Dissecting CTC Phenotypes: Insights into Mechanisms of Breast Cancer Dormancy

Uncovering phenotypes of patient-derived Circulating Tumor Cells (CTCs) offers the promise to dissect CTC heterogeneity in relation to metastatic competence, and to determine biomarkers of therapeutic utility.

We hypothesized that breast cancer CTC subsets possessing markers of pluripotency avoid organ arrest with extreme efficiency by the concomitant presence of quiescence and stem cell properties; and that expression of urokinase plasminogen activator receptor (uPAR) and beta-1 integrin (β1int) are relevant in controlling the recurrence of breast cancer brain metastasis (BCBM). First, we captured EpCAM-negative/CD45- /ALDH1+/CD44+/CD24- breast cancer CTC subsets that possessed combinatorial uPAR and β1int expression, and characterized these subsets using DEPArray^TM, a CTC platform able to dissect CTC heterogeneity at a single-cell level. Second, CTC subsets generated in vitro tumorspheres that were characterized for human embryonic stem cell markers by RT² PCR arrays. Gene expression profiling was consistently distinct among uPAR+/β1int+ vs. uPAR-/β1int- CTC subset ratios and dependent upon patients’ BCBM clinical status. Expression of genes implicated in cell cycle progression, angiogenesis, and pluripotency was >30-fold higher than controls in uPAR/β1int ratios of CTC subsets from patients diagnosed with BCBM. Of note, expression of RIF-1, a protein that counteracts actions of the breast cancer suppressor BRCA1, was highest (>50-fold) with presence of RIF-1 nuclear foci in BCBM CTC subsets. Lastly, BRCA1 gene was found to be wild-type in CTC subsets following single-cell whole genome amplification and DNA sequencing.

In summary, we have linked EpCAM-negative uPAR/β1int CTC subsets and their properties to clinical BCBM; and will assess the therapeutic inhibition of uPAR/β1int CTC biomarkers on BCBM development and RIF-1/BRCA1 interplays in cell cycle-dependent mechanisms of DNA repair as key biomarkers delineating dormancy vs. BCBM competence.