Lymph Liquid Biopsy for Detection of Cancer Stem Cells

Abstract

Collection of a blood sample defined by the term "blood liquid biopsy" is commonly used to detect diagnostic, prognostic, and therapeutic decision-making markers of metastatic tumors including circulating tumor cells (CTCs). Many tumors also release CTCs and other markers into lymph fluid, but the utility of lymphatic markers largely remains unexplored. Here, we introduce lymph liquid biopsy through collection of peripheral (afferent) and central (thoracic duct [TD]) lymph samples and demonstrates its feasibility for detection of stem-like CTCs potentially responsible for metastasis development and tumor relapse. Stemness of lymphatic CTCs (L-CTCs) was determined by spheroid-forming assay in vitro. Simultaneously, we tested blood CTCs by conventional blood liquid biopsy, and monitored the primary tumor size, early metastasis in a sentinel lymph node (SLN) and distant metastasis in lungs. Using a mouse model at early melanoma stage with no distant metastasis, we identified stem-like L-CTCs in lymph samples from afferent lymphatic vessels. Since these vessels transport cells from the primary tumor to SLN, our finding emphasizes the significance of the lymphatic pathway in development of SLN metastasis. Surprisingly, in pre-metastatic disease, stem-like L-CTCs were detected in lymph samples from the TD, which directly empties lymph into blood circulation. This suggests a new contribution of the lymphatic system to initiation of distant metastasis. Integration of lymph and blood liquid biopsies demonstrated that all mice with early melanoma had stem-like CTCs in at least one of three samples (afferent lymph, TD lymph, and blood). At the stage of distant metastasis, spheroid-forming L-CTCs were detected in TD lymph, but not in afferent lymph. Altogether, our results demonstrated that lymph liquid biopsy and testing L-CTCs holds promise for diagnosis and prognosis of early metastasis. © 2020 International Society for Advancement of Cytometry.

Keywords: metastasis circulating tumor cells cancer stem cells central thoracic duct lymph peripheral afferent lymphlymph liquid biopsy.

Fig 1.

Principle of lymph liquid biopsy of afferent and TD lymph.

Fig 2.

Mouse model of metastatic melanoma. (a) Diagram of the experiment (left) and intravital images (right) of primary tumor (top right) and blue-colored afferent lymphatic vessel (LV) after EB lymphography (bottom right). (b) Individual (diamond plot) and average volume of primary tumor (column, mean ± SEM, n = 11) in the group of mice at week 1 after melanoma inoculation. (c) Percentage of early SLN involvement. (d) H&E histology of intact SLN. (e) Histologically (H&E) detectable early SLN metastasis (left) and the enlarged fragment of the metastatic nodule with melanoma-specific brown pigment melanin (right).

Fig 3.

Sampling lymph using cannulation of lymphatic vessels. (a) Cannula (C) in afferent lymphatic vessel (LV). (b) TD after 30 min of milk cream gavage in vivo before cannulation. (c) Optical images of intact (i.e., from healthy mouse) TD lymph sample on the slide in vitro obtained at 10× magnification of the objective (right) and 60× magnification of the objective (oil immersion). (d) Identification of CD45+ cells from the total population of TD lymphatic cells using flow cytometry; the representative experiment is shown.

Fig 4.

Lymph liquid biopsy of stem L-CTCs. (a) High-resolution optical images (60× magnification of the objective, oil immersion) of individual L-CTC in the lymph sample on the slide immediately after lymph liquid biopsy. (b) Incidence (% mice and % wells on the plate; each plate contained cells from the same afferent lymph sample of one mouse) of growing melanoma spheroids from afferent L-CTCs sampled from the mice at Week 1 post-inoculation; insert: optical image (20× magnification of the objective) of the afferent L-CTC originated spheroid. (c) Difference in primary tumor sizes between group of mice (n = 5), which had afferent L-CTC originated spheroids and the group of mice, which afferent L-CTCs did not form spheroids; diamond plots are individual results, black columns is mean ± SEM. (d) Incidence (% mice and % wells on the plate) of growing melanoma spheroids from TD L-CTCs sampled from the mice at Week 1 post-inoculation; insert: optical image (20× magnification of the objective) of the TD L-CTC originated spheroid. (e) Incidence (% mice) of growing melanoma spheroids from B-CTCs sampled from the mice at Weeks 1 and 3 post-inoculation. (f) Incidence (% mice) of growing melanoma spheroids from afferent and TD L-CTCs sampled from the mice at Week 3 post-inoculation.