RESUMO
Subcutaneous (s.c.) tumor models are widely used in pre-clinical cancer metastasis research. Despite this, the dynamics and natural progression of circulating tumor cells (CTCs) and CTC clusters (CTCCs) in peripheral blood are poorly understood in these models. In this work, we used a new technique called 'diffuse in vivo flow cytometry' (DiFC) to study CTC and CTCC dissemination in an s.c. Lewis lung carcinoma (LLC) model in mice. Tumors were grown in the rear flank and we performed DiFC up to 31 days after inoculation. At the study endpoint, lungs were excised and bioluminescence imaging (BLI) was performed to determine the extent of lung metastases. We also used fluorescence macro-cryotome imaging to visualize infiltration and growth of the primary tumor. DiFC revealed significant heterogeneity in CTC and CTCC numbers amongst all mice studied, despite using clonally identical LLC cells and tumor placement. Maximum DiFC count rates corresponded to 0.1 to 14 CTCs per mL of peripheral blood. In general, CTC numbers did not necessarily increase monotonically over time and were poorly correlated with tumor volume. However, there was a good correlation between CTC and CTCC numbers in peripheral blood and lung metastases. We attribute the differences in CTC numbers primarily due to growth patterns of the primary tumor. This study is one of the few reports of CTC shedding dynamics in sub-cutaneous metastasis models and underscores the value of in vivo methods for continuous, non-invasive CTC monitoring.
RESUMO
PURPOSE: We recently developed a new instrument called "diffuse in vivo flow cytometry" (DiFC) for enumeration of rare fluorescently labeled circulating tumor cells (CTCs) in small animals without drawing blood samples. Until now, we have used cell lines that express fluorescent proteins or were pre-labeled with a fluorescent dye ex vivo. In this work, we investigated the use of a folate receptor (FR)-targeted fluorescence molecular probe for in vivo labeling of FR+ CTCs for DiFC. PROCEDURES: We used EC-17, a FITC-folic acid conjugate that has been used in clinical trials for fluorescence-guided surgery. We studied the affinity of EC-17 for FR+ L1210A and KB cancer cells. We also tested FR- MM.1S cells. We tested the labeling specificity in cells in culture in vitro and in whole blood. We also studied the detectability of labeled cells in mice in vivo with DiFC. RESULTS: EC-17 showed a high affinity for FR+ L1210A and KB cells in vitro. In whole blood, 85.4 % of L1210A and 80.9 % of KB cells were labeled above non-specific background with EC-17, and negligible binding to FR- MM.1S cells was observed. In addition, EC-17-labeled CTCs were readily detectable in circulation in mice with DiFC. CONCLUSIONS: This work demonstrates the feasibility of labeling CTCs with a cell-surface receptor-targeted probe for DiFC, greatly expanding the potential utility of the method for pre-clinical animal models. Because DiFC uses diffuse light, this method could be also used to enumerate CTCs in larger animal models and potentially even in humans.
