In Vivo Lymphatic Circulating Tumor Cells and Progression of Metastatic Disease

Abstract

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.

Keywords: circulating tumor cells; in vivo flow cytometry; lymphatic vessels; lymphography; marker; metastasis; personalized prognosis; photoacoustics; sentinel lymph node.

Figure 1

Design of the lymphatics circulating tumor cell (L-CTC0 detection in preclinical animal models of metastatic tumors in vivo.

Figure 2

Optimization of photoacoustic (PA) flow cytometry (PAFC) to assess L-CTCs in mice in vivo. (a) Evans Blue (EB) lymphography of ear afferent lymphatic vessels. (b) The PA trace recorded from the intact (control) lymph without false signals before (left) and after (right) high-pass filtration. (c) The PA trace recorded from the intact (control) lymph with one false signal and from the lymph with melanoma L-CTCs. (d) The representative peak shapes from PA traces identified as artifact-associated false signal (top) and CTC-associated signal (bottom). The examples of eight independent experiments of control mice and five melanoma-bearing mice are shown in b–e.

Figure 3

Progress of primary melanoma in the mouse ear. (a) Primary tumor size (mean ± standard error of the mean (SEM)). (b) Satellite lesions growing along the afferent lymphatic vessels, connecting primary tumor and sentinel lymph node (SLN).

Figure 4

Metastatic disease in melanoma-bearing mice. (a) Percentage of mice with histologically detectable SLN and distant metastasis; inset: intravital photos of intact (left) and metastatic (right; week 2 after inoculation) cervical lymph node. (b) Histology and immunohistochemistry of organs from mice bearing ear melanoma.

Figure 5

In vivo monitoring of L-CTCs over melanoma progression in groups of mice at week 1 (n = 33), week 2 (n = 21), week 3 (n = 15) and week 4 (n = 6) after inoculation. (a) Average counts of L-CTCs (mean ± SEM), as determined by PAFC; inset: 30-min PAFC trace from the afferent lymph in the mouse with early melanoma (week 1 after inoculation). (b) Representative examples of individual patterns of L-CTC counts.

Figure 6

Parallel dissemination of blood CTCs (B-CTCs) and L-CTCs. (a) Average counts of B-CTCs (mean ± SEM) in groups of mice at week 1 (n = 33), week 2 (n = 21), week 3 (n = 15) and week 4 (n = 6) after inoculation; inset; 30-min PAFC trace from the ear artery in the mouse with early melanoma (week 1 after inoculation). (b) Representative examples of individual interrelations between L-CTCs and B-CTCs over disease progression.

Figure 7

Statistical analysis. (a,b) Scatterplot matrices of correlation between L-CTC count, B-CTC count and primary tumor volume, n = 56 records. (c) Scatterplot matrices of correlation between L-CTCs and metastasis.

Figure 8

Assessment of mice with early breast cancer. (a) Integrated transmission and fluorescence image of primary breast cancer in the mouse ear. (b) Primary tumor size. (c) The average counts of L-CTCs in afferent lymphatic vessels and B-CTCs in ear blood vessels. Results in “b” and “c” panels are presented as means ± SEM.

Figure 9

Peak detection threshold selection.