Microglia-Derived Olfactomedin-like 3 Promotes Pro-Tumorigenic Microglial Function and Malignant Features of Glioma Cells

Under the influence of transforming growth factor-beta (TGFβ), glioma-associated microglia produce molecules that promote glioma growth and invasion. Olfactomedin-like 3 (Olfml3), a novel, secreted glycoprotein, is known to promote several non-CNS cancers. While it is a direct TGFβ1 target gene in microglia, the role of microglia-derived OLFML3 in glioma progression is unknown. Here, we tested the hypotheses that microglial Olfml3 is integral to the pro-tumorigenic glioma-associated microglia phenotype and promotes glioma cell malignancy. Using an Olfml3 knockout microglial cell line (N9), we demonstrated that Olfml3 is a direct target gene of all TGFβ isoforms in murine microglia. Moreover, loss of Olfml3 attenuated TGFβ-induced restraint on microglial immune function and production of cytokines that are critical in promoting glioma cell malignancy. Importantly, microglia-derived OLFML3 directly contributes to glioma cell malignancy through increased migration and invasion. While exposure to conditioned medium (CM) from isogenic control microglia pre-treated with TGFβ increased mouse glioma cell (GL261) migration and invasion, this effect was abolished with exposure to CM from TGFβ-treated Olfml3-/- microglia. Taken together, our data suggest that Olfml3 may serve as a gatekeeper for TGFβ-induced microglial gene expression, thereby promoting the pro-tumorigenic microglia phenotype and glioma cell malignancy.


Introduction
Glioblastoma (GBM) is the most common and aggressive primary brain tumor of adults, with a 5-year survival rate of approximately 5% [1]. Although immunotherapy has advanced the treatment of non-central nervous system (CNS) tumors, it has failed to overcome the substantial barrier of immune resistance in the glioma microenvironment. While immunoresistance and tumor progression are conferred by a confluence of factors, glioma-associated microglia/macrophages (GAM) play a critical role. As the most abundant infiltrating cells [2], GAM infiltration has been positively correlated with glioma grade [3], invasiveness [4], and resistance to therapy [5,6].
While the glioma-GAM signaling axis is complex, transforming growth factor-beta (TGFβ) isoforms have been recognized to substantially influence the pro-tumorigenic effects of GAM. β1 stimulates GAM to produce cytokines and growth factors promoting glioma growth [7] and invasion [8,9], whereas β2 suppresses GAM immune responses [10]. β3 promotes tumor invasion and augments β1 and β2 signaling [11]. A variety of approaches to inhibit TGFβ signaling [12] and glioma recruitment of GAM [13] have failed to show therapeutic efficacy in GBM [12], underscoring the need for refined therapeutic targets.
While the role of OLFML3 in GBM has just begun to be explored, depletion of OLFML3 in human glioma cells reduced GAM infiltration and extended survival in a glioma xenograft mouse model [23]. However, the function of microglia-derived Olfml3, and its contribution to the TGF-induced pro-tumorigenic GAM phenotype, is unknown. Therefore, this study aimed to (1) define the function of Olfml3 in microglia phenotype determination and (2) determine the effect of microglia-derived OLFML3 on the malignant phenotype of murine glioma cells.

OLFML3 Is Up-Regulated in GBM and Is a TGF Target Gene in Microglia
Examination of The Cancer Genome Atlas (TCGA) transcriptomic datasets revealed that OLFML3 mRNA expression increased with increasing glioma tumor malignancy. While low-grade gliomas (LGG, n = 592) had increased OLFML3 mRNA levels relative to normal brain ((Normal, n = 1141; p < 0.001), glioblastomas (GBM, n = 166) had increased OLFML3 mRNA expression relative to both LGG (p < 0.001) and normal brain (p < 0.001) ( Figure 1A).

Figure 1. OLFML3 is increased in GBM and is regulated by TGF in microglia. (A) OLFML3
mRNA is increased in low-grade glioma (LGG; n = 529) and glioblastoma (GBM; n = 166) relative to normal brain (Normal; n = 1141) in TCGA patient datasets. (B) Exposure to 1 (5 ng/mL; 48 h) increased Olfml3 mRNA 20-fold in a microglial cell line (N9) but did not affect mRNA expression in a mouse glioma cell line (GL261) or primary mouse brain endothelial cells. Fold was calculated via Ct and normalized to GAPDH; *** p < 0.001. (C) Exposure to each TGF isoform increased Olfml3 mRNA (5 ng/mL; 48 h); *** p < 0.001. (D) Representative immunoblot for OLFML3 protein in N9 cell lysate following exposure to vehicle (Veh) and TGF isoforms (5 ng/mL; 48 h). The optical density of OLFML3 protein in cell lysates was measured and normalized to the Ponceau stain. Relative optical densities (ROD) were expressed relative to vehicle-treated cells. No differences Figure 1. OLFML3 is increased in GBM and is regulated by TGFβ in microglia. (A) OLFML3 mRNA is increased in low-grade glioma (LGG; n = 529) and glioblastoma (GBM; n = 166) relative to normal brain (Normal; n = 1141) in TCGA patient datasets. (B) Exposure to β1 (5 ng/mL; 48 h) increased Olfml3 mRNA 20-fold in a microglial cell line (N9) but did not affect mRNA expression in a mouse glioma cell line (GL261) or primary mouse brain endothelial cells. Fold was calculated via ∆∆Ct and normalized to GAPDH; *** p < 0.001. (C) Exposure to each TGFβ isoform increased Olfml3 mRNA (5 ng/mL; 48 h); *** p < 0.001. (D) Representative immunoblot for OLFML3 protein in N9 cell lysate following exposure to vehicle (Veh) and TGFβ isoforms (5 ng/mL; 48 h). The optical density of OLFML3 protein in cell lysates was measured and normalized to the Ponceau stain. Relative optical densities (ROD) were expressed relative to vehicle-treated cells. No differences were measured between groups (p = 0.17). Comparisons based on one-way ANOVA with Tukey's Multiple Comparison Test. Bars represent group mean with standard error of the mean (SEM); data represent one of three independent experiments.

CRISPR/Cas9-Mediated Knockout of Olfml3 in Microglia
To determine the function of Olfml3 and its contribution to the TGFβ-induced protumorigenic phenotype in mouse microglial cells, we performed CRISPR-Cas9-mediated Olfml3 gene editing in N9 cells. Due to alternative splicing within the Olfml3 gene ( Figure 2A), exon 1 was targeted using the guide RNA, as outlined in Table 1. Forty base-pairs were deleted in Exon 1 ( Figure 2A) and verified via Sanger sequencing. This deletion resulted in an immediate stop codon. Knockout of Olfml3 was validated via qRT-PCR and Western blot ( Figure 2B,C). To begin to explore putative sources for increased OLFML3 in GBM, we confirmed Olfml3 expression in a mouse microglia cell line (N9) [24], a mouse glioma cell line (GL261) [25], and primary mouse brain endothelial cells. As previously demonstrated [26], exposure to 1 increased Olfml3 mRNA 22-fold in N9 cells relative to vehicle-treated cells (p < 0.001). However, neither GL261 nor endothelial cell Olfml3 mRNA levels were affected by 1 treatment ( Figure 1B). Importantly, exposure to all three TGF isoforms increased N9 Olfml3 mRNA (1: 20-fold, p < 0.001; 2: 13-fold, p < 0.001; 3: 33-fold, p < 0.001) ( Figure  1C). Exposure to TGF isoforms did not alter OLFML3 protein in N9 cell lysate (p = 0.17; Figure 1D). Given these findings, it is possible that increased OLFML3 mRNA expression in GBM is derived from microglia.

CRISPR/Cas9-Mediated Knockout of Olfml3 in Microglia
To determine the function of Olfml3 and its contribution to the TGF-induced protumorigenic phenotype in mouse microglial cells, we performed CRISPR-Cas9-mediated Olfml3 gene editing in N9 cells. Due to alternative splicing within the Olfml3 gene ( Figure  2A), exon 1 was targeted using the guide RNA, as outlined in Table 1. Forty base-pairs were deleted in Exon 1 ( Figure 2A) and verified via Sanger sequencing. This deletion resulted in an immediate stop codon. Knockout of Olfml3 was validated via qRT-PCR and Western blot ( Figure 2B,C).

Discussion
In this study, we began to uncover the role of Olfml3 in microglial function and glioma cell malignancy. Our data showed that microglial Olfml3 is a direct target gene of all TGFβ isoforms and plays a key role in TGFβ-induced, pro-tumorigenic microglia phenotype determination. Importantly, our data suggest that OLFML3 may directly contribute to glioma cell malignancy through increasing migration and invasion capacity. The myriad pro-tumorigenic effects of microglia-derived Olfml3 illuminates the potential for therapeutic development targeting the TGFβ-GAM-Olfml3 signaling axis in GBM.
OLFML3 is a secreted glycoprotein that belongs to the family of the olfactomedin domain-containing proteins [15]. It has been identified as an extracellular matrix protein [20], suggesting that the majority of OLFML3 is secreted. This aligns well with our observation that TGFβ exposure dramatically increases Olfml3 mRNA but not protein expression in the cell lysate.
While the biological function of olfactomedin domain-containing proteins remains incompletely characterized, growing evidence indicates that they are important for intercellular signaling and protein-protein interaction during development and disease. In particular, olfactomedin 4 (OLFM4), a member of a closely related subfamily of OLFML3, negatively regulates pro-inflammatory responses. OLFM4 knockout mice have enhanced bacterial clearance of Staphylococcus aureus and Escherichia coli through modulation of neutrophil killing [38], as well as Helicobacter pylori through disinhibition of NF-kB [39]. Moreover, Olfm4 deletion exacerbated inflammation and mucosal damage in a mouse model of colitis [40], further supporting its role in immune restraint. Similarly, our study suggests that Olfml3 may restrict microglial immune responses, thereby contributing to the markedly immunosuppressed tumor microenvironment of GBM.
Anti-tumor immune responses in GBM are limited through the combination of GAM and T cell dysfunction. Within the glioblastoma microenvironment, GAMs exert immunosuppressive functions through direct cell-cell interactions and release of soluble factors. Importantly, microglia function as antigen-presenting cells in the CNS, requiring up-regulation of MHC II for T cell activation [41]. However, this activity is suppressed in GBM [34]. In fact, MHC I and MHC II molecules were absent in 50% of GBM samples [42], with specific suppression of GAM MHC II occurring through TGFβ signaling. In line with these findings, we demonstrated that Olfml3 deletion abolished β1-mediated transcriptional suppression of MHC II, which may improve microglial antigen presentation function. Additionally, loss of Olfml3 may mitigate T cell turnover. In the glioma microenvironment, GAM perpetuate CD4 + /CD8 + T cell apoptosis through secretion of CD95 [35], the ligand for the T cell death receptor Fas, and IL-6, a potent inducer of Fas [43]. Strikingly, Olfml3 deletion abolished microglial secretion of CD95. While exposure to TGFβ increased secretion in isogenic control cells, CD95 was undetectable in the media of Olfml3 -/in all conditions. Moreover, loss of Olfml3 attenuated secretion of IL-6. These findings, coupled with the dependency of microglial Olfml3 expression upon TGFβ1-SMAD2-mediated de novo protein synthesis [26], suggest that Olfml3 functions as a gatekeeper for TGFβ-induced effects on microglia-mediated immunity.
Importantly, targeting the immunomodulatory effects of Olfml3 may enhance efficacy of currently available immunotherapies. Expression of the immune checkpoint molecule programmed cell death ligand-1 (PD-L1) is inversely correlated with overall patient survival in GBM [44]. While there are many ongoing Phase I and II clinical trials targeting PD-1/PD-L1, preliminary results in patients with recurrent GBM demonstrate unpredictable efficacy, with meager to no survival benefit compared to standard therapies [45][46][47]. As IL-6 is necessary and sufficient for PD-L1 induction [48], we speculate that therapeutic targeting of Olfml3 may enhance current immunotherapeutic approaches for GBM patients. In support of this hypothesis, recent work has demonstrated that anti-OLFML3 therapy in conjunction with anti-PD1 immunotherapy increased overall survival in a mouse model of colorectal cancer [21]. Thus, inhibition of microglial Olfml3, in tandem with immune checkpoint blockade, may yield improved patient survival in GBM.
Treatment resistance is also governed by the diffuse infiltrative capacity of glioblastoma. Our results support the hypothesis that microglia-derived OLFML3 acts as a paracrine factor facilitating glioma cell invasion. Glioma cell migration and invasion were only affected following 48 h exposure to rhOLFML3, suggesting that OLFML3 may regulate key signaling pathways in glioma cells. This is consistent with general properties of the olfactomedin protein family, which are known to interact with multiple protein binding partners and regulate several cell signaling pathways [16]. This effect is in contrast to recent work that demonstrated that glioma-derived OLFML3 is a GAM chemoattractant [23]. Thus, OLFML3 may have cell type-specific functions within the glioma microenvironment that collectively support tumor growth. Moreover, OLFML3 expression is likely regulated by multiple molecules. The circadian regulator CLOCK and its partner BMAL1 have been identified to promote transcriptional upregulation of OFLML3 in GBM cells [23]. Remarkably, TGFβ signaling is necessary for normal circadian clock function [49]. In fact, TGFβ induces expression of the core clock gene Per1 [50]. The interaction between CLOCK, BMAL1, and molecules of the canonical TGFβ signaling pathway in GBM is unknown. However, it is interesting to consider the interconnectedness of these systems and their possible synergistic promotion of OLFML3 expression in microglia and glioma cells alike.
Herein, our data demonstrated that microglia-derived Olfml3 may contribute to glioma cell malignancy through intrinsic and extrinsic mechanisms. Silencing of Olfml3 attenuated the pro-tumorigenic microglial secretome, as well as mitigating glioma cell malignancy in vitro. Together, these results provide a rationale for further exploration of anti-OLFML3 therapeutic strategies in GBM.

Cell Culture and Reagents
The N9 microglial cell line [24] was generously donated from Jyoti Watters at The University of Wisconsin School of Veterinary Medicine. N9 cells were submitted to ATCC for authentication and confirmed to be of murine origin. N9 cells were maintained in DMEM (Gibco TM , ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco TM , ThermoFisher Scientific, Waltham, MA, USA) and 1% Penicillin/Streptomycin (Gibco TM , ThermoFisher Scientific, Waltham, MA, USA). The GL261 mouse glioma cell line was obtained from the Developmental Therapeutics Program Repository at the National Cancer Institute. GL261 cells were maintained in RPMI 1640 (Gibco TM , ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco TM , ThermoFisher Scientific, Waltham, MA, USA). All cells were confirmed to be Mycoplasma-free and maintained at 37 • C in a humidified incubator with 5% CO 2 . All cells were used below passage 15 and within 1 month after thawing.

CRISPR/Cas9-Mediated Olfml3 Knockout
Generation of the Olfml3-knockout (Olfml3 -/-) microglial cell line was achieved using the CRISPR-Cas9 gene editing system. All reagents were purchased from Integrated DNA Technologies (IDT; Coralville, IA, USA) and used according to the manufacturer's recommendations. Briefly, a guide-RNA (Table 1), targeted to exon 1 of Olfml3 and the tracrRNA-ATTO-550, was duplexed and mixed with recombinant Cas9 enzyme (IDT, Coralville IA, USA) to form the ribonucleoprotein (RNP) complex. The RNP complex was transfected into cells using Lipofectamine CRISPRMAX (ThermoFisher Scientific, Waltham, MA, USA) transfection reagent. Then, 24 h following transfection, cells were subjected to fluorescence-activated cell sorting and individual ATTO-550-positive cells were sorted into a single well of a 96-well plate. Each single cell created a clonal population, whereby Sanger sequencing confirmed Olfml3 editing within the defined region of exon 1. Western blot analysis confirmed successful Olfml3 knockout. An isogenic control line was generated using the same parameters described above without the addition of the gRNA for Olfml3.

Generation of Anti-OLFML3 Antibody
We generated an anti-OLFML3 polyclonal antibody using the commercially available service from Cocalico Biologicals (Reamstown, PA, USA). Briefly, recombinant OLFML3 protein was generated as described above, purified, and electrophoresed on a 12% SDS-PAGE gel. The OLFML3 band was excised and sent to Cocalico for inoculation of rabbit host. Serum antibody titer was tested until endogenous OLFML3 was detectable using wild-type N9 microglia and rhOLFML3 as a positive control. Final exsanguination was carried out and antibody was purified from the final serum volume.

Quantitative Real-Time PCR
Cells were grown to 80% confluency and treated with human recombinant TGFβ isoforms (5 ng/mL; β1: 100-21, PeproTech, Cranbury, NJ, USA; β2: PHG9114, Life Technologies, ThermoFisher Scientific, Waltham, MA, USA; β3: SRP3171, Sigma-Aldrich, St. Louis, MO, USA) or vehicle (PBS; ThermoFisher Scientific, Waltham, MA, USA) once every 24 h for a total of two treatments (48-h total incubation). We evaluated Olfml3 mRNA expression in murine microglia cells following 24-, 48-, and 72-h exposure to TGFβ. We observed the greatest increase in Olfml3 mRNA at 48 h; thus, all subsequent experiments were performed at this timepoint. Cells were pelleted and RNA was isolated with the Direct-zol MiniPrep kit (Zymo Research, Irvine, CA, USA) according to manufacturer's specifications. Using one microgram-purified DNase-treated RNA, cDNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher-Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA). Primer sets were designed using NCBI primer design (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi) (accessed on 4 August 2019) and purchased through IDT (Table 1). Primer validation was performed using a 4× cDNA serial dilution series from isogenic control microglia as template. The efficiency and fit of the generated curves were evaluated; primer sets that did not produce efficiency of at least 0.9 and R 2 value of 0.95 from the cDNA dilution series were rejected. Only experimental quantification cycle (Cq) values that fell within the boundaries of the validated curves were used for analysis.
The qPCR reactions consisted of primer pairs at a final concentration of 200 nM, 50 ng cDNA template, and 2× SSoAdvanced Universal SYBR Green Superix (Bio-Rad, Hercules, CA, USA) per manufacturer's protocol on a CFXConnect (Bio-Rad, Hercules, CA, USA) machine as previously described [51]. All reactions were run as 20-µL triplicates, and the average Cq was used as the data point for a given sample. The mRNA expression values were quantified by the 2 −∆∆Ct method, whereby ∆CT = 18S Ct−gene of interest Ct.

Immunofluorescence and Confocal Microscopy
Isogenic control and Olfml3 -/microglia were cultured on sterile glass coverslips treated with fibronectin. Cells were fixed using 4% (w/v) paraformaldehyde (Millipore Sigma, Burlington, MA, USA), washed three times for 5 min at RT, and permeabilized with 0.1% Triton X-100-Tris-buffered saline (TBST) for 15 min at RT and blocked for 2 hours at RT with normal goat serum (5% w/v) and bovine serum albumin (1% w/v) in TBST. Cells were incubated in primary antibody solution (mouse monoclonal anti-TMEM119 (BioLegend, San Diego, CA, USA #853302; 1:1000) in fresh blocking buffer) overnight at 4 • C. Cells were washed three times for 5 min at RT and incubated in secondary antibody solution for 1 hour at RT (IgG (heavy and light) anti-mouse Alexa Fluor 555 (Molecular Probes, Invitrogen, Carlsbad, CA, USA; 1:1000) in fresh blocking buffer). Cells were washed three times for 5 min at RT and mounted with Vectashield with 4 5-diamidino-2phenylindole (DAPI) (Vector Labs, Burlingame, CA, USA). Images were captured via Leica TCS Sp8 STED 3× confocal microscope.

Western Blot Analysis
Whole cell protein samples were lysed using RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 1% SDS, 1× protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA)). The cellular homogenate was rotated for 30 min at 4 • C and centrifuged at 12,000× g for 10 min. Protein concentrations of the resultant supernatants were determined using the BCA assay (Pierce Biotechnology, Rockford, IL, USA). Forty micrograms of protein were loaded, electrophoresed on 15% SDS-PAGE gels, and transferred to nitrocellulose membranes overnight. All blots were incubated with Ponceau S (Sigma, St. Louis, MO, USA) to validate equal loading and transfer across all lanes. Membranes were blocked overnight at 4 • C in 5% fat-free milk. Anti-OLFML3 primary antibody was diluted (1:1000) in Tris-buffered saline + Tween-20 (TBST) with 1% fat-free milk and applied to the membrane overnight at 4 • C with gentle rocking. The membranes were washed three times in TBST and incubated in horseradish peroxidase (HRP)-conjugated secondary antibody (1:20,000; Cell Signaling Technology, Danvers, MA, USA) for 1 h at RT with gentle rocking. The HRP substrate for enhanced chemiluminescence (ThermoFisher Scientific, Waltham, MA, USA) was applied immediately prior to exposure. Band densitometry was performed using Image Lab (Bio-Rad, Hercules, CA, USA) and normalized to the Ponceau as a protein loading and transfer control. Optical densities were normalized to vehicle-treated conditions and expressed as relative optical densities (ROD). All experiments were independently repeated in triplicate.

Murine Protein Arrays
Isogenic control and Olfml3 -/microglia were grown to 80% confluency and treated with human recombinant TGFβ isoforms as described above (5 ng/mL; 48 h), followed by serum starvation for 12 h (0.1% FBS). The cell media were aspirated, centrifuged at 350 g for 5 min, and concentrated using Pierce PES protein concentrator columns (ThermoFisher Scientific, Waltham, MA, USA). Whole cell protein samples were treated as described for Western blot. Cell lysate and media samples were sent for analysis by RayBiotech Life (Peachtree Corners, GA, USA) with standard quality control. In brief, Quantibody®Mouse Full Testing Service (QAH-INF-1) utilized two non-overlapping arrays of antibody pairs to quantify selected molecules. RayBiotech confirmed no cross reactivity between antibody pairs and standard controls.

Cell Viability
Cell viability was performed using the Cell Titer Glo®2.0 Assay (Promega, Madison, WI, USA) according to the manufacturer's protocol. Cells (2.5 × 10 4 ) were seeded in 96-well, black-sided plates. Titer Glo®reagent was added to each well and the plate was incubated for 10 min at RT on a plate shaker, followed by luminescence recording via plate reader (BioTek800TS). Optical densities were recorded for six replicates per condition and the average optical density of media alone (blank) was subtracted from all experimental conditions. Three independent experiments were performed.
Similar to Cell Titer Glo®, cells (2.5 × 10 4 ) were seeded in 96-well, black-sided plates and cultured for 48 h. MTS reagent was added to each well and the plate was incubated for 10 min at RT on a plate shaker, followed by absorbance reading via plate reader (BioTek800TS) at 590 mm. Optical densities were recorded for six replicates per condition. Three independent experiments were performed.

Transwell Migration and Invasion Assays
The modified Boyden chamber assay was used for analysis of cell migration and invasion. Migration assays were performed using cells (microglia: 2 × 10 5 ; GL261: 5 × 10 4 ) suspended in serum-free culture medium and seeded into 24-well Transwell inserts with