Simultaneous expression of epithelial and immune cell markers in circulating tumor cells identified in patients with stage 4 breast cancer.

Abstract

Background: Heterogeneous circulating tumor cells (CTCs) have been implicated in the formation of new metastases. However, circulating cells expressing both tumor and immune cell proteins are often dismissed as insignificant findings in CTC studies.

Methods: Two non-contemporaneous blood samples from a metastatic breast cancer patient were analyzed using an enrichment-free platform to identify canonical, epithelial-only CTCs (CD45-/cytokeratin + , epi.CTCs) and CD45 + /cytokeratin+ immune-like CTCs (im.CTCs). Single cells from both samples were subjected to copy number and protein expression profiling. A cohort of 36 metastatic breast cancer patients was then analyzed to search for additional cases with im.CTCs.

Results: Here, we identified and characterized a population of CTCs exhibiting an immune-like state. In two samples from an index patient, im.CTCs outnumbered epi.CTCs, comprising >97% of the CTC population. Single-cell copy number analysis of 43 im.CTCs and 30 epi.CTCs revealed clonal alterations across both populations, confirming a shared tumor origin. Furthermore, im.CTCs contained pseudo-diploid profiles that did not reflect dilution from the addition of a normal diploid genome, indicating that they were unlikely to have originated from tumor-immune cell fusion. Protein expression analysis showed that im.CTCs express CD45 as well as other immune-related markers, such as CD3 and CD4, and the cancer stemness marker, CD44. Subsequent analysis of a metastatic breast cancer cohort identified an additional patient harboring im.CTCs with the same tumor-derived, non-fusion genome as in the index case.

Conclusions: Collectively, these genomic and proteomic features distinguish im.CTCs from previously reported circulating cells may represent a novel form of tumor cell plasticity.