Lymph node metastases can invade local blood vessels, exit the node, and colonize distant organs in mice

Abstract

Lymph node metastases in cancer patients are associated with tumor aggressiveness, poorer prognoses, and the recommendation for systemic therapy. Whether cancer cells in lymph nodes can seed distant metastases has been a subject of considerable debate. We studied mice implanted with cancer cells (mammary carcinoma, squamous cell carcinoma, or melanoma) expressing the photoconvertible protein Dendra2. This technology allowed us to selectively photoconvert metastatic cells in the lymph node and trace their fate. We found that a fraction of these cells invaded lymph node blood vessels, entered the blood circulation, and colonized the lung. Thus, in mouse models, lymph node metastases can be a source of cancer cells for distant metastases. Whether this mode of dissemination occurs in cancer patients remains to be determined.

Fig. 1. Circulating tumor cells (CTCs) that transited through the lymph node detected in mouse models

(A) Dendra2H2B positive cancer cells were injected orthotopically into syngeneic recipients. Approximately 20 days later, the primary tumor was resected and tumor draining lymph nodes were photoconverted using a 405nm diode for 5 consecutive days. Blood was analyzed for the presence of green/red fluorescent CTCs using an Amnis Imagestream flow cytometer. (Photoconverted animals: 4T1 model n=11, B16F10 model n=7, SCCVII model n=5. Control animals: 4T1 model n=5, B16F10 model n=7, SCCVII n=3) (B) Dendra2H2B-4T1 cells and Dendra2H2B-B16F10 cells but not Dendra2H2B-SCCVII cells photoconverted in the draining lymph node were detected in the blood. Data are represented as the percentage of red CTCs (photoconverted) to total detected CTCs. Green CTCs were detected in all three models. *p<0.05 comparing 405nm light exposure to sham exposure for individual cells lines. (C) Representative images obtained by Imagestream of CTCs from 4T1/BALB/c and B16F10/C57BL/6 mouse models show positive photoconverted cancer cells verified by nuclear localization of Dendra2H2B. Scale bar=10μm.

Fig. 2. Lymph node metastatic cancer cells can colonize the lung

(A, C) 100μm sections of fresh frozen lungs were obtained from Dendra2H2B-4T1 and Dendra2H2B-B16F10 tumor-bearing mice that either had their lymph node photoconverted with a 405nm diode or had no photoconversion. The top panels are representative images of micrometastatic disease (arrowheads) in the lung from control animals (no photoconversion), whereas the bottom panels show photoconverted isolated cancer cells that have colonized the lung via the lymph node. Scale bar=20μm. (B, D) The percentage of cancer cells detected in the lungs of Dendra2H2B-4T1 (n=8) and Dendra2H2B-B16F10 (n=8) tumor-bearing mice that were green (not photoconverted) versus red (photoconverted cells) are shown. *p<0.05 comparing 405nm light exposure (Photoconverted) to sham light exposure (Unconverted) for individual cells lines. (E) Schematic of experiment to determine whether cancer cells injected directly in the lymph node and in the mammary fat pad (MFP) can both form large metastases in the lungs. (F) Image of a lung with red lesions (originating from MFP) and green lesions (originating from lymph node) marked by arrowheads. Scale bar=1mm. (G) Lung metastases are represented as a percentage of lymph node origin metastasis (green) to total macroscopic lesions (red + green) (n=10). Using a one-sample Student’s t-test, both lymph node and MFP origin tumors had an incidence greater than zero (p<0.001).

Fig. 3. Cancer cells in the lymph node associate with blood vessels and invade the vascular basement membrane

(A) (Ai, Aiv) Immunofluorescence staining of metastatic lymph nodes with isolated 4T1 cancer cells (anti-cytokeratin, green), blood vessels (anti-CD31, red) and basement membrane (anti- collagen IV, blue) shows cancer cells associating with blood vessels (Aii and Av, arrowheads) and the vascular basement membrane (Aiii and Avi, arrowheads). A cancer cell is observed inside a blood vessel (Aii and Aiii) (arrowhead). Scale bar= 50μm (Ai), 20μm (Aiv), 10μm (Aii,iii,v,vi) (B) Quantification of the fraction of cancer cells within 5μm of a blood vessel (Sample) in a lymph node compared to a theoretical random distribution (Reference) of the same number of cells in the same lymph node shows cancer cell association with lymph node blood vessels. n=11 individual lymph nodes *p<0.05. (C) Representative histogram of the fraction of cancer cells at varying distances from the nearest blood vessel in a given lymph node with isolated cancer cells (red bars-Sample) compared to the reference distribution for that lymph node (white bars-Reference). The measured distribution shows an association of cancer cells with blood vessels in the lymph node. (D) Quantification of the fraction of cancer cells inside a blood vessel in lymph nodes containing macro-metastatic lesions (Lesions) or isolated tumor cells (ITC).

Fig. 4. Time-lapse intravital imaging of lymph node metastasis shows slow cancer cell migration toward blood vessels

(A) 4T1 Dendra2 expressing cancer cells (green fluorescence) form a large colony in the subcapsular sinus of the draining lymph node. Scale bar=60μm. (B to C) Cancer cells (green) that invaded the lymph node cortex wrapped around blood vessels (arrowheads), which were labeled by intravenous injection of rhodamine-dextran, 2million MW (red). Images were obtained by multiphoton microscopy at a depth of 80-110μm below the surface of the lymph node. Scale bar=20μm. (D) Time-lapse intravital imaging of cancer cells (green) in association with blood vessels (red) and collagen fibers (blue-detected by second harmonic generation) over the course of 75 min shows slow movement (arrowheads) of some cancer cells toward blood vessels. Images were obtained every 2 mins, with a 50μm z-stack. Scale bar=50μm. See also Supplementary Movies. (E) Quantification of the fraction of 4T1 tumor cells per field that associate with blood vessels or were inside blood vessels, analyzed by intravital microscopy in a tumor-draining lymph node. (F) Quantification of the speed of individual cancer cells in 4T1 and SCCVII lymph node metastases. (G) Quantification of the percentage of motile tumor cells in the image field over 75 min in 4T1 and SCCVII lymph node metastases. Quantification for (E to G) was done on 4-6 individual mice. Where indicated NS=not significant and *p<0.05.