Inducible Nitric Oxide Synthase and CD11b+Gr1+ Cells Impair Lymphatic Contraction of Tumor-Draining Lymphatic Vessels

Abstract

Background: Metastatic tumor cells spread through lymphatic vessels and colonize draining lymph nodes (LNs). It is known that tumors induce lymphangiogenesis to enhance lymphatic metastasis and that metastatic cancer cells are carried by lymph flow to LNs. Methods and Results: Here, we investigated the molecular and cellular regulation of collecting lymphatic vessel contraction in vessels draining a metastatic tumor using intravital microscopy. In tumor-draining collecting lymphatic vessels, we found vessel contraction was suppressed. The infiltration of peritumor tissue by inducible nitric oxide synthase positive and CD11b⁺Gr1⁺ myeloid cells played a critical role in the suppression of lymphatic contraction. Depletion of Gr1⁺ cells with an anti-Gr1 antibody improved contraction of tumor-draining lymphatic vessels. In addition, inducing tumor cell death restored lymphatic contraction in nude mice. Conclusions: These findings indicate that tumors contribute to regulation of lymphatic transport in a reversible manner, warranting further investigation into the role of impaired lymphatic transport in cancer progression.

Keywords: inducible nitric oxide synthase; lymph node metastasis; lymphatic contraction; lymphatic vessels; nitric oxide; tumor draining.

FIG. 1.

Lymphatic vessels are dilated and lymphatic contraction is inhibited in vessels draining B16F10 melanomas. B16F10 melanoma was implanted into the hind footpad of C57Bl/6 mice. Lymphatic contraction analysis of the afferent lymphatic vessel to the popliteal lymph node was performed in nontumor-bearing control animals (A, top) and in animals when the footpad reached 6–8 mm in thickness after B16F10 tumor implantation (A, bottom). Representative lymphatic diameter measurements of control and tumor-bearing mice are shown (B).

FIG. 2.

Lymphatic contraction is maintained in iNOS^−/− mice. B16F1 or B16F10 melanoma was implanted into the hind footpad of C57Bl/6 WT or iNOS^−/− mice before undergoing lymphatic contraction analysis. There is no difference in tumor size between WT and iNOS^−/− mice implanted with B16F1 or B16F10 melanoma (A and B, left). Ejection fraction is increased in iNOS^−/− mice versus WT (A and B, right). TDLNs were collected to assess LN metastasis. Representative H&E sections of a nonmetastatic LN and an LN with a B16F1 metastasis are shown (C, D). Scale bar = 40 μm. There is no significant difference in the percentage of LN metastasis between iNOS^−/− and WT mice with B16F1 or B16F10 melanoma (E, F). Bars shown ± SEM, n = 5–14, *p < 0.05 by unpaired t-test with a Welch's correction. H&E, hematoxylin and eosin; iNOS, inducible nitric oxide synthase; LN, lymph node; SEM, standard error of the mean; TDLNs, tumor-draining lymph nodes; WT, wild-type.

FIG. 3.

Lymphatic contraction is increased by depleting Gr1 cells. WT C57Bl/6 mice were implanted with B16F10 melanoma and underwent treatment with anti-Gr1 IgG or nonspecific rat IgG (control) to determine whether depletion of Gr1⁺ cells would restore lymphatic contraction. Anti-Gr1 IgG treatment had no significant effect on B16F10 melanoma growth or tumor size (A, B). The ejection fraction is significantly higher in anti-Gr1 IgG-treated animals (C). Bars shown ± SEM, n = 12, *p < 0.05 by unpaired t-test with a Welch's correction. IgG, immunoglobulin G.

FIG. 4.

Depleting tumor cells increase lymphatic contraction in HSTS26T human sarcoma. HSTS26T was implanted in the footpad of nude mice and DT was used to reduce tumor burden. DT treatment directly killed tumor cells, reducing tumor size (A). Maximum lymphatic diameter and ejection fraction were measured after depletion of tumor cells (B, C). Correlation between tumor size and ejection fraction is shown (D). This cumulative data support the model that iNOS expressing CD11b⁺Gr1⁺ cells (green) associated with tumors (blue cells) contribute to the inhibition of lymphatic contraction (E). Bars shown ± SEM, *p < 0.05 by unpaired t-test with a Welch's correction. DT, diphtheria toxin.