**4. Discussion**

CS invasion is a commonly demonstrated aggressive behavior exhibited by PitNETs [24–26], and this property has been recommended to describe aggressive PitNETs in the revised 2017 World Health Organization (WHO) classification [1]. Recently, Rutkowski et al. [27] re-emphasized the importance of classical histological characteristics. They demonstrated that mitotic activity, extensive p53 staining, and Ki-67 index were associated with poor prognosis [27]. However, in the present study, these classical histological characteristics did not show a correlation with CS invasion.

In contrast, our data suggested that VEGF-A/VEGFR1 expressions could be associated with CS invasion. The relationship between the expressions of VEGF-A/VEGFR1 and the prognosis of PitNETs has been previously discussed [28,29]. VEGF-A and VEGFR1 are known to contribute to the tumor cell growth of PitNETs [28,30,31]. Some studies have demonstrated that VEGFR2 is widely expressed in NF-PitNETs, with aggressive behavior such as suprasellar extension in NF-PitNETs [3,32]. MVD, characterized by CD31 immunopositivity and VEGF-A expression, reflected poor prognosis of NF-PitNETs [4]. Our findings corroborate with the findings of these studies. Importantly, VEGF-A and VEGFR1 were expressed on not only endothelial cells, but also on tumor cells, which have been previously confirmed using PitNETs cell line HP75 [33,34]. Tumor cells expressing VEGFR1 themselves release VEGF-A, and an autocrine regulatory function for VEGF in tumor growth in PitNETs is plausible.

Xiao et al. demonstrated rapid and hemorrhagic transformation in PitNETs via the HIF-1α hypoxic signaling pathway [35]. Interestingly, there was no significant correlation in the expression levels of HIF-1α and VEGF mRNA in PitNETs, although VEGF-A is mainly induced by HIF-1α [35]. RSUME, a small RWD-domain containing protein, was reported to play an important role in tumor neovascularization by regulating VEGF-A production in PitNETs [36–39]. The lack of correlation between VEGF-A and HIF-1α observed in the present study was in accordance with previous observations [35]. It is noteworthy that Barbagallo et al. [40] demonstrated that circSMARCA5, which acts as circular RNA for the splicing factor Serine and Arginine Rich Splicing Factor 1 SRSF1 in glioblastomas, is an upstream regulator of VEGF-A. Other regulators, such as circSMARCA5, might be involved in the VEGF-A expression of PitNETs.

Other aggressive characteristics, such as cystic change, were previously correlated with upregulated VEGF-A [29]. However, controversy exists over the relationship between hemorrhagic change and VEGF-A expression [29,41]. VEGF-A was not associated with cystic or hemorrhagic change in the present study. The cause for the discrepancy in the status of cystic and hemorrhage change between previous relevant studies and this study remains unclear. It could be attributed to the small sample size, highly heterogeneous PitNETs, and the difference between the analytical methods of immunohistochemistry and quantitative analysis (RT-PCR and western blot). Although VEGF-A is widely considered as a marker of poor prognosis in PitNETs, Takada et al. could not find significant correlations between vascularity and other clinical and endocrinological parameters, suggesting that angiogenesis is not essential for growth or invasiveness of PitNETs [42]. Further analysis using a large number of patients might elucidate the role of VEGF-A in PitNETs.

There is a lack of studies related to the tumor microenvironment of PitNETs. PD-L1 RNA and protein expression were significantly increased in recurrent functioning (growth

hormone and prolactin-expressing) PitNETs compared with in NF-PitNETs (null cell and silent gonadotroph). Tumor infiltrating CD8 (+) lymphocytes were positively correlated with increased PD-L1 expression [43,44]. In the present study, most NF-PitNETs without CS invasion showed low PD-L1 expression score and low CD8 (+) lymphocyte count, which was compatible with previous studies [43,44]. However, some NF-PitNETs with CS invasion demonstrated a high PD-L1 expression score and a high number of CD8 (+) lymphocyte counts. Interestingly, PD-1/PD-L1 expressions are known to be associated with VEGF-A exposure [45,46].

Tumor size in NF-PitNETs is positively correlated with the number of CD68+ macrophages [47]. Macrophages express different functional programs in response to microenvironmental signals, which is defined as M1/M2 polarization [48]. CD68 antigen is expressed on both M1 and M2 macrophages, and CD163 is a specific marker for M2 macrophages [48]. Although the number of CD163 + M2 macrophages (TAMs) was not associated with the tumor volume, TAMs were associated with CS invasion in the present study. TAMs produce matrix metalloproteinase (MMP)-9 [48] that might promote the invasive behavior of PitNETs. Furthermore, VEGF-A is known to promote the immunosuppressive microenvironment [49], as well as the migration and differentiation of TAMs from immature myeloid cells [50,51].

Upregulation of VEGF-A induces VEGFR-2-expressing Tregs and also promotes their recruitment to the tumor microenvironment via over-expression of chemokine—chemokine ligand 28 by tumor cells [52]. Foxp3/CD8 ratio are known to correlate with the immunosuppressive microenvironment [46, 53]. In the present study, the Foxp3/CD8 ratio was strongly associated with CS invasion, which might serve as a new biomarker of invasive NF-PitNETs.

The results obtained in the present study suggest that VEGF-A/VEGFR1 expression can be a treatment target. Blocking VEGF-A can regulate immunosuppressive cells such as TAMs. However, PitNETS with high PD-L1 expression deserve special attention as they correlate to poor outcomes of certain chemo- and immunotherapies [45,54–57].

A limitation of this study was the paucity of the number of patients. Other invasive markers such as MMP-9 and -14 were previously correlated with the hemorrhage and invasive behavior of PitNETs [41,58]. Future studies should analyze the role of these MMPs in a large number of patients to confirm the findings of this study. In addition, NF-PitNETs are morphologically heterogeneous. The new classification byWHO in 2017 was based on hormone immunohistochemistry and pituitary transcription factors. Although gonadotroph adenoma is the most common subtype among non-functional adenomas [1,59], some cases with thyroid stimulating hormone (TSH), growth hormone (GH), adrenocorticotropic hormone (ACTH), or prolactin (PRL) stainings behave as silent adenomas with no secretion [1]. The relationship between VEGF/VEGFR signaling, tumor microenvironment, and the above-mentioned hormonal and transcriptional characteristics should be investigated in future studies.
