Abstract
Simple SummaryDeadly metastases occur when tumor cells are shed from primary tumor into lymph and blood that circulate in distinct networks of vessels and disseminate circulating tumor cells (CTCs) through the body. Therefore, detection of CTCs at potentially treatable early disease stage might improve patient survival. However, most efforts have been made to test CTCs in blood only. Here, we explored the clinically relevant photoacoustic and fluorescent flow cytometry for early in vivo detection of lymphatic CTCs using metastatic melanoma and breast cancer mouse models. We demonstrated the presence of detectable lymphatic CTCs at pre-metastatic disease, estimated correlation between CTCs, primary tumor, and metastasis, and observed parallel CTC dissemination by blood and lymph. Our findings suggest the use of lymphatic CTC testing in vivo as a new indicator of metastasis initiation, and combined assessment of two body fluids as a more promising diagnostic platform compared to existing mono-detection of blood CTCs. The dissemination of circulating tumor cells (CTCs) by lymph fluid is one of the key events in the development of tumor metastasis. However, little progress has been made in studying lymphatic CTCs (L-CTCs). Here, we demonstrate the detection of L-CTCs in preclinical mouse models of melanoma and breast cancer using in vivo high-sensitivity photoacoustic and fluorescent flow cytometry. We discovered that L-CTCs are be detected in pre-metastatic disease stage. The smallest primary tumor that shed L-CTCs was measured as 0.094mm×0.094mm, its volume was calculated as 0.0004 mm3; and its productivity was estimated as 1 L-CTC per 30 minutes. As the disease progressed, primary tumors continued releasing L-CTCs with certain individual dynamics. The integrated assessment of lymph and blood underlined the parallel dissemination of CTCs at all disease stages. However, the analysis of links between L-CTC counts, blood CTC (B-CTC) counts, primary tumor size and metastasis did not reveal statistically significant correlations, likely due to L-CTC heterogeneity. Altogether, our results showed the feasibility of our diagnostic platform using photoacoustic flow cytometry for preclinical L-CTC research with translational potential. Our findings also demonstrated new insights into lymphatic system involvement in CTC dissemination. They help to lay the scientific foundation for the consideration of L-CTCs as prognostic markers of metastasis and to emphasize the integrative assessment of lymph and blood.
Highlights
Metastasis arises from individual tumor cells that are shed from the primary lesion into distant organs, usually by way of body fluids, including blood, lymph and cerebrospinal fluid [1,2,3]
We primarily focused on melanoma because (1) melanoma is one of the most aggressive metastatic tumors in humans, (2) current therapy often has low efficacy, (3) multiple clinical observations indicate that melanoma uses the lymphatic system to metastasize, and (4) melanoma circulating tumor cells (CTCs) provide well-readable PA signals in label-free mode [25,30,35,36,41,42]
Our study demonstrated that in vivo photoacoustic and fluorescent flow cytometry platform provides highly sensitive diagnosis of lymphatic CTCs (L-CTCs) with the ability to identify and count the earliest L-CTCs at the pre-metastatic stage of disease
Summary
Metastasis arises from individual tumor cells that are shed from the primary lesion into distant organs, usually by way of body fluids, including blood, lymph and cerebrospinal fluid [1,2,3]. Further experimental and clinical studies of metastatic tumors (e.g., melanoma, breast and prostate cancer) have demonstrated controversial results. Preclinical studies from ours and other laboratories using clinically relevant in vivo photoacoustic (PA) flow cytometry (PAFC) platforms and experimental in vivo fluorescent flow cytometry (FFC) demonstrated the ability to count extremely rare and early B-CTCs with a sensitivity unachievable by in vitro methods [11]. PAFC identifies single cells with endogenous (e.g., melanin in melanoma CTCs) or exogenous (e.g., low toxic gold nanoparticles, spasers, and layered composite structures) PA contrast agents in multicellular flow without skin incision and extraction of blood samples (i.e., noninvasively) [10,11,12,18,19,20,21,22,23]. We demonstrated the successful PAFC detection of weakly absorbing tumor cells labeled with only
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