Abstract Rationale Neuroblastoma is a devastating pediatric malignancy, for which surgical resection is a key factor in long-term survival. However, there are significant challenges in its resection, particularly in high-risk disease, as neuroblastoma encases surrounding critical structures, is often difficult to distinguish from desmoplastic or scar tissue, and can carry occult deposits of disease not readily identified on preoperative imaging or intraoperative visualization. Building on the principles of fluorescent and radio-guided surgery, in combination with the known overexpression of GD2 in neuroblastoma, we sought to develop and optimize 111 In-Dinutuximab-IRDye800, a dual-modality GD2-targeted intraoperative molecular imaging agent, for use in pediatric neuroblastoma to help enhance patient safety while facilitating a more complete resection. Methods Dinutuximab was conjugated to IRDye800 and DTPA, then radiolabeled with Indium-111 to yield 111 In-Dinutuximab-IRDye800. Optimization occurred through ELISA assay to assess binding affinity, fluorescence intensity analysis to determine the optimal fluorescent degree of labeling, and phototoxicity testing through flow cytometry. Rodent models of neuroblastoma were then generated through injection of SK-N-BE(2) human neuroblastoma cells into the left adrenal glands of nude mice or RNU rats. A series of fluorescent and gamma biodistributions was performed, varying the dose, timing, and specific activity of the tracer. Tumor and organ uptake of the tracer was compared with one- or two-way ANOVA as appropriate, with Sidak’s multiple comparison test to compare tumor uptake to individual organs. Once optimization was complete, a clinically significant events study modeled after human clinical trials was performed to evaluate the in vivo capabilities of 111 In-Dinutuximab-IRDye800. Results Increased ratios of IRDye800 per antibody led to decreased binding affinity for GD2 and was associated with formulation instability without significant return on fluorescence intensity. Specific activity of the tracer was not found to impact overall biodistribution of the tracer. A 45-50 µg dose of 111 In-Dinutuximab-IRDye800 with ratios around 1 DTPA and 1-1.5 IRDye800 per antibody imaged 4 days after tracer administration was found to be the optimal combination that maximized detectable tumor-specific signal. In the clinically significant events study mirroring human IMI clinical trials, fluorescent guidance identified additional malignant lesions not originally detected under white light in 64% of rodents. Conclusions 111 In-Dinutuximab-IRDye800 is a dual-modality GD2-targeted intraoperative imaging agent that is well-poised for clinical translation. As it preserves tumor specificity, yields clinically meaningful radiofluorescent signal, and is well-tolerated without adverse events after optimization was completed, it carries the potential to positively impact the safety and completeness of neuroblastoma resection.

Myeloid cells are key mediators of immunosuppression and treatment resistance in primary brain tumors, including glioblastoma (GBM). This study aims to eradicate CD11b+ immunosuppressive cells at the tumor site to enhance overall survival in a model of GBM using an α-emitting radiopharmaceutical therapy targeted to tumor-associated myeloid cells as a monotherapy or in combination with immune checkpoint inhibitors. An anti-CD11b (αCD11b) antibody was modified for radiolabeling with diagnostic (zirconium-89) or therapeutic (actinium-225) radioisotopes. Initial PET imaging and biodistribution studies using 89Zr-αCD11b found that an antibody concentration of ∼5 mg/kg of αCD11b (100 μg) was effective in saturating on-target/off-site sinks, such as the spleen, but effective in increasing tumor accumulation. The estimated maximum tolerable activity of [225Ac]Ac-DOTA-αCD11b (225Ac-αCD11b) was determined by biodistribution and dosimetry studies, including the free in vivo-generated decay daughters. The dose-limiting tissue was the bone marrow, and an estimated maximum tolerable activity (∼0.55 kBq, 100 μg) was determined. The therapeutic efficacy of 225Ac-αCD11b was evaluated by survival studies, both as a monotherapy and in combination with immune checkpoint inhibitors. Combination therapy resulted in increased survival in the GBM model compared with the monotherapy and controls; in addition, long-term survival was observed in 50% of the mice receiving combination therapy as well as in a single mouse receiving 225Ac-αCD11b alone. No long-term surviving mice were observed in the control groups. Long-term surviving mice were rechallenged, and potential antitumor immunity was observed, as no tumors developed over 120 days after rechallenge. Overall, these results validate the preclinical relevance of CD11b-targeted image-guided α-emitting radiopharmaceutical therapy.

Supplementary Data - Results section

Abstract Lung cancer is the second most common cancer in both men and women in the US and the leading cause of cancer death. Among younger US women, lung cancer incidence rates have in recent years become higher than rates seen in men, despite comparable histories of tobacco use. The possible contribution of stress to higher lung cancer risk in young women has received little research attention despite preclinical evidence that exposure to stress-related neuroendocrine responses can cause DNA damage. The present study used a well-established lung cancer model in young female mice (in vivo exposure to NNK) along with a mouse stress model to explore their independent and combined effects on lung cancer development. In addition, we explored one possible biological pathway by chronic administration of a beta-adrenergic receptor blocker (propranolol). We also tested the use of in vivo imaging for repeated monitoring of tumor development over time. Cohort 1 (n=60) had 4 groups: Home cage control, NNK treatment alone, repeated stress (RS) alone and NNK+RS. Cohort 2 (n=60) had the same 4 groups with the addition of administration of propranolol in drinking water. Cohort 3 (n=48, imaging cohort) had 3 groups: Home cage control, NNK alone and NNK+RS. FVB/N (4-6 wk) were purchased from a commercial supplier and acclimated with gentle handling for two weeks. The repeated stress protocol began week-3 and continued for the duration of the study. It combined two well-established rodent models: social disruption stress via changes in cage mates at the time of cage cleaning (2x/week) and a 90-minute restraint stress immediately prior to cage change (1x/week). NNK or saline treatment began on week-4 (IP; 2x/week; 3 mg/injection) and continued for 4 weeks. 21 weeks after stress initiation, the mice in cohorts 1 and 2 were sacrificed, lungs removed, inflated and tumors counted and sized. Mice in cohort 3 were imaged by Hounsfield-calibrated micro-CT (μCT) at baseline, 14, 19 and 23 weeks. The entire lung space, excluding the heart, was defined as a volumetric region of interests (VOIs). The VOIs were subjected to thresholding to segment the lungs into air space and tissue space compartments. The volume of tissue space of each animal was ratioed against its own baseline tissue space volume. Results from Cohort 1 revealed significantly greater numbers and overall size of lung tumors in stressed mice that received NNK. No significant effects were seen in the absence of NNK. Results from Cohort 2 revealed no significant differences between stressed and unstressed groups treated with propranolol. Cohort 3 revealed a pattern of results with stressed mice generally showing the highest lung tumor volume levels (Hounsfield Units), which reached significance at 19 weeks in preliminary analyses. Conclusion: In this study, we found that repeated stress increased lung tumor burden in female mice treated with NNK, and that a beta-adrenergic pathway may be involved. Citation Format: Frank Jenkins, Laura P. Stabile, Jessie R. Nedrow, Kathryn Day, Joseph D. Latoche, Patrick M. Tarwater, Jessica Manculich, Dana H. Bovbjerg. Effects of repeated psychological stress on NNK-induced lung cancer development in young female mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3391.

Precision management of fibrotic lung diseases is challenging due to their diverse clinical trajectories and lack of reliable biomarkers for risk stratification and therapeutic monitoring. Here, we validated the accuracy of CMKLR1 as an imaging biomarker of the lung inflammation-fibrosis axis. By analyzing single-cell RNA sequencing datasets, we demonstrated CMKLR1 expression as a transient signature of monocyte-derived macrophages (MDMφ) enriched in patients with idiopathic pulmonary fibrosis (IPF). Consistently, we identified MDMφ as the major driver of the uptake of CMKLR1-targeting peptides in a murine model of bleomycin-induced lung fibrosis. Furthermore, CMKLR1-targeted positron emission tomography in the murine model enabled quantification and spatial mapping of inflamed lung regions infiltrated by CMKLR1-expressing macrophages and emerged as a robust predictor of subsequent lung fibrosis. Last, high CMKLR1 expression by bronchoalveolar lavage cells identified an inflammatory endotype of IPF with poor survival. Our investigation supports the potential of CMKLR1 as an imaging biomarker for endotyping and risk stratification of fibrotic lung diseases.

Glioblastoma (GBM) is the most common primary malignant brain tumor. Currently, there are few effective treatment options for GBM beyond surgery and chemo-radiation, and even with these interventions, median patient survival remains poor. While immune checkpoint inhibitors (ICIs) have demonstrated therapeutic efficacy against non-central nervous system cancers, ICI trials for GBM have typically had poor outcomes. TIGIT is an immune checkpoint receptor that is expressed on activated T-cells and has a role in the suppression of T-cell and Natural Killer (NK) cell function. As TIGIT expression is reported as both prognostic and a biomarker for anti-TIGIT therapy, we constructed a molecular imaging agent, [89Zr]Zr-DFO-anti-TIGIT (89Zr-αTIGIT), to visualize TIGIT in preclinical GBM by immunoPET imaging. PET imaging and biodistribution analysis of 89Zr-αTIGIT demonstrated uptake in the tumor microenvironment of GBM-bearing mice. Blocking antibody and irrelevant antibody tracer studies demonstrated specificity of 89Zr-αTIGIT with significance at a late time point post-tracer injection. However, the magnitude of 89Zr-αTIGIT uptake in tumor, relative to the IgG tracer was minimal. These findings highlight the features and limitations of using 89Zr-αTIGIT to visualize TIGIT in the GBM microenvironment.

Abstract Background Surgical resection is integral for the treatment of neuroblastoma, the most common extracranial solid malignancy in children. Safely locating and resecting primary tumor and remote deposits of disease remains a significant challenge, resulting in high rates of complications and incomplete surgery, worsening outcomes. Intraoperative molecular imaging (IMI) uses targeted radioactive or fluorescent tracers to identify and visualize tumors intraoperatively. GD2 was selected as an IMI target, as it is highly overexpressed in neuroblastoma and minimally expressed in normal tissue. Methods GD2 expression in neuroblastoma cell lines was measured by flow cytometry. DTPA and IRDye® 800CW were conjugated to anti-GD2 antibody to generate DTPA-αGD2-IR800. Binding affinity (Kd) of the antibody and the non-radiolabeled tracer were then measured by ELISA assay. Human neuroblastoma SK-N-BE(2) cells were surgically injected into the left adrenal gland of 3.5-5-week-old nude mice and the orthotopic xenograft tumors grew for 5 weeks. 111In-αGD2-IR800 or isotype control tracer was administered via tail vein injection. After 4 and 6 days, mice were euthanized and gamma and fluorescence biodistributions were measured using a gamma counter and ImageJ analysis of acquired SPY-PHI fluorescence images of resected organs (including tumor, contralateral adrenal, kidneys, liver, muscle, blood, and others). Organ uptake was compared by one-way ANOVA (with a separate analysis for each tracer/day combination), and if significant, Sidak’s multiple comparison test was used to compare the uptake of each organ to the tumor. Handheld tools were also used to detect and visualize tumor in situ, and to assess for residual disease following non-guided resection. Results 111In-αGD2-IR800 was successfully synthesized with 0.75-2.0 DTPA and 2–3 IRDye® 800CW per antibody and retained adequate antigen-binding (Kd = 2.39 nM for aGD2 vs. 21.31 nM for DTPA-aGD2-IR800). The anti-GD2 tracer demonstrated antigen-specific uptake in mice with human neuroblastoma xenografts (gamma biodistribution tumor-to-blood ratios of 3.87 and 3.88 on days 4 and 6 with anti-GD2 tracer), while isotype control tracer did not accumulate (0.414 and 0.514 on days 4 and 6). Probe accumulation in xenografts was detected and visualized using widely available operative tools (Neoprobe® and SPY-PHI camera) and facilitated detection ofputative residual disease in the resection cavity following unguided resection. Conclusions We have developed a dual-labeled anti-GD2 antibody-based tracer that incorporates In-111 and IRDye® 800CW for radio- and fluorescence-guided surgery, respectively. The tracer adequately binds to GD2, specifically accumulates in GD2-expressing xenograft tumors, and enables tumor visualization with a hand-held NIR camera. These results encourage the development of 111In-αGD2-IR800 for future use in children with neuroblastoma, with the goal of improving patient safety, completeness of resection, and overall patient outcomes.

Abstract Pediatric high-grade gliomas (HGGs) are aggressive tumors and are one of the drivers of pediatric brain tumor mortality. Few effective treatment options exist for pediatric HGGs beyond surgery and chemoradiation. Additionally, some HGGs, such as diffuse midline glioma, are often surgically inaccessible and respond poorly to chemotherapy leaving radiotherapy as the only treatment for this disease. While immune checkpoint inhibitors (ICIs) have seen success in treating cancers outside the CNS, ICIs for gliomas have shown limited efficacy. TIGIT is an IC receptor that is expressed on activated T-cells and has a critical role in suppressing T-cell and NK cell function. Prior work by our group has shown TIGIT to be a promising target for adult gliomas. These studies included analysis of transcriptomic data, and preclinical studies in glioma-bearing mice. We therefore assessed pediatric glioma transcriptomic datasets and identified via optimal cutoff analysis that at a specific mRNA threshold, TIGIT expression is associated with worsened survival. As TIGIT expression may be both prognostic and a biomarker for anti-TIGIT ICI therapy, we created a radiolabeled immunoPET tracer capable of binding TIGIT antigen that could be visualized via PET imaging. Referred to as “89Zr-DFO-TIGIT”, we found this tracer bound TIGIT with moderate immunoreactivity (57.7% ± 3.5%) as determined by bead-based immunoreactivity assays. As proof of feasibility, we injected glioma-bearing mice (GL261 cell line) with 89Zr-DFO-TIGIT which enabled the visualization of TIGIT in the tumor microenvironment via PET imaging. Thereafter, we performed biodistribution studies which found there to be significantly increased 89Zr-DFO-TIGIT in mouse tumor-bearing hemispheres as compared to non-tumor-bearing hemispheres. PET imaging also showed tracer-to-tumor uptake increased from day one to day eight. Our findings demonstrate the capability of 89Zr-DFO-TIGIT to visualize and quantify TIGIT in the glioma microenvironment, highlighting how this tracer may be of value in pediatric brain tumor immunotherapy studies.

Immuno-PET of 89Zr-DFO-CD69 Ab as a prognostic predictor after ICI treatment in a GBM mouse model. Survival follow-up of mice inoculated with GL261 cells, and evaluated by immuno-PET of 89Zr-DFO-CD69 Ab, comparing treatment (ICI) group to control group (represented by orange and blue dots, respectively). A, Schematic showing timeline of tumor inoculation, ICI treatment, MRI, tail vein injection of 89Zr-DFO-CD69 Ab, immuno-PET, and survival follow-up. B, The overall survival (OS) rates of the ICI-treated group and control group are plotted using Kaplan–Meier survival curves, with log-rank (Mantel–Cox) curve comparison test. C, Right, heat map display of Pearson correlation coefficient analysis between immuno-PET signals and survival in the two groups. Results are shown for each SUV measurement method (SUVmax, SUVmax TBR, SUVmean, and SUVmean TBR) in each immuno-PET designated time point (days 1, 2, 3, 4, and 6). Shown are representative data of two independent experiments, n = 4–5 mice per group (*, P < 0.05; **, P <0.01). Left, examples of scatter diagrams of Pearson correlation coefficient taken from immuno-PETs on day 2 of SUVmax and SUVmax TBR. Scattered dots represent individual mice, n = 4–5 mice per group.

Objective: We aimed to evaluate an early marker of T-cell activation, CD69, for its use as an imaging biomarker of response to immunotherapy for GBM. Background: Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Immunotherapy may be promising for the treatment of some GBM patients, however, there is a need for non invasive neuroimaging techniques to predict immunotherapeutic responses. The effectiveness of most immunotherapeutic strategies requires T-cells activation. Design/Methods: We performed CD69 immunostaining on human and mouse T-cells following in vitro activation and post-immune checkpoint inhibitors (ICI) in an orthotopic syngeneic mouse glioma model. CD69 expression on tumor-infiltrating leukocytes was assessed using single-cell RNA sequence (scRNA-seq) data from recurrent GBM patients receiving ICI. Radiolabeled CD69 antibody (Ab) positron emission tomography/computed tomography (PET/CT) imaging (CD69 immuno-PET) was performed on GBM-bearing mice longitudinally to quantify CD69 and its association with survival following immunotherapy Results: CD69 expression was upregulated upon T-cell activation and on tumor-infiltrating lymphocytes (TILs) in response to immunotherapy. Similarly, scRNA-seq data demonstrated elevated CD69 on TILs from ICI-treated recurrent GBM patients as compared with TILs from a control cohort. CD69 immuno-PET studies showed a significantly higher tracer uptake in the tumors of ICI-treated mice compared with controls. Importantly, we observed a strong positive correlation between survival and CD69 immuno-PET signals in immunotherapy-treated animals and established a trajectory of T-cell activation by virtue of CD69-immuno-PET measurements. Conclusions: Our study supports the use of CD69 immuno-PET as an early immunotherapy response assessment imaging tool for GBM patients Disclosure: Michal Nisnboym has nothing to disclose. Miss Vincze has nothing to disclose. The institution of Dr. Raphael has received research support from Walter L. Copeland fund of The Pittsburgh foundation. Dr. Xiong has nothing to disclose. Mr. Sneiderman has nothing to disclose. Mrs. Raphael has nothing to disclose. Dr. Li has nothing to disclose. Dr. Jaswal has nothing to disclose. Miss Sever has nothing to disclose. Ms. Day has nothing to disclose. Mr. Latoche has nothing to disclose. Ms. Foley has nothing to disclose. Dr. Hitchenes has nothing to disclose. Prof. Agnihotri has nothing to disclose. Dr. Hu has nothing to disclose. Dr. Rajasundaram has nothing to disclose. Prof. Anderson has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Novartis. Prof. Anderson has received personal compensation in the range of $500-$4,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for Society of Nuclear Medicine and Molecular Imaging. Prof. Anderson has stock in Lumiphore. Prof. Anderson has stock in Focus-X. The institution of Prof. Anderson has received research support from Cancer Targeted Technology. Dr. Blumenthal has nothing to disclose. Thomas Pearce has nothing to disclose. Dr. Uttam has nothing to disclose. Dr. Nedrow has received personal compensation in the range of $500-$4,999 for serving as a Consultant for ChemImage. The institution of Dr. Nedrow has received research support from Guerbet. Prof. Panigrahy has nothing to disclose. Dr. Pollack has nothing to disclose. The institution of Dr. Lieberman has received research support from Novocure. The institution of Dr. Lieberman has received research support from Black Diamond. The institution of Dr. Lieberman has received research support from Chimerix. The institution of Dr. Lieberman has received research support from Abbvie. Dr. Drappatz has stock in Pfizer. Dr. Drappatz has stock in Vertex. Dr. Drappatz has stock in GSK. The institution of Dr. Drappatz has received research support from Servier. Dr. Drappatz has received publishing royalties from a publication relating to health care. An immediate family member of Dr. Edwards has received personal compensation in the range of $0-$499 for serving as a Consultant for Lumiphore. The institution of Dr. Kohanbash has received research support from NIH.

Immuno-PET of 89Zr-DFO-CD69 Ab visualizes the TME after ICI treatment in a GBM mouse model. Mice were inoculated with GL261 cells, and evaluated by immuno-PET of 89Zr-DFO-CD69 Ab, comparing the treatment (ICI) group to the control group (represented by orange and blue dots, respectively). A, Schematic showing timeline of tumor inoculation, ICI treatment, tail vein injection of 89Zr-DFO-CD69 Ab, immuno-PET, and BioD. B, Representative coronal head images from immuno-PET of 89Zr-DFO-CD69 Ab of ICI-treated and control mice at designated time points. Scales show SUVs of PET (SUV; colored) and CT (HU; gray). C and D, Comparison between ICI-treated mice and control at designated time points of tumor-specific regions’ SUVmax (C) and SUVmax TBR (D). E, Representative coronal-ventral and sagittal 3D whole-body MIP PET images of 89Zr-DFO-CD69 Ab of control and ICI-treated mice acquired 6 days after tracer administration. The scale shows SUVs of PET. F, Day 6 BioD results of blood- and tumor site–associated radioactivity, assessed as %ID/g. In C, D, and F, bars show means ± SEM. Scattered dots represent individual mice. Shown are representative data of three independent experiments, n = 5 per group. C and D, Multiple unpaired t test with Welch correction. F, Two-way ANOVA with multiple comparison test (*, P < 0.05; **, P <0.01; ***, P <0.001). IP, intraperitoneal.

Background The lack of noninvasive methods for assessment of dysregulated inflammation as a major driver of fibrosis (i.e., inflammation-fibrosis axis) has been a major challenge to precision management of fibrotic lung diseases. Here, we determined the potential of very late antigen-4 (VLA-4)-targeted positron emission tomography (PET) to detect inflammation in a mouse model of bleomycin-induced fibrotic lung injury. Method Single time-point and longitudinal VLA-4-targeted PET was performed using a high-affinity peptidomimetic radiotracer, 64 Cu-LLP2A, at weeks 1, 2, and 4 after bleomycin-induced (2.5 units/kg) lung injury in C57BL/6J mice. The severity of fibrosis was determined by measuring the hydroxyproline content of the lungs and expression of markers of extracellular matrix remodeling. Flow cytometry and histology was performed to determine VLA-4 expression across different leukocyte subsets and their spatial distribution. Results Lung uptake of 64 Cu-LLP2A was significantly elevated throughout different stages of the progression of bleomycin-induced injury. High lung uptake of 64 Cu-LLP2A at week-1 post-bleomycin was a predictor of poor survival over the 4-week follow up, supporting the prognostic potential of 64 Cu-LLP2A PET during the early stage of the disease. Additionally, the progressive increase in 64 Cu-LLP2A uptake from week-1 to week-4 post-bleomycin correlated with the ultimate extent of lung fibrosis and ECM remodeling. Flow cytometry revealed that LLP2A binding was restricted to leukocytes. A combination of increased expression of VLA-4 by alveolar macrophages and accumulation of VLA-4-expressing interstitial and monocyte-derived macrophages as well as dendritic cells was noted in bleomycin-injured, compared to control, lungs. Histology confirmed the increased expression of VLA-4 in bleomycin-injured lungs, particularly in inflamed and fibrotic regions. Conclusions VLA-4-targeted PET allows for assessment of the inflammation-fibrosis axis and prediction of disease progression in a murine model. The potential of 64 Cu-LLP2A PET for assessment of the inflammation-fibrosis axis in human fibrotic lung diseases needs to be further investigated.

ICIs influence T-cell distribution in tumor and spleen. Mice (n = 28) were inoculated with GL261 cells, treated with ICI or vehicle control, and analyzed at the designated time points relative to treatment. A, Schematic showing tumor inoculation day, treatment days, and time points relative to treatment in which mice were analyzed. B, Percentage of CD8+ TILs (red dots) and CD4+ TILs (blue dots) in each time point relative to treatment, for vehicle-control group (left panel) and ICI-treated group (right panel) C, Absolute number of TILs per mm3 of tumor. CD8+ cells × 102 (upper panels; red dots) and CD4+ cells × 102 (bottom panels; blue dots) in each time point relative to treatment, for ICI-treated group and vehicle-control group. D, Frequencies of CD8+ (red dots) and CD4+ T-cells (blue dots) in spleen in each time point relative to treatment, for vehicle-control group and ICI-treated group. B–D, Shown are means ± SEM. Scattered dots represent individual mice. Shown are representative data of two independent experiments. n = 3–4 per group. CD8+ and CD4+ were gated from CD3+CD45+ cells. One-way ANOVA with multiple comparison test (*, P < 0.05; **, P <0.01; ***, P <0.001).

CD69 validation by tissue staining.

Abstract Malignant gliomas are one of the drivers of brain tumor patient mortality as these tumors respond poorly to conventional therapies and are known to grow aggressively. Many clinical trials have evaluated the efficacy of novel immuno-therapeutics targeted to gliomas; however, these trials have largely been unsuccessful. It is believed that one of the primary reasons immunotherapies have been unsuccessful in gliomas is due to the highly immunosuppressive nature of the tumor microenvironment. T cell immunoreceptor with Ig and ITIM domain (TIGIT) is an IC receptor that is expressed on activated T cells, Natural Killer (NK) cells, and Regulatory T cells (Tregs). It has been well established that TIGIT has a primary role in down-regulating T cell and NK cell function and can serve as an inhibitor of anti-tumor immune responses. Our laboratory and others have shown that anti-TIGIT therapy may be promising for the treatment of adult gliomas via glioma patient transcriptomic data and preclinical models. In this study, our team created a radiolabeled tracer (referred to as 89Zr-DFO-TIGIT) that bound TIGIT antigen with moderate immunoreactivity (57.7% ± 3.5%). Binding affinity was determined by performing bead-based immunoreactivity assays. To create 89Zr-DFO-TIGIT, a TIGIT monoclonal antibody was conjugated to the metal chelator DFO and radiolabeled with 89Zr. Following tracer preparation, glioma-bearing mice (GL261 cell line) were injected with 89Zr-DFO-TIGIT allowing for the visualization of TIGIT in the tumor microenvironment. PET imaging analyses revealed tracer-to-tumor uptake that increased from day one to day eight while biodistribution studies confirmed that there were significantly higher amounts of 89Zr-DFO-TIGIT detected in the glioma-bearing mouse brain hemisphere compared to the non-tumor bearing hemisphere. We now intend to evaluate 89Zr-DFO-TIGIT in a glioma-bearing large animal model to determine if this method demonstrates similar efficacy in visualizing immunosuppression in the tumor microenvironment before proceeding to clinical studies.