breast cancer (BC). Efforts to improve IT’s effect in ER+ tumors are important as IT could be a valuable clinical tool to treat advanced ER+ BC. Neoadjuvant radiation could augment IT through multiple mechanisms. We sought to assess the impact of radiation and IT in a spontaneous mouse model of BC while also using genetic lineage tracing to monitor clonal dynamics of tumor and immune cells through treatment.

Materials/Methods:

A transgenic mouse model of BC with lineage tracing capability, the MARC1-iCas9-PyMT line, was created. This model combines the spontaneous, well-characterized MMTV-PyMT BC model with the Mouse for Actively Recording Cells 1 (MARC1) lineage tracing system. Doxycycline was used to induce Cas9 expression, and therefore initiate lineage recording, starting during mammary gland development in utero. Palpable tumors formed within 13-19 weeks for all mice and cells were collected for single-cell RNA (scRNA) sequencing via needle biopsy when tumors were >50mm³. For untreated mice, a needle biopsy was repeated at least a week later, and a variety of tumor and adjacent mammary gland samples were collected at endpoint for scRNA analysis. To compare natural tumor progression to radiation and IT, treated mice received one fraction of 5Gy focally to the tumor, followed by 3 doses of 5mg/kg anti-PD1. One week after treatment, tumor and adjacent mammary gland tissues were collected for scRNA sequencing. In addition to scRNA sequencing, DNA from tumors, mammary glands, and other organs were collected at endpoint from all mice for tissue-level MARC1 lineage information.

Results:

In treated mice, all tumors regressed following radiation and anti-PD1 therapy. PyMT tumors are typically resistant to single-agent IT, so the strong effect of our treatment regimen demonstrates the utility of neoadjuvant radiation to boost IT’s effect. Increased CD4+ T cell fraction was observed in treated and untreated tumors with time; in treated tumors, CD4+ follicular helper T cells specifically expanded. Transcriptomic changes were also detected in treated tumor cells with decreased expression of many oncogenic signaling pathways. Using the MARC1 lineage system, tumor clones with metastatic potential were identified. No clear pattern of gene expression differentiated these clones. Instead, transcriptomic heterogeneity was detected within clones highlighting the plasticity of tumor cell states.

Conclusion:

Our results show neoadjuvant radiation increases the efficacy of anti-PD1 in a transgenic mouse model of ER+ BC. Based on scRNA analysis of tumor and immune cells, it appears this combined therapy causes cancer regression by acting both directly on tumor cells and indirectly via altering immune cell populations within the tumor.