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The effects of tumor-associated neutrophils (TANs) on glioblastoma biology remain poorly understood. Flow cytometric analysis of 5 newly diagnosed glioblastoma fresh tissue specimens surprisingly revealed a high fraction (21.0-34.1%) of TANs expressed MHCII, a marker of antigen-presenting cells not classically associated with neutrophils and not expressed in matched peripheral blood (PBNs). Transcriptomic profiling confirmed that patient TANs upregulated expression of MHCII subunits (HLA-DR), chaperones (HLA-DM), and costimulatory ligands (CD86/83). Ex vivo cocultures further demonstrated that TANs activated patient-matched naïve T cells in an MHCII-dependent manner, while PBNs did not (CD25 MFI fold-change: 1.1 vs 3.2, p< 0.001). The antitumoral relevance of this property was confirmed in a syngeneic mouse glioma model, wherein αLy6G-mediated neutrophil depletion in T-cell-competent mice (Balb/c, n= 13) yielded endpoint tumors that had reduced CD8+ T cell infiltration (p= 0.0024) and were 2.4-fold larger by BLI (p= 0.0383) than controls, but had no bearing on tumor burden in T-cell-deficient (athymic) mice. Given the absence of MHCII+ neutrophils in circulation, we interrogated the inducibility of this phenotype by tumor-conditioned media in murine marrow/blood, finding that only immature Ly6Glow bone marrow neutrophils had sufficient plasticity to express MHCII and process foreign antigen as measured by DQ-ovalbumin uptake/proteolysis. Because of the non-inducibility of PBNs, we investigated the hypothesis that MHCII+ TANs were recruited to the glioblastoma microenvironment from adjacent skull bone marrow by labeling this space with CFMDA in tumor-implanted mice; at 72h, skull marrow-derived neutrophils contributed disproportionately to MHCII+ TANs compared to all TANs (41.7% vs 9.3%). As confirmation, in the first-ever scRNA-seq of human TANs, we demonstrated via pseudotime analysis that MHCII+ neutrophils represent a developmental lineage seen in TANs but not matched PBNs. Given the immunostimulatory effects of this population, mechanisms to promote neutrophil egress and tumor infiltration from the skull marrow may have therapeutic value.

Cancer-associated fibroblasts (CAFs) constitute a key component of the tumor microenvironment. Pro-tumoral cancer-associated fibroblasts were presumed absent in glioblastoma given the lack of brain fibroblasts. Using single-cell RNA sequencing we identified CAFs in patient GBMs. CAFs were identified using a negative selection strategy to filter endothelial, epithelial, immune cells and pericytes and for the positive expression of previously defined CAF markers. Copy number variation (CNV) analysis was performed to distinguish CAFs from malignant cells. Single-cell spatial transcriptomics from 16 GBM patients confirmed the proximity of CAFs to mesenchymal GBM stem cells (GSCs), endothelial cells, and M2-macrophages. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and transcriptomic profile. CAFs were chemotactically attracted to GSCs and CAFs induced GSC proliferation. To identify CAF and GSC interaction mediators, we created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGFB as mediators of GSC effects on CAFs, and osteopontin and HGF as mediators of CAF-induced GSC enrichment. Furthermore, CAFs were found to induce M2-macrophage polarization by producing the EDA fibronectin variant which binds macrophage toll-like receptor 4 (TLR4) in a targetable manner. Glioblastoma CAFs were enriched in the subventricular zone which houses the neural stem cells that houses GSCs. Including CAFs in GSC-derived xenografts induced in vivo tumor growth and reduced survival in two different xenograft models. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target for Glioblastomas.

Objective The 2017 World Health Organization classification of pituitary adenomas identified the plurihormonal PIT-1–positive (PP1) adenoma as a distinct subtype. The reported data suggest that PP1 adenomas encompass the former class of silent subtype 3 (SS3) adenomas and might have an aggressive phenotype. In the present study, we summarized the current clinical data on PP1 and SS3 adenomas and compared the reported data with the data from a single institutional cohort. Methods Medline and Google Scholar were searched from 1990 to 2020 for clinical series of PP1 and SS3 adenomas in accordance with the PRISMA (preferred reporting items for systematic reviews and meta-analyses) guidelines. Studies were included if they had reported pituitary pathology as PP1 or SS3 adenomas and had reported the clinical outcomes after surgical intervention. To better define the PP1 phenotype compared with non-PP1 adenomas, we also reviewed the adenomas treated surgically at our institution from 2012 to 2019. Results Of all the tumors reported in the studies as PP1 or SS3, 99% were macroadenomas and 18% were giant adenomas (>4 cm). Of the reported patients, 31.8% had received radiotherapy, and 22.9% had undergone multiple surgeries for their pituitary tumor. In our single-center experience, 20 patients had an adenoma that met the criteria for a PP1 adenoma. Compared with the 1146 non-PP1 tumors, the PP1 tumors did not show statistically significant differences in the extent of resection, size, number of previous surgeries, future reoperations, rate of radiotherapy, p53 staining, or MIB-1 labeling index. Conclusions The findings from the present large, single-center study comparing PP1 and non–PP1 adenomas do not suggest that PP1 tumors are more aggressive. Further work is warranted to identify the pathologic subtypes of pituitary adenomas that are consistently more clinically aggressive.

INTRODUCTION Life expectancy has increased over the past century, shifting the demographic distribution towards older age groups. Elderly patients comprise up to 14% of patients with pituitary tumors, with most lesions being nonfunctioning adenomas (NFPAs). Here, we compare clinical and surgical outcomes and post-operative complications between non-elderly adult (age ≥ 18 years and ≤ 65 years) and elderly (age > 65 years) NFPA patients. METHODS Retrospective review of 908 patients undergoing transsphenoidal surgery for NFPA at a single institution from 2007-2019. RESULTS Elderly patients represented 32.4% of patients. Both groups were similar in gender (57.3% vs 60.5% male;P=0.4), tumor size (2.56 vs 2.46 cm;P=0.2), and cavernous sinus invasion (35.8% vs 33.7%;P=0.6). Regarding post-operative outcomes, length of stay (1 vs 2 days; P=0.5), extent of resection (59.8% vs 64.8% GTR;P=0.2), CSF leak requiring surgical revision (4.3% vs 1.4%;P=0.06), 30-day readmission (8.1% vs 7.3%;P=0.7), infection (3.1% vs 2.0%;P=0.5), and new hypopituitarism (13.9% vs 12.0%;P=0.3) were similar between both groups. Elderly patients were less likely to have adjuvant radiation (8.7% vs 16.3%;P=0.009), future re-operation (3.8% vs 9.5%;P=0.003), and post-operative diabetes insipidus (DI) (3.7% vs 9.4%;P=0.002), and more likely to have post-operative hyponatremia (26.7% vs 16.4%;P< 0.001) and new cranial nerve deficit (1.9% vs 0.0%;P=0.01). Elderly patients’ post-operative sodium peaked and troughed on POD3 (mean=138.7 mEq/L) and POD9 (mean=130.8 mEq/L), respectively, compared to non-elderly patients (peak POD2 mean=139.9 mEq/L, trough POD8 mean=131.3 mEq/L). CONCLUSION Our analysis revealed that elderly patients experienced more post-operative hyponatremia, while non-elderly patients experienced more post-operative DI. Elderly patients also experience later peak and trough in serum sodium, suggesting age-related differences in stalk-related morbidities of NFPA resection. Overall, our results show that transsphenoidal surgery for NFPA in elderly patients is safe with low complication rates. We hope our results will guide discussions with elderly patients regarding possible risks and outcomes.

Cancer-associated fibroblasts (CAFs) constitute a key component of the tumor microenvironment. While pro-tumoral CAFs have been identified in some cancers, CAFs had been presumed absent in glioblastoma given the lack of brain fibroblasts. We found that serial trypsinization of primary glioblastoma cultures yields cells that morphologically resemble fibroblasts and transcriptomically resemble CAFs as shown by bulk RNA-seq and single-cell RNA-seq. Moreover, Single-cell RNA-seq from patient GBMs showed a mesenchymal lineage for CAFs. We demonstrate that Glioblastoma CAFs are chemotactically attracted to glioblastoma stem cells (GSCs) and CAFs enriched GSCs. To identify CAF/GSC interaction mediators, we created a resource of inferred crosstalk by mapping the expression of receptors to their cognate ligands/agonists. This analysis suggested PDGF-b and TGF-b as mediators of GSC recruitment and proliferation of CAFs, and osteopontin and hepatocyte growth factor (HGF) as mediators of CAF-induced GSC enrichment, hypothesis confirmed by blocking antibodies. Glioblastoma CAFs also induce hypertrophied vessels and M2 macrophage polarization, the latter through unique CAF production of the EDA fibronectin variant which binds macrophage toll-like receptor 4 (TLR4) in a targetable manner. Glioblastoma CAFs were enriched in the subventricular zone which houses the neural stem cells that produce GSCs. Depleting CAFs in GSC-derived xenografts slowed their in vivo growth. These findings are among the first to identify glioblastoma CAFs and reveal their involvement with GSCs, making them an intriguing target.

Among clinical analyses, elevated neutrophil-lymphocyte ratio has been correlated with poor outcomes of glioblastoma patients independent of other prognostic factors. Additionally, our flow cytometric studies of primary patient samples found neutrophil percentage to be significantly higher in higher-grade glioma versus lower-grade glioma. Tumor-associated neutrophils (TANs) comprise less than 2% of the glioblastoma microenvironment. While TANs were initially considered passive bystanders due to their short-lived nature, investigation of TANs in other cancers revealed distinct pro-tumoral roles. Therefore, we transcriptomically characterized glioblastoma TANs and defined their oncologic effects. Transcriptomic analysis of patient-matched TANs versus peripheral blood neutrophils revealed that functionally quiescent circulating neutrophils infiltrate IDH1-wild type glioblastoma via leukotriene B4 chemoattraction, where tumor cells morphologically and transcriptomically activate them to become TANs. Single-cell RNA-sequencing of patient-matched TANs and peripheral blood neutrophils revealed a subset of tumor-activated neutrophils which adopt a pro-tumoral secretory phenotype, marked by activation of the IL-17 signaling pathway and high osteopontin production. Using immunofluorescence stains of primary patient glioblastoma sections, we demonstrated that activated, myeloperoxidase-positive TANs reside in the perivascular niche of glioblastoma in close proximity to glioblastoma stem-like cells (GSCs) and CD31-positive endothelial cells. Further analysis in culture demonstrated that TAN-secreted osteopontin drives the formation, self-renewal, and proliferation of GSC-containing neurospheres. These results were validated using a syngeneic stem cell-derived IDH1-wild type murine glioblastoma model in vivo. Thus, while TANs are rare in glioblastoma, their enrichment in the glioblastoma perivascular niche uniquely positions them to support the GSCs that are crucial to therapeutic resistance of GBM.

Glioblastoma is a primary malignant brain tumor with a median survival under two years. The poor prognosis glioblastoma caries is largely due to cellular invasion, which enables escape from resection and drives inevitable recurrence. While numerous factors have been proposed as the primary driving forces behind glioblastoma’s ability to invade adjacent tissues rapidly, little attention has been paid to the alterations in tumor cell metabolism needed for tumor cells to thrive in isolation in the peritumoral white matter. To improve on biased 2D cell culture studies, we defined the links between glioblastoma metabolism in invasion using unbiased CRISPR screens and metabolomics performed in biomimetic 3D hydrogels and regional biopsies of patient glioblastomas. Through these platforms, we identified targetable metabolic factors which drive cellular invasion in glioblastoma. Metabolomics revealed cystathionine to be selectively enriched in the invasive tumor front of both site-directed biopsies (6-fold change), and 3D organoid models (14-fold change). RNA sequencing revealed 7/30 (23%) metabolic genes upregulated in the invasive tumor front were involved in cysteine or glutathione metabolism. These results highlight a clear role of the transsulfuration pathway in glioblastoma invasion, revealing a targetable alteration unique to invading glioblastoma cells.

INTRODUCTION Prolactinomas are common pituitary adenomas managed medically or surgically. METHODS Reviewed 181 prolactinomas resected transsphenoidally 2012-2019. Tumor volumes were quantified using BrainLab Smartbrush. Pearson correlation analysis and linear regression were used to identify associations between tumor volumes and serum prolactin. Tumor density was defined as serum prolactin divided by tumor volume. RESULTS Mean tumor volume was 6.33cm3 and mean pre-op prolactin was 803.4ug/L, with men having larger (12.11 vs 2.93cm3;p< 0.001) and women having denser (173.9 vs 107.6ug/L/cm3;p=0.011) prolactinomas. Pearson correlation (R=0.688;p< 0.001) and linear regression revealed a strong association between pre-op volume and prolactin levels, with 96.9g/L increase in prolactin/cm3 increase in volume (p< 0.001); this holds true for men (R=0.584;p< 0.001) and women (R=0.939;p< 0.001), with women demonstrating greater prolactin/cm3 tumor density (186.5 vs 75.0ug/L;p< 0.001). MiB index did not correlate with pre-op volume (p=0.449) or pre-op prolactin (p=0.452). Logistic regression showed decreased biochemical remission with increasing pre-op volume (OR=0.891;p< 0.001). Increased MiB index (p=0.971) and p53 (p=0.525) staining did not affect remission rates. Positive PIT-1 staining was associated with higher remission rates (OR=2.508;p=0.005). Patients without remission had denser tumors (149.9 vs. 100.6ug/L/cm3;p=0.013), with Pearson correlation yielding R=0.736 between pre-op volume and pre-op prolactin (p< 0.001), and R=0.476 between residual volume and post-op prolactin (p< 0.001). Patients without remission exhibited 142.9ug/L increase in prolactin/cm3 of pre-op volume (p< 0.001), higher than the 58.9ug/L increase in prolactin/cm3 in patients with remission (p< 0.001). Patients without remission had residual tumors with 68.4ug/L increase in prolactin/cm3 of remaining volume after resection (p< 0.001). CONCLUSION Our analysis revealed significant correlation between prolactinoma volume and serum prolactin levels. Patients without remission had greater tumor cellular density than those with remission. The volume-prolactin correlation persisted post-operatively, although surgery reduced tumor density. These results could identify prolactinomas for which surgery could achieve biochemical remission.