(10a) Biomimetic Scaffolds Recapitulate Immune Cell Anti-Tumor Phenotypes in the Early Breast Cancer Metastatic Niche

Background: One in eight women will be diagnosed with breast cancer. Although therapeutic advances have drastically improved outcomes for localized disease, treatments for metastatic cancer focus on prolonging life rather than a finding a cure. Metastasis diagnoses often originate through patient-reported symptoms, such as difficulties breathing or coughing. Once secondary tumors are detected, millions of tumor cells have colonized the tissue and compromised organ function. Furthermore, common metastatic sites, such as the brain, liver, and lung, are painful and risky to biopsy repeatedly. Early detection provides an opportunity to intervene while tumor burden and heterogeneity are low. Our research group has developed a biomaterial scaffold that can be used as a surrogate metastatic niche and readily accessed. We previously demonstrated their utility in detecting early metastatic disease and recruiting tumor cells that are phenotypically identical to those at the native metastatic niche.

We aim to dissect the heterogeneity and temporal transitions of the lung metastatic niche to investigate how changing phenotypes influence tumor cell behavior. Clinical samples, which can only be collected from late-stage metastatic tumors, have shown that immune cells at the metastatic niche promote tumor growth. However, the phenotypic evolution of these cells from healthy tissue to lethal tumors is largely unstudied. The combined findings from the native metastatic niche (lung) and surrogate niche (scaffold) can be used to identify diagnostic markers and actionable targets for the treatment of early metastatic disease.

Methods: Polycaprolactone (PCL) porous scaffolds were implanted in female BALB/c mice two weeks before orthotopic inoculations of 4T1 triple-negative tumor cells. Tumors were allowed to progress for 7, 14, or 21 days, equating to the pre-metastatic, early metastatic, and metastatic niche, respectfully. Lungs and scaffolds were collected, with healthy mice serving as controls (day 0). Tissues were broken down to a single cell suspension, library preparation was completed using Drop Seq, and samples were analyzed for single cell RNA sequencing. We used the Seurat pipeline to identify cell types, phenotypes, and differential gene expression. Pseudotime analysis was completed with Monocle3. Enrichment of signaling pathways was determined with Gene Set Enrichment Analysis. Receptor-ligand interactions, transcription factor expression, and metabolic pathways were assessed with a combination of CellPhoneDB, DoRothEA, NicheNet, and COBRA.

Results: We initially characterized the lung, the native metastatic niche, to understand the physiological dynamics in metastatic development. We identified key phenotypic shifts in the lung as disease progressed. Specifically, days 7 and 14 exhibited increased anti-tumor signaling, while day 21 was highly pro-tumor. For example, the enrichment of pathways indicative of metastasis-associated immune dysregulation drastically decreased between days 14 and 21. Neutrophils and monocytes acted as the primary drivers of this phenotypic shift. The percentage of pro-tumor neutrophils increased from 21% at day 7 to 74% at day 21. Moreover, day 7 and day 14 neutrophils correlated to pathways associated with T cell activation and interferon gamma, further identifying their role in suppressing tumor cells. Although the percentage of classical monocytes increased from 23% to 70% from days 0 to 14, tumor-associated monocytes (Chi3l3+ classical monocytes) were undetected until day 21 (36%). These day 21 monocytes were enriched for genes (e.g. Tgfbi, Lcn2, S100a4) and pathways correlating to extracellular matrix remodeling. Importantly, pseudotime analyses were unable to correlate the neutrophils at day 21 to those collected at the other time points. The dramatic transition at day 21 may be associated with the tumor cell expansion that is observed at this time. Unlike the neutrophils, the transitions in monocytes could be tracked over time, suggesting that late-stage monocytes evolve but do not radically change.

The temporal dynamics in the lung were compared to the scaffold to determine if the trends were paralleled, which would allow the scaffold to be used as a diagnostic. Neutrophils collected from the healthy scaffolds had an inherently anti-tumor phenotype relative to the lung (Figure 1A). Neutrophils from healthy scaffolds were more pro-inflammatory than those collected from the lungs as a result of the foreign body response. Yet, as disease progressed, the phenotypes of the neutrophils in the lung and scaffold converged, becoming increasingly pro-tumor, and were identical by day 21. Whereas monocyte phenotypes were more comparable between the two tissues at all time points (Figure 1B). This held particularly true at day 21 where monocytes collected from the lungs and scaffolds were enriched for the same extracellular matrix-associated genes. These data show incredible promise for the scaffolds to model the specific phenotypic changes associated with the progression of metastatic breast cancer.

Conclusions: We have identified anti-tumor immune cells at the metastatic niche in early-stage metastatic disease. Anti-tumor neutrophils and monocytes could be leveraged to pursue new therapeutic strategies and improve our understanding of the early events in metastatic disease. Markers derived from such cells may also help stratify metastatic disease to help direct providers to the best course of treatment, specifically in the context of immunotherapies. Importantly, the recapitulation of these phenotypes in the accessible biomaterial scaffold could allow us to identify potential cases of metastasis prior to the formation of macroscopic secondary tumors. Earlier detection and treatment of metastatic breast cancer has the potential to revolutionize patient outcomes.