Neuropilin-2 Isoforms Regulate Distinct Functions of Tumor-associated Macrophages in Breast Cancer

Abstract

Abstract Introduction Neuropilins are neural guidance molecules which contribute to tissue development. We have shown that the two isoforms of neuropilin-2 endow opposing functionality to tumor cells due to distinct signaling pathways, with Nrp2b promoting metastatic behavior. Due to the role of macrophages (Mθ) in organogenesis and metastasis, we examine the role of Nrp2 isoforms in these cells. Methods Stable shRNA knockdown of Nrp2a or Nrp2b in Raw264.7 Mθ were generated. Phagocytosis, cytokine production, and migration were assessed in knockdowns in response to stimuli (TGFβ, HGF, VEGF, IL-10, IFNγ, LPS, β-glucan). Nrp2 isoforms in Mθ from mouse mammary tissue or EO771-induced mammary tumors were measured by FACS and RT-PCR. Mθ were phenotyped via FACS for wound-healing or inflammatory markers. Single-cell (sc)qPCR for a 96 gene panel examining components of signaling pathways, autophagy, metabolism, and pro/anti-tumor responses was performed on 576 CD11b+F4/80+ TAMs FACS-sorted from EO771 tumors. Results Nrp2b expression was significantly upregulated in TAMs compared to Mθ of the blood, spleen, or mammary tissues. 56% of the scqPCR transcripts analyzed were significantly altered in Nrp2bHigh vs. Nrp2bLow TAMs, and computation analysis (PCA/tSNE) revealed two distinct TAM subsets enriched for Nrp2b. Nrp2b+ Raw264.7 cells showed decreased ability to phagocytose tumor cells, but increased rates of division and migration in response to growth factors compared to Nrp2a+ counterparts. Conclusions We demonstrate, for the first time, that the principle neuropilin-2 isoforms are present in Mθ, regulate unique functionality, and that Nrp2b+ TAMs are both upregulated in mammary tumors and represent a phenotypically unique subtype.