Heterogeneity of circulating tumor cell dissemination and lung metastases in a subcutaneous Lewis lung carcinoma model

Abstract

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.

Short-term circulating tumor cell dynamics in mouse xenograft models and implications for liquid biopsy

“…We previously used DiFC to measure CTC dissemination in an LLC-tumor bearing mouse model (29), where CTCs were consistently rare throughout tumor growth. Specifically, in 102 DiFC scans, the CTC count rates corresponded to approximately 0.…”

“…The DiFC instrument (figure 1a) and data processing algorithms were described in detail previously (26,27,29). Briefly, two specially designed fiber-optic probes ( fig.…”

“…In this work, we analyzed five data sets as follows: i) Lewis lung carcinoma (LLC) metastasis model: We re-analyzed previously reported DiFC data measured in sub-cutaneous (s.c.) Lewis lung carcinoma tumor bearing mice (29). As noted above, we previously considered only the mean CTC numbers on each day but not short-term fluctuations as we do here.…”

“…'In vivo flow cytometry' (IVFC) is a general term for optical instrumentation designed to detect and enumerate circulating cells directly in vivo, most often using either fluorescence or photoacoustic contrast (19,24). We recently developed 'diffuse in vivo flow cytometry' (DiFC) specifically for enumeration of rare green fluorescent protein (GFP)-expressing CTCs in mouse models of metastasis (25)(26)(27)(28)(29). DIFC uses diffuse light to non-invasively and continuously interrogate PB flowing in large, deeply-seated vessels (19).…”

“…DIFC uses diffuse light to non-invasively and continuously interrogate PB flowing in large, deeply-seated vessels (19). We recently used DiFC to monitor CTC dissemination in multiple myeloma (MM) and Lewis lung carcinoma (LLC) (27,29) mouse xenograft models. We showed that DiFC permitted longitudinal study of CTCs and CTC multi-cellular clusters (CTCCs) in individual mice at burdens below 1 CTC per mL of PB.…”