High-grade gliomas are a major health challenge with poor prognosis and high morbidity. Immune-checkpoint inhibitors (ICI) have emerged as promising therapeutic options for several malignancies yet show little efficacy against central nervous system (CNS) tumors. CD200 is a newly recognized immune checkpoint that modulates immune homeostasis. CD200 protein is expressed by a variety of cells, including immune cells and stromal cells, and is overexpressed by many tumors. The shedding of CD200 from tumor cells can create an immunosuppressive environment that dampens anti-tumor immunity by modulating cytolytic activity and cytokine expression both within and outside the tumor microenvironment (TME). While it is well-accepted that CD200 induces a pro-tumorigenic environment through its ability to suppress the immune response, we sought to determine the role of glioma-specific expression of CD200. We show that CD200 is expressed across glioma types, is shed from tumor cells, and increases over time in the serum of patients undergoing immunotherapy. Using CD200 knockout (KO) glioma models, we demonstrated that glioma cell-derived CD200 promotes tumor growth in vivo and in vitro. Notably, CD200 KO gliomas are spontaneously rejected by their host, a process that required a fully functional immune system, including NK and T-cells. Moreover, we report that glioma-derived or brain-injected soluble CD200 contributes to the suppression of antigen-specific CD8 T-cells in the draining lymph nodes (dLNs). Our work provides new mechanistic insights regarding CD200-mediated immunosuppression by gliomas. Statement of significance:We demonstrate mechanisms of the druggable glioma-derived CD200 checkpoint on tumor growth and immune suppression.

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.

Significance:Intraoperative molecular imaging (IMI) enables the detection and visualization of cancer tissue using targeted radioactive or fluorescent tracers. While IMI research has rapidly expanded, including the recent Food and Drug Administration approval of a targeted fluorophore, the limits of detection have not been well-defined. Aim:The ability of widely available handheld intraoperative tools (Neoprobe and SPY-PHI) to measure gamma decay and fluorescence intensity from IMI tracers was assessed while varying characteristics of both the signal source and the intervening tissue or gelatin phantoms. Approach:Gamma decay signal and fluorescence from tracer-bearing tumors (TBTs) and modifiable tumor-like inclusions (TLIs) were measured through increasing thicknesses of porcine tissue and gelatin in custom 3D-printed molds. TBTs buried beneath porcine tissue were used to simulate IMI-guided tumor resection. Results:Gamma decay from TBTs and TLIs was detected through significantly thicker tissue and gelatin than fluorescence, with at least 5% of the maximum signal observed through up to 5 and 0.5 cm, respectively, depending on the overlying tissue type or gelatin. Conclusions:We developed novel systems that can be fine-tuned to simulate variable tumor characteristics and tissue environments. These were used to evaluate the detection of fluorescent and gamma signals from IMI tracers and simulate IMI surgery.

Abstract Neuroblastoma (NB) is the most common pediatric extracranial solid tumor, with a 5-year survival rate under 50% for those with high-risk disease. Complete surgical resection is essential for these patients and is associated with improved outcomes. However, NB resection presents significant technical challenges due to encasement of vital structures that must be preserved, as well as remote deposits of disease. The current approach to find and safely resect tumor depends on the surgeon’s eyes and hands alone. Fluorescence tracers allow enhanced tumor visualization, but most are not molecularly targeted which leads to poor tumor to background signal. Further, fluorescent signal does not penetrate more than a few millimeters, a limitation that can be overcome by inclusion of a radiotracer. We, therefore, generated and evaluated a dual radio and fluorescent antibody-based probe targeting the GD2 tumor antigen to improve the identification of NB with widely available intraoperative handheld instruments. We generated an intraoperative probe (111In-αGD2-IRDye800CW) with retained affinity and stability. In vivo- Nu/j mice were injected with SK-N-BE(2) NB cells. After tumors grew for five weeks, mice were given tail vein injections of the probe. Gamma and optical biodistribution studies were performed to determine the specificity of binding and tumor to background signal four- and six-days post probe injection. Probe uptake was compared to an isotype. Gross dissections and surgeries aimed at improving completeness of resection were performed using a handheld fluorescent camera (SPY-PHI) and a gamma probe (Neoprobe). The handheld gamma probe detected xenografts with great sensitivity (average in vivo 818 cps of In111 in tumor region versus 34.8 cps in the tail). The fluorescent imaging showed high specific binding to the tumor region. Gamma biodistribution results indicated high accumulation within tumor (13.5 %ID/g) with lower accumulation in blood (3.49 %ID/g) and muscle (0.45 %ID/g). Uptake of the probe greatly exceeded isotype control antibody on days 4 and 6 post-agent injection, for both gamma and optical biodistributions. Initial tumor resection using 111In-DTPA-aGD2-IRDye800CW in an NB bearing mouse identified residual disease left behind when resection was performed using white light alone; the residual disease was then removed, guided by fluorescence imaging. Our dual-labeled probe demonstrated antigen specificity and allowed for the sensitive detection and visualization of NB in vivo with widely available intraoperative tools. This targeted probe could provide a means to improve surgical NB resection, by enabling localization of the all regions of tumor using radio-guidance, followed by clear definition of tumor margins with fluorescent signal, to maximize preservation of vital tissues. Ultimately, we believe this approach can improve safety, extent of NB resection, and patient outcomes. Citation Format: ReidAnn E. Sever, Lauren Taylor Rosenblum, Kayla C. Stanley, Dominic M. Menendez, W Barry Edwards, Marcus M. Malek, Gary Kohanbash. GD2 targeted dual-labeled intraoperative molecular imaging probe for neuroblastoma [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 2575.

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.

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).

Neuroblastoma, the most common extracranial solid malignancy in children, accounts for 15% of pediatric cancer deaths despite multimodal therapy including surgical resection. Unfortunately, complete surgical resection remains challenging due to encasement of major neurovascular structures, unclear tumor margins, and remote nodal disease. While mouse models of neuroblastoma are extremely valuable for studying tumor biology and medical treatments, the small size renders the mouse model insufficient to evaluate novel surgical therapy.Here, we have developed a novel rat model of neuroblastoma to facilitate further development of surgical treatment. Human neuroblastoma cells (SK-N-BE([2][1])) were injected into the adrenal gland of RNU nude rats. They developed 2 cm xenograft tumors at 5 weeks which were easily identifiable on MRI imaging and on visual inspection. The rats began losing weight and neared end stage at 7 weeks, at which point surgical resection was attempted. While surgical resection was technically feasible, the rats were too frail to survive surgery at the late stage. The pathology of the tumors was consistent with neuroblastoma: small round blue cells with strong PHOX2B staining. Thus, we present a novel rat neuroblastoma model that can be used for development of surgical techniques, such as the use of intraoperative contrast agents.### Competing Interest StatementThe authors have declared no competing interest. [1]: #ref-2

CD69 validation by tissue staining.

MRI analysis of tumor volume before and after treatment.

MRI analysis of tumor volume prior to immuno-PET.

CD69 analysis in single-cell RNA-seq data.

Production, quality control, and immunoreactivity of 89Zr-DFO-CD69 Ab.

Equilibrium dissociation constants of anti-CD69 Abs clone CD69.2.2 and H1.2F3.

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Immunotherapy may be promising for the treatment of some patients with GBM; however, there is a need for noninvasive neuroimaging techniques to predict immunotherapeutic responses. The effectiveness of most immunotherapeutic strategies requires T-cell activation. Therefore, 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. Herein, 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 sequencing (scRNA-seq) data from patients with recurrent GBM receiving ICI. Radiolabeled CD69 Ab PET/CT imaging (CD69 immuno-PET) was performed on GBM-bearing mice longitudinally to quantify CD69 and its association with survival following immunotherapy. We show CD69 expression is upregulated upon T-cell activation and on tumor-infiltrating lymphocytes (TIL) in response to immunotherapy. Similarly, scRNA-seq data demonstrated elevated CD69 on TILs from patients with ICI-treated recurrent GBM as compared with TILs from control cohorts. CD69 immuno-PET studies showed a significantly higher tracer uptake in the tumors of ICI-treated mice compared with controls. Importantly, we observed a 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. Our study supports the potential use of CD69 immuno-PET as an immunotherapy response assessment imaging tool for patients with GBM.Significance: Immunotherapy may hold promise for the treatment of some patients with GBM. There is a need to assess therapy responsiveness to allow the continuation of effective treatment in responders and to avoid ineffective treatment with potential adverse effects in the nonresponders. We demonstrate that noninvasive PET/CT imaging of CD69 may allow early detection of immunotherapy responsiveness in patients with GBM.