Stratifying nonfunctional pituitary adenomas into two groups distinguished by macrophage subtypes

// Garima Yagnik 1, * , Martin J. Rutowski 1, * , Sumedh S. Shah 1 and Manish K. Aghi 1 1 Department of Neurosurgery, University of California San Francisco (UCSF), San Francisco, CA, USA * These authors contributed equally to this work Correspondence to: Manish K. Aghi, email: manish.aghi@ucsf.edu Keywords: macrophages; pituitary adenomas; M1; M2 Abbreviations: CD: Cluster of differentiation; EZH2: Enhancer of zeste homologue 2; NFPA: Non-functional pituitary adenoma; siRNA: small interference ribonucleic acid; TAM: Tumor-associated macrophage. Received: March 09, 2018      Accepted: February 21, 2019      Published: March 15, 2019 ABSTRACT Tumor-associated macrophages (TAMs) polarize to M1 and M2 subtypes exerting anti-tumoral and pro-tumoral effects, respectively. To date, little is known about TAMs, their subtypes, and their roles in non-functional pituitary adenomas (NFPAs). We performed flow cytometry on single cell suspensions from 16 NFPAs, revealing that CD11b + myeloid cells comprise an average of 7.3% of cells in NFPAs (range = 0.5%–27.1%), with qPCR revealing most CD11b + cells to be monocyte-derived TAMs rather than native microglia. The most CD11b-enriched NFPAs (10–27% CD11b + ) were the most expansile (size>3.5 cm or MIB1>3%). Increasing CD11b + fraction was associated with decreased M2 TAMs and increased M1 TAMs. All NFPAs with cavernous sinus invasion had M2/M1 gene expression ratios above one, while 80% of NFPAs without cavernous sinus invasion had M2/M1<1 ( P = 0.02). Cultured M2 macrophages promoted greater invasion ( P < 10 -5 ) and proliferation ( P = 0.03) of primary NFPA cultures than M1 macrophages in a manner inhibited by siRNA targeting S100A9 and EZH2, respectively. Primary NFPA cultures were of two types: some recruited more monocytes in an MCP-1-dependent manner and polarized these to M2 TAMs, while others recruited fewer monocytes and polarized them to M1 TAMS in a GM-CSF-dependent manner. These findings suggest that TAM recruitment and polarization into the pro-tumoral M2 subtype drives NFPA proliferation and invasion. Robust M2 TAM infiltrate may occur during an NFPA growth phase before self-regulating into a slower growth phase with fewer overall TAMs and M1 polarization. Analyses like these could generate immunomodulatory therapies for NFPAs.