The Role of Neuropilin 2 In The Angiogenesis and Lymphangiogenesis In Oral Squamous Cell Carcinoma

Abstract

Background: Oral squamous cell carcinoma (OSCC) remains a major clinical challenge, with five-year overall survival largely unchanged over four decades despite advances in surgery, radiotherapy, and immunotherapy. Approximately 60% of patients present with regional or distant metastasis at diagnosis, and cervical lymph node involvement reduces survival by half. Anti-angiogenic agents targeting VEGF/VEGFR signaling have produced only modest, inconclusive benefit in head and neck cancer trials, suggesting that strategies addressing the blood vasculature alone, without simultaneously disrupting the lymphangiogenic pathways that drive metastatic spread, are insufficient. Neuropilin-2 (NRP2) is a transmembrane co-receptor that uniquely enhances both VEGFR-2–mediated angiogenesis and VEGFR-3–mediated lymphangiogenesis, and is therapeutically modulated by its endogenous ligand semaphorin 3F (SEMA3F). This dual function positions NRP2 at the intersection of tumor growth and lymphatic dissemination. Hypothesis and Aims: We hypothesized that NRP2 is upregulated in OSCC-associated vessels and is necessary for tumor-associated angiogenesis and lymphangiogenesis, and that systemic SEMA3F inhibits OSCC vascularization. Aim 1 examined NRP2 expression and vascular density in human OSCC and tested the functional role of NRP2 in orthotopic mouse models. Aim 2 assessed the effect of exogenous SEMA3F on tumor angiogenesis. Methods: A Bergen HNSCC tissue microarray (n=188) was stained for NRP2, CD31 (blood vessels), and D2-40 (lymphatics) and quantified by QuPath digital image analysis, with markers dichotomized at the median and correlated with clinicopathological variables and five-year overall survival. Two syngeneic orthotopic OSCC isograft models were used: WT3 cells implanted in global Nrp2–/– mice and 4MOSC2 cells implanted in tamoxifen-inducible R26CreERT2;Nrp2f/f conditional knockout mice. Tumor microvessel density (MVD) and lymphatic vessel density (LVD) were quantified by CD31 and LYVE-1 immunohistochemistry. For Aim 2, mini-osmotic pumps delivered SEMA3F (0.3 mg/kg/day) or vehicle to wildtype mice bearing orthotopic WT3-Luc tumors, with response monitored by bioluminescence imaging. Results: In the human cohort, high tumor NRP2 expression was significantly associated with cancer recurrence (p=0.041) and predicted markedly reduced five-year overall survival (Log-rank p=0.001), whereas BVD and LVD did not predict survival. High LVD was significantly associated with advanced TNM stage (p=0.002) and lymph node metastasis (p=0.003). In the 4MOSC2 conditional knockout model, NRP2 loss produced a statistically significant 51% reduction in tumor-associated LVD (9.0 vs. 18.5 vessels/mm²; p=0.0465), with a non-significant trend toward reduced MVD (p=0.2354). The WT3 global knockout cohort showed concordant directional reductions in both compartments. Pooled cross-model analysis confirmed a significant 43% reduction in LVD (p=0.0112) with NRP2 loss and a trend toward reduced MVD (p=0.0781). Preliminary SEMA3F pump data demonstrated tumor regression in 3 of 4 treated mice versus 1 of 4 controls by bioluminescence and histology, with formal MVD comparison precluded by the resulting cohort size. Conclusions: These findings establish NRP2 as a clinically meaningful prognostic biomarker in OSCC and as a functionally relevant regulator of tumor-associated lymphangiogenesis across two independent orthotopic models. The lymphatic compartment appears preferentially sensitive to NRP2 loss, and preliminary SEMA3F data support further investigation of NRP2-axis blockade as a dual anti-angiogenic and anti-lymphangiogenic therapeutic strategy in OSCC.