Polymorphonuclear myeloid-derived suppressor cells and phosphatidylinositol-3 kinase gamma are critical to tobacco-mimicking oral carcinogenesis in mice.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a devastating disease most often associated with tobacco consumption that induces a field of mutations from which a tumor arises. Identification of ways to prevent the emergence of cancer in high-risk patients is an ultimate goal for combatting all types of cancer, including OSCC. METHODS: Our study employs a mouse model of tongue carcinogenesis induced by tobacco carcinogen mimetic, 4-nitroquinoline 1-oxide (4NQO), to establish tongue dysplasia and OSCC. We use conventional histology, immunohistochemistry, multispectral imaging, mass cytometry, novel cell lines, pharmaceutical inhibition of PI3Kγ, T-cell suppression assays and mouse transplant models in our functional experimentation. RESULTS: In our study, we identify Ly6G+ granulocytes as the most abundant immune cell type in a model of tongue carcinogenesis induced by tobacco carcinogen mimetic 4NQO. Targeting Ly6G+ granulocytes with a pharmacologic inhibitor of PI3Kγ, an isoform of PI3K exclusively expressed by myeloid cells, resulted in reduced tongue dysplasia severity, and reduced rates of OSCC. Importantly, we performed functional assays with the Ly6G+ granulocytes induced in cell line models of 4NQO carcinogenesis to demonstrate that these granulocytes have increased polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) activity against T-cell proliferation and these PMN-MDSCs play a functional role in promoting tumor formation by inhibiting tumor regression in a PI3Kγ-dependent manner. CONCLUSIONS: Overall, our data suggest that recruitment of PMN-MDSCs to sites of dysplasia is critical to immune suppression of CD8 T cells, thereby permitting malignancy, and PI3Kγ inhibitors are one mechanism to reduce PMN-MDSC recruitment, immunosuppression and tumorigenesis in OSCC.

Incidence, etiology and time course of delays to adult spinal deformity surgery: a single-center experience.

PURPOSE: We sought to determine the incidence, origin, and timeframe of delays to adult spinal deformity surgery so that institutions using preoperative multidisciplinary patient assessment teams might better anticipate and address these potential delays. METHODS: Complex spine procedures for treatment of adult spinal deformity from 1/1/18 to 8/31/21 were identified. Procedures for infection, tumor, and urgent/emergent cases were excluded. Operations delayed due to COVID or those that were performed outside of our established perioperative care pathway were also excluded. The electronic health record was used to identify the etiology and timeline of all pre- and peri-operative delays. RESULTS: Of 235 patients scheduled for complex spine surgery, 193 met criteria for inclusion. Of these patients, 35 patients experienced a surgical delay (18.1%) with a total of 41 delays recorded. Reasons for delay include medically unoptimized (25.6%), intraoperative complication (17.9%), patient directed delay (17.9%), patient illness/injury (15.4%), scheduling complication (10.3%), insurance delay/denial (5.1%), and unknown (2.6%). Twenty-four delays experienced by 22 individuals occurred within 7 days of their scheduled surgery date. CONCLUSION: At a single multidisciplinary center, most delays to adult spinal deformity surgery occur before a patient is admitted to the hospital, and for recommendations of additional medical workup/clearance. We suspect that the preoperative protocol might increase pre-admission delays for unoptimized patients, as the protocol is intended to ensure patients receive surgery only when they are medically ready. Further research is needed to determine the economic and system impact of delays related to a preoperative optimization protocol weighed against the reduction in adverse events these protocols can provide.

Detection of Human Papillomavirus Integration in Brain Metastases from Oropharyngeal Tumors by Targeted Sequencing.

Human papillomavirus (HPV) positive and negative head and neck squamous cell carcinoma (HNSCC) are known to have differential phenotypes, including the incidence and location of metastases. HPV positive (HPV+) HNSCC are more likely to metastasize to distant sites, such as the lung, brain, and skin. Among these locations, metastasis to the brain is a rare event, and little is known about specific risk factors for this phenotype. In this report, we describe two patients who developed brain metastases from HNSCC. Both patient tumors had p16INK4a overexpression, suggesting these tumors were HPV+. This was confirmed after PCR, in situ hybridization, and mass spectrometry detected the presence of HPV type 16 (HPV16) DNA, RNA and protein. To further characterize the presence of HPV16, we used a target enrichment strategy on tumor DNA and RNA to isolate the viral sequences from the brain metastases. Analysis by targeted next generation sequencing revealed that both tumors had the HPV genome integrated into the host genome at known hotspots, 8q24.21 and 14q24.1. Applying a similar target enrichment strategy to a larger cohort of HPV+ HNSCC brain metastases could help to identify biomarkers that can predict metastasis and/or identify novel therapeutic options.

Glioma Stem Cells as Immunotherapeutic Targets: Advancements and Challenges.

Glioblastoma is the most common and lethal primary brain malignancy. Despite major investments in research into glioblastoma biology and drug development, treatment remains limited and survival has not substantially improved beyond 1-2 years. Cancer stem cells (CSC) or glioma stem cells (GSC) refer to a population of tumor originating cells capable of self-renewal and differentiation. While controversial and challenging to study, evidence suggests that GCSs may result in glioblastoma tumor recurrence and resistance to treatment. Multiple treatment strategies have been suggested at targeting GCSs, including immunotherapy, posttranscriptional regulation, modulation of the tumor microenvironment, and epigenetic modulation. In this review, we discuss recent advances in glioblastoma treatment specifically focused on targeting of GCSs as well as their potential integration into current clinical pathways and trials.

Development of syngeneic murine cell lines for use in immunocompetent orthotopic lung cancer models.

BACKGROUND: Immunocompetent animal models are required to study tumor-host interactions, immunotherapy, and immunotherapeutic combinations, however the currently available immunocompetent lung cancer models have substantial limitations. While orthotopic models potentially help fill this gap, the utility of these models has been limited by the very small number of murine lung cancer cell lines capable of forming orthotopic tumors in immunocompetent C57BL/6 hosts. METHODS: Primary lung tumors with specific genetic alterations were created in C57BL/6 background mice. These tumors were then passaged through other animals to increase tumorigenicity and select for the ability to grow in a non-self animal. Once tumors demonstrated growth in a non-self host, cell lines were established. Successful cell lines were evaluated for the ability to produce orthotopic lung tumors in immunocompetent hosts. RESULTS: We produced six murine lung cancer lines capable of orthotopic lung tumor formation in immunocompetent C57BL/6 animals. These lines demonstrate the expected genetic alterations based on their primary tumor genetics. CONCLUSIONS: These novel cell lines will be useful for evaluating tumor-host interactions, the impact of specific oncogenic alterations on the tumor microenvironment, and immunotherapeutic approaches. This method of generating murine lines capable of orthotopic growth can likely be applied to other tumors and will broaden the applicability of pre-clinical testing of immunotherapeutic treatment regimens.

Enzymatic and Mutational Analysis of the PruA Pteridine Reductase Required for Pterin-Dependent Control of Biofilm Formation in Agrobacterium tumefaciens.

Pterins are ubiquitous biomolecules with diverse functions including roles as cofactors, pigments, and redox mediators. Recently, a novel pterin-dependent signaling pathway that controls biofilm formation was identified in the plant pathogen, Agrobacterium tumefaciens A key player in this pathway is a pteridine reductase termed PruA, where its enzymatic activity has been shown to control surface attachment and limit biofilm formation. Here, we biochemically characterize PruA to investigate the catalytic properties and substrate specificity of this pteridine reductase. PruA demonstrates maximal catalytic efficiency with dihydrobiopterin and comparable activities with the stereoisomers dihydromonapterin and dihydroneopterin. Since A. tumefaciens does not synthesize or utilize biopterins, the likely physiological substrate is dihydromonapterin or dihydroneopterin, or both. Notably, PruA does not exhibit pteridine reductase activity with dihydrofolate or fully oxidized pterins. Site-directed mutagenesis studies of a conserved tyrosine residue, the key component of a putative catalytic triad, indicate that this tyrosine is not directly involved in PruA catalysis but may be important for substrate or cofactor binding. Additionally, mutagenesis of the arginine residue in the N-terminal TGX3RXG motif significantly reduces the catalytic efficiency of PruA, supporting its proposed role in pterin binding and catalysis. Finally, we report the enzymatic characterization of PruA homologs from Pseudomonas aeruginosa and Brucella abortus, thus expanding the roles and potential significance of pteridine reductases in diverse bacteria.Importance Biofilms are complex multicellular communities that are formed by diverse bacteria. In the plant pathogen, Agrobacterium tumefaciens, the transition from a free-living motile state to a non-motile biofilm state is governed by a novel signaling pathway involving small molecules called pterins. The involvement of pterins in biofilm formation is unexpected and prompts many questions about the molecular details of this pathway. This work biochemically characterizes the PruA pteridine reductase involved in the signaling pathway to reveal its enzymatic properties and substrate preference, thus providing important insight into pterin biosynthesis and its role in A. tumefaciens biofilm control. Additionally, the enzymatic characteristics of related pteridine reductases from mammalian pathogens are examined to uncover potential roles of these enzymes in other bacteria.

Human cytomegalovirus seropositivity is associated with decreased survival in glioblastoma patients.

BACKGROUND: Human cytomegalovirus (HCMV) is an oncomodulatory human herpesvirus that has been detected in glioblastoma (GBM) and is associated with worse prognosis in patients with the disease. The effects of HCMV systemic infection on survival in GBM patients, however, are largely unknown. We aimed to determine the association between HCMV serostatus at diagnosis and survival via a retrospective cohort study of GBM patients. METHODS: Plasma from 188 GBM patients treated at the Ben and Catherine Ivy Center (Seattle, WA) was tested for HCMV serostatus via enzyme-linked immunosorbent assays of anti-HCMV immunoglobulin (Ig)G. HCMV IgG serostatus was analyzed with respect to each patient's progression-free and overall survival (OS) via log-rank and multivariable Cox regression analysis. RESULTS: Ninety-seven of 188 (52%) patients were anti-HCMV IgG seropositive. Median OS was decreased in the IgG+ cohort (404 days) compared to IgG- patients (530 days; P = .0271). Among O 6-methylguanine-DNA methyltransferase (MGMT) unmethylated patients (n = 96), median OS was significantly decreased in IgG+ patients (336 days) compared to IgG- patients (510 days; P = .0094). MGMT methylation was associated with improved OS in IgG+ patients versus those who were unmethylated (680 vs 336 days; P = .0096), whereas no such association was observed among IgG- patients. CONCLUSIONS: In this study, HCMV seropositivity was significantly associated with poorer OS in GBM patients. This finding suggests prior infection with HCMV may play an important role in GBM patient outcomes, and anti-HCMV antibodies may, therefore, prove a valuable prognostic tool in the management of GBM patients.