**Mizuto Sato, Ryota Tamura, Haruka Tamura, Taro Mase, Kenzo Kosugi, Yukina Morimoto, Kazunari Yoshida and Masahiro Toda \***

Department of Neurosurgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; mizuto.sato@gmail.com (M.S.); moltobello-r-610@hotmail.co.jp (R.T.); rovin124th@gmail.com (H.T.); mstr.komed05241996@gmail.com (T.M.); kensan03977@yahoo.co.jp (K.K.); yukinaxnashiko@yahoo.co.jp (Y.M.); kazrmky@keio.jp (K.Y.) **\*** Correspondence: todam@keio.jp; Tel.: +81-3-3353-1211

Received: 15 April 2019; Accepted: 14 May 2019; Published: 16 May 2019

**Abstract:** Cavernous sinus (CS) invasion is an aggressive behavior exhibited by pituitary neuroendocrine tumors (PitNETs). The cause of CS invasion in PitNETs has not been fully elucidated. The tumor immune microenvironment, known to promote aggressive behavior in various types of tumors, has not been examined for PitNETs. Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) signaling is strongly associated with the tumor immune microenvironment. In the present study, these molecular and histopathological characteristics were examined in invasive non-functional PitNETs (NF-PitNETs). Twenty-seven patients with newly diagnosed NF-PitNETs (with CS invasion: 17, without CS invasion: 10) were analyzed by immunohistochemistry for VEGF-A/VEGFR1 and 2, hypoxia-inducible Factor (HIF), tumor-infiltrating lymphocytes, immunosuppressive cells including regulatory T cells (Tregs) and tumor-associated macrophages (TAMs), and immune checkpoint molecules. Previously validated tumor proliferation markers including mitotic count, Ki-67 index, and p53 were also analyzed for their expressions in NF-PitNETs. VEGF-A and VEGFR1 were expressed on not only vascular endothelial cells, but also on tumor cells. The expressions of VEGF-A and VEGFR1 were significantly higher in NF-PitNETs with CS invasion. The number of TAMs and the expression of PD-L1 were also significantly higher in NF-PitNETs with CS invasion than in NF-PitNETs without CS invasion. The high expression of VEGF-A and VEGFR1 and associated immunosuppressive microenvironment were observed in NF-PitNETs with CS invasion, suggesting that a novel targeted therapy can be applied.

**Keywords:** pituitary neuroendocrine tumors; VEGF; Treg; TAM; PD-1; PD-L1
