Tumor Vascular Remodeling Affects Molecular Dissemination to Lymph Node and Systemic Leukocytes

Abstract

Angiogenic and lymphangiogenic remodeling has long been accepted as a hallmark of cancer development and progression; however, the impacts of this remodeling on immunological responses, which are paramount to the responses to immunotherapeutic treatments, are underexplored. As immunotherapies represent one of the most promising new classes of cancer therapy, in this study, we explore the effects of angiogenic and lymphangiogenic normalization on dissemination of molecules injected into the tumor microenvironment to immune cells in lymph nodes draining the tumor as well as in systemically distributed tissues. A system of fluorescent tracers, size-matched to biomolecules of interest, was implemented to track different mechanisms of tumor transport and access to immune cells. This revealed that the presence of a tumor, and either angiogenic or lymphangiogenic remodeling, altered local retention of model biomolecules, trended toward normalizing dissemination to systemic organs, and modified access to lymph node-resident immune cells in manners dependent on mechanism of transport. More specifically, active cell migration by skin-derived antigen presenting cells was enhanced by both the presence of a tumor and lymphangiogenic normalization, while both angiogenic and lymphangiogenic normalization restored patterns of immune cell access to passively draining species. As a whole, this work uncovers the potential ramifications of tumor-induced angiogenesis and lymphangiogenesis, along with impacts of interrogation into these pathways, on access of tumor-derived species to immune cells. Impact Statement Angiogenic and lymphangiogenic normalization strategies have been utilized clinically to interrogate tumor vasculature with some success. In the age of immunotherapy, the impacts of these therapeutic interventions on immune remodeling are unclear. This work utilizes mouse models of angiogenic and lymphangiogenic normalization, along with a system of fluorescently tagged tracers, to uncover the impacts of angiogenesis and lymphangiogenesis on access of tumor-derived species to immune cell subsets within various organs.

Keywords: angiogenic normalization; biodistribution; growth factor; immune remodeling; lymphangiogenesis; tissue engineering.

FIG. 1.

Tumor-mediated alterations in vasculature alter dissemination from the local microenvironment. Tumor vasculature remodels (a). (b) Tracer system used to assess changes in tumor-disseminated transport. Concentration of 500, 30, and 10 nm tracers within dLNs assessed on a bulk basis (c) and by flow cytometry (d). (e) Frequency of live dLN-resident cells accessing each size tracer across time. (f) Counts for tracer-positive cells expressing CD11c, F4/80, B220, or CD3 in local dLN and systemic tissues, including the spleen, lungs, and liver. n = 6 mice per group. dLNs, draining lymph nodes.

FIG. 2.

Model for interrogation of angiogenesis and lymphangiogenesis within melanomas. (a) Tumor growth after implantation of WT B16F10 tumors in immunocompetent mice. (b) Intratumoral VEGF-A and VEGF-C concentrations of B16F10 (WT) tumors over the course of tumor progression. (c) VEGF-C measurements for B16F10-VC compared with B16F10 cells in culture per 1000 cells plated. (d, e) Growth of B16F10-VC (d) and B16F10 tumors with R2 inhibition (e) after implantation in immunocompetent mice. (f) Concentration of VEGF-A and VEGF-C in naive skin, WT, VC, and R2 tumors at day 7 of tumor growth. Blood and lymphatic densities quantified from immunohistochemistry staining and images; white arrows indicate vessel structures; scale bars = 200 μm (g). (h) Total blood (CD31⁺Lyve-1⁻) and lymphatic (Lyve-1⁺) structures in naive skin, and WT < VC, and R2 tumors, assessed by immunohistochemistry at day 7 of tumor growth. (i) 3D reconstructions of microcomputed tomography of the tumor blood vasculature at day 7 postimplantation for naive skin, WT, VC, and R2 tumors at day 7 of tumor growth; scale bars = 1 mm. (j) Total number of cells within LNs draining naive skin, and WT, VC, and R2 B16F10 tumors, assessed by flow cytometry. ^†Indicates significance for VEGF-A WT days 5–9 grouped versus naive by t-test; *indicates significance by multiple t-tests; *indicates p < 0.05; **indicates p < 0.01; ***indicates p < 0.005; ****indicates p < 0.001; n = 4–12 animals per group. 3D, three dimensional; LNs, lymph nodes; VC, VEGF-C overexpressing; VEGF, vasoendothelial growth factor; WT, wild type.

FIG. 3.

(Lymph)angiogenic normalization effects on retention of tracers at tissue site of injection. Retention at site of injection as area under curve of measurements taken 4, 24, and 72 h after administration in naive skin, or WT, VC, or R2 B16F10 tumors. *Indicates significance by two-way ANOVA with Tukey's post hoc test; *indicates p < 0.05; ***indicates p < 0.005; ****indicates p < 0.001. ANOVA, analysis of variance.

FIG. 4.

Local (lymph)angiogenic normalization normalizes systemic dissemination of tumor-derived molecules to an extent. (a) Accumulation of 500, 30, and 10 nm tracers in systemic tissues (liver, kidneys, spleen, and lung), after injection into naive skin or day 7 melanoma. Comparison of cellular subtype exposure versus whole tissue exposure per tracer in the spleen (b), lungs (c), and liver (d). n = 4–6 mice per group. *Indicates significance by multiple t-tests (a) or linear fit of slope through all points is significantly nonzero; *indicates p < 0.05; ***indicates p < 0.005. (b–d); n = 4–9 animals.

FIG. 5.

(Lymph)angiogenic normalization normalizes dissemination of tumor-derived molecules to dLNs. (a) Total accumulation of each tracer within dLN as AUC from 4 to 72 h. (b) Proportional tracer exposure represented as AUC of tracer-positive cell counts from 0 to 72 h in dLN. *Indicates significance by multiple t-tests; *indicates p < 0.05; **indicates p < 0.01; ^{^}indicates slope significantly different from 0 with p < 0.05; n = 4–6 mice per group. AUC, area under the curve.

FIG. 6.

(Lymph)angiogenic normalization normalizes dissemination of tumor-derived molecules to systemic tissues. Ratio of dLN 500, 30, and 10 nm concentration relative to systemic concentration across 72 h after tracer injection into naive skin or day 7 melanoma. *Indicates significance by two-way ANOVA with Tukey's post hoc comparison; *indicates p < 0.05; ***indicates p < 0.005; n = 4–6 animals.

FIG. 7.

(Lymph)angiogenic remodeling impacts cellular distribution and exposure to actively transported model antigen in draining lymph nodes. (a) Number of 500 nm+migratory cells in dLN at 72 h relative to naive dLN cells. (b) Five hundred nanometer mean fluorescence index in 500 nm+migratory cells in dLN at 72 h, fold change relative to dLN cells. (c) Five hundred nanometer signal in CD45⁺ cells in LNs draining naive skin, and WT and VC melanomas. *Indicates significance by one-way ANOVA compared with naive value with p < 0.05; n = 4–6 animals.

FIG. 8.

(Lymph)angiogenic remodeling impacts cellular distribution and exposure to passively draining model antigen in draining lymph nodes. (a) Frequency of cells containing tracer relative to total 10 nm tracer accumulation in the dLN 4, 24, and 72 h after injection in naive skin or WT, VC, and R2 tumors. (b) Efficiency of tracer accumulation via comparison of % of cells containing 10 nm tracer relative to total accumulation within tumor draining lymph nodes. (c) Ten or thirty nanometer MFI of tracer+pDCs from 4 to 72 h after injection. (d) Ten and thirty nanometer MFI over time within CD11c⁺ and F4/80⁺ cells. *Indicates significance by two-way ANOVA with Tukey's post hoc test; *indicates p < 0.05; **indicates p < 0.01; ***indicates p < 0.005; ****indicates p < 0.001; ^#indicates significance by one-way ANOVA with Tukey's post hoc; ^#indicates p < 0.05; ^##indicates p < 0.01; n = 4–6 animals (per time point). MFI, mean fluorescence index; pDC, plasmacytoid dendritic cells.