Tumor Regulation of Lymph Node Lymphatic Sinus Growth and Lymph Flow in Mice and in Humans

Abstract

The lymphatic vasculature collects and drains fluid and cells from the periphery through lymph nodes (LNs) for immune monitoring, and then returns lymph to the bloodstream. During immune responses LNs enlarge and remodel, featuring extensive growth of lymphatic sinuses (lymphangiogenesis). This LN lymphangiogenesis also arises in cancer, and is associated with altered lymph drainage through LNs. Studies of mouse solid tumor models identified lymphatic sinus growth throughout tumor-draining LNs (TDLNs), and increased lymph flow through the expanded sinuses. Mice developing B cell lymphomas also feature LN lymphangiogenesis and increased lymph flow, indicating that these changes occur in lymphoma as well as in solid tumors. These LN alterations may be key to promote tumor growth and metastasis to draining LNs and distant organs. Lymphatic sinus growth within the TDLN may suppress anti-tumor-immune responses, and/or the increased lymph drainage could promote metastasis to draining LNs and distant organs. Investigations of human cancers and lymphomas are now identifying TDLN lymphatic sinus growth and increased lymph flow, that correlate with metastasis and poor prognosis. Pathology assessment of TDLN lymphangiogenesis or noninvasive imaging of tumor lymph drainage thus could potentially be useful to assist with diagnosis and treatment decisions. Moreover, the expanded lymphatic sinuses and increased lymph flow could facilitate vaccine or drug delivery, to manipulate TDLN immune functioning or to treat metastases. The insights obtained thus far should encourage further investigation of the mechanisms and consequences of TDLN lymphatic sinus growth and lymph flow alterations in mouse cancer models, and in human cancer patients.

Keywords: cancer; lymph flow; lymphangiogenesis; lymphatic endothelium; lymphography; tumor-draining lymph node.

Figure 1

Lymph node lymphatic sinus growth in E-µ-c-myc mice. a). Schematic of lymph node gross anatomy, illustrating the location of the cortex that contains thin subcapsular and cortical lymphatic sinuses and B lymphocytes, the paracortex containing conduits and T lymphocytes, and the medulla containing medullary lymphatic sinuses and T lymphocytes. b). Immunostaining with LYVE-1 antibody demonstrates subcapsular (arrow) and medullary (arrowhead) purple-stained lymphatic sinuses in popliteal LN of normal 6 week-old WT C57Bl6/J mouse, with Methyl Green nuclear counterstaining. c). LYVE-1 immunostaining identifies extensive lymphatic sinus growth throughout the LN from a preneoplastic 6 week-old E-µ-c-myc mouse, so that the cortex and medulla cannot be distinguished. Scale bar 50 µm.

Figure 2

Lymph drainage through lymphatic sinuses of tumor-draining lymph nodes. B16-F10 tumors were injected in one rear footpad of 5-week-old C57Bl/6J mice and grown for 3 weeks. Some mice (panels a-f) were anesthetized and injected in both dorsal toes with Texas Red dextran (Lysine-fixable 10,000 MW) to label the popliteal LN lymph drainage for 20 min before euthanasia (n = 9), while other mice were not injected with dextran (panels g,h). The lymphatic sinuses of the draining popliteal LNs were examined by immunostaining paraformaldehyde-fixed cryosections with 10.1.1 antibody (green). A small amount of Texas Red dextran is confined to the green lymphatic sinuses in the NTDLN (a). In the TDLN, much larger amounts of dextran are identified in the expanded green lymphatic sinuses (b). The white dashed boxes outline cortical regions shown at higher magnification in (c) and (d), while the outlined solid white boxes outline medullary regions shown at higher magnification in (e) and (f). Texas Red dextran is largely confined to the subcapsular and cortical lymphatic sinuses of the NTDLN (c) and TDLN (d). In the medulla, Texas Red dextran is largely confined to the sinuses, and is greatly increased in TDLNs (f), relative to the NTDLN (e). Red perlecan and green LYVE-1 lymphatic sinus immunostaining and Deltavision microscope imaging (Applied Biosciences) of the paracortex region adjacent to the medulla (g) identifies perlecan fibers surrounding lymphatic sinuses (L), high endothelial venules (H), capillaries, and conduits (arrows). Green perlecan immunostaining in combination with red SMA-1 immunostaining of FRCs (h) identifies the conduits of the paracortex (arrows). Scale bars for each image are 50 µm.

Figure 3

Lymph node lymph flow increases in reneoplastic E-µ-c-myc mice. Near infrared imaging of 52 nm diameter quantum dots (800 nm emission, Invitrogen) using a Xenogen IVIS CCD camera, 10 min after injecting 25 microliters of quantum dots into the dorsal rear toe of the right leg of 6-week-old WT (a) or E-µ-c-myc mouse (b), draining to the popliteal LN (arrows). c). Total fluorescence efficiency was measured in a region of interest drawn over the popliteal LN of each mouse and pre-injection fluorescence efficiency of the same region of interest was subtracted at each time point. Standard errors are shown. Differences between WT and E-µ-c-myc mice at each time point are statistically significant by Mann-Whitney test (N = 10; *, p < 0.05; **, p < 0.01).

Figure 4

Collecting lymphatic vessels show similar morphology in wild-type and E-µ-c-myc mice. Anesthetized 6-week-old WT (a) and E-µ-c-myc mice (b) were injected with 5% Evans Blue dye (80 µl) in the tailbase and in the rear dorsal toe (Sigma), followed by euthanasia 30 minutes later. The skin of the flank was reflected from the body wall, placed flat, and photographed in a stereomicroscope. The arrows indicate the afferent LV from the gluteal LN to the inguinal LN. The arrowheads indicate the efferent LV that travels along the milk line from the inguinal LN to the axillary LNs. While much more Evans Blue is taken up in E-µ-c-myc mice, the morphology of the afferent and efferent LVs is similar.