Chimeric antigen receptor CAR T cell therapy faces notable limitations in treatment of solid tumors. The suppressive tumor microenvironment TME, characterized by complex interactions among immune and stromal cells, is gaining recognition in conferring resistance to CAR T cell therapy. Despite the abundance and diversity of macrophages in the TME, their intricate involvement in modulating responses to CAR T cell therapies remains poorly understood. Here, we conducted single-cell RNA sequencing scRNA seq on tumors from 41 glioma patients undergoing IL13Ra2-targeted CAR T cell therapy, identifying elevated suppressive SPP1 signatures predominantly in macrophages from patients who were resistant to treatment. Further integrative scRNA seq analysis of high-grade gliomas as well as an interferon-signaling deficient syngeneic mouse model both resistant to CAR T therapy demonstrated the role of congruent suppressive pathways in mediating resistance to CAR T cells and a dominant role for SPP1+ macrophages. SPP1 blockade with an anti-SPP1 antibody abrogates the suppressive TME effects and substantially prolongs survival in IFN signaling-deficient and glioma syngeneic mouse models resistant to CAR T cell therapy. These findings illuminate the role of SPP1+ macrophages in fueling a suppressive TME and driving solid tumor resistance to CAR cell therapies. Targeting SPP1 may serve as a universal strategy to reprogram immune dynamics in solid tumors mitigating resistance to CAR T therapies.

Abstract In the evolving landscape of glioblastoma (GBM) treatment, clinical trials exploring chimeric antigen receptor (CAR) T cell therapies have shown promise; however, their therapeutic efficacy remains constrained, particularly due to the immunosuppressive tumor microenvironment (TME). Emerging evidence highlights that the TME, especially myeloid-driven suppressive networks, plays a central role in resistance to CAR T cell therapy. Despite progress in elucidating these mechanisms, significant ambiguities endure, particularly regarding patient-specific TME heterogeneity and its impact on therapeutic outcomes. Radiation therapy remains a cornerstone in GBM management, known not only for its direct cytotoxic effects but also for its ability to modulate the TME and potentially enhance immune responses. However, the combination of radiation with CAR T cell therapy has not been fully explored, particularly in the context of targeting suppressive myeloid subsets. Recent studies suggest that radiation may induce immunogenic changes within the TME, potentially enhancing CAR T cell infiltration and activity. Our study seeks to investigate how targeting key suppressive mediators, such as SPP1-expressing macrophages, can reshape the TME, setting the stage for combinatorial approaches that leverage the immunomodulatory effects of radiation therapy In this study, we utilized single-cell RNA sequencing to analyze tumors from 41 glioma patients undergoing IL13Rα2-targeted CAR T cell therapy. Our findings revealed heightened suppressive extracellular matrix (ECM) remodeling and immunosuppressive signatures driven by SPP1-expressing myeloid cells, predominantly in non-responsive tumors. Elevated SPP1 expression and increased infiltration of SPP1+ myeloid cells were associated with poor clinical outcomes. Functional transcriptomic analyses demonstrated that SPP1-high macrophages exhibited reduced antigen presentation, impaired phagocytosis, and enhanced ECM biogenesis, coupled with elevated lipid metabolism and non-glycolytic ATP production. These metabolic adaptations were linked to a suppressive immunophenotype that hindered CAR T cell efficacy. To explore therapeutic strategies, we assessed the impact of SPP1 blockade in preclinical syngeneic glioma models. Pre-treatment with anti-SPP1 antibodies prior to CAR T cell infusion significantly reprogrammed the TME, enhancing CAR T cell efficacy and reversing resistance in previously non-responsive models. These results highlight the potential of targeting SPP1 to disrupt suppressive macrophage networks, thereby augmenting CAR T cell responses. Our findings underscore SPP1 as a promising target to overcome resistance in CAR T cell therapies for GBM. This study lays the groundwork for future integration of targeted therapies, such as SPP1 inhibition, with radiation therapy to potentially improve therapeutic outcomes in resistant gliomas, offering new avenues for combinatorial therapeutic innovation. Citation Format: Sharareh Gholamin, Heini Natri, Yuqi Zhao, Shengchao Xu, Maryam Aftabizadeh, Begonya CominAnduix, Supraja Saravanakumar, Christian Masia, Robyn Wong, Lance Peter, Mei-i Chung, Evan D. Mee, Brenda Aguilar, Davis Y. Torrejon, Anusha Kalbasi, Darya Alizadeh, Xiwei Wu, Antoni Ribas, Stephen Forman, Behnam Badie, Terence M. Williams, Nicholas E. Banovich, Christine E. Brown.Targeting SPP1 to enhance CAR T cell therapy in glioblastoma: Implications for integrating radiation therapy in resistant solid tumors.[abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P015

Abstract In the evolving landscape of glioblastoma (GBM) therapy, the recent proliferation of clinical trials exploring chimeric antigen receptor (CAR) T cell interventions has garnered attention, although their therapeutic efficacy remains limited. Despite recent progress in elucidating the role of the GBM microenvironment in immunotherapy resistance, particularly regarding intercellular networks and macrophages, significant ambiguities endure, impeding a comprehensive understanding of resistance mechanisms amidst the patient-specific heterogeneity of the tumor microenvironment (TME). In this study, we utilized single-cell RNA sequencing to analyze tumors from 41 glioma patients undergoing IL13Rα2-targeted CAR T cell therapy. Our findings unveiled heightened suppressive extracellular network signatures, particularly SPP-1, predominantly observed within myeloid cells from non-responsive patients. The clinical significance of both SPP1+ myeloid cell abundance and overall expression levels revealed associations with patient outcomes. Subsequently, transcriptomic insights were translated to myeloid cell functions in patients exhibiting high versus low SPP1 expression. SPP1-expressing macrophages displayed diminished antigen presentation, phagocytosis, and cell-mediated cytotoxicity, along with augmented extracellular matrix biogenesis and degradation, iron efflux, and suppressive interleukin pathways. Moreover, SPP1-high macrophages exhibited enhanced ligand-receptor interactions, implying a predisposition towards suppressive activities. Immunometabolism pathways, including lipid and ketone body biogenesis and regeneration, were upregulated in SPP1-expressing macrophages, indicating an increased reliance on non-glycolysis-mediated ATP production. To assess translational implications, the therapeutic efficacy of SPP1 blockade was evaluated in a syngeneic glioma model. Blockade of SPP1 with an anti-SPP1 antibody prior to CAR T cell infusion effectively mitigated the immunosuppressive milieu, reversing resistance in preclinical GBM models to murine IL13Rα2-targeted CAR T cell therapy. Subsequent profiling of tumors post-treatment provided insights into TME alterations following SPP1 blockade therapy. This investigation illuminates the intricate interplay between the TME and CAR T cell therapy resistance in GBM, underscoring SPP1 as a promising therapeutic target to surmount CAR T cell resistance and enhance treatment outcomes.

INTRODUCTION:Cancer immunotherapy has revolutionized the field of oncology, offering new hope to patients with advanced malignancies. Tumor-induced immunosuppression limits the effectiveness of current immunotherapeutic strategies, such as PD-1/PDL-1 checkpoint inhibitors. Adenosine, a purine nucleoside molecule, is crucial to this immunosuppression because it stops T cells from activating and helps regulatory T cells grow. Targeting the adenosine pathway and blocking PD-1/PDL-1 is a potential way to boost the immune system's response to tumors. AREAS COVERED:This review discusses the current understanding of the adenosine pathway in tumor immunology and the preclinical and clinical data supporting the combination of adenosine pathway inhibitors with PD-1/PDL-1 blockade. We also discuss the challenges and future directions for developing combination immunotherapy targeting the adenosine pathway and the PD-1/PDL-1 axis for cancer treatment. EXPERT OPINION:The fact that the adenosine signaling pathway controls many immune system processes suggests that it has a wide range of therapeutic uses. Within the next five years, there will be tremendous progress in this area, and the standard of care for treating malignant tumors will have switched from point-to-point therapy to the integration of immunological networks comprised of multiple signaling pathways, like the adenosine axis.

Background Glioblastoma (GBM) is the most aggressive form of glioma with a median survival rate of less than two years. Despite aggressive standard treatments, GBM remains uniformly fatal with a poor prognosis. Achieving responsiveness of GBMs to chimeric antigen receptor (CAR) T cell therapy has been a significant challenge due to the heterogeneity and evasive mechanisms employed by solid tumors, particularly GBM, to resist therapy. Recent studies have highlighted the substantial role of cancer-associated fibroblasts (CAFs) in GBM invasion by depositing various collagen subunits, including COL1A1, COL1A2, COL5A1, COL5A2, and COL8A1, within the extracellular matrix (ECM). This collagen deposition leads to increased ECM stiffness, modulating the tumor microenvironment, inducing immune suppression, and hindering T cell trafficking, ultimately worsening clinical outcomes and patient survival. Additionally, COL1A1 has been implicated in promoting tumor aggressiveness, particularly in GBM cases with wildtype isocitrate dehydrogenase (IDH-wt) status and a poor prognosis. Based on these findings, we investigated whether the expression level of COL1A1 could influence the response to CAR T cell therapy. Methods We performed immunofluorescent staining on tissue biopsies obtained from GBM patients to identify a subpopulation of CAFs expressing ACTA2 (alpha smooth muscle actin), PDGFRβ, and COL1A1. Confocal microscopy was used to visualize the stained CAFs. ACTA2 and PDGFRβ serve as markers for a specific subset of CAFs involved in the epithelial-to-mesenchymal transition (EMT), a process associated with immune suppression and tumor growth. The presence of these CAF subpopulations was previously confirmed in GBM patients enrolled in an IL13Ra2 targeted CAR T cell trial through single-cell RNA sequencing. Subsequently, we compared our immunostaining images with flow cytometry data obtained from cerebrospinal fluid (CSF) or tumor fragment (TF) biopsies collected from the same patients after CAR T cell therapy. Results Our preliminary analysis revealed an increase in the number of infiltrating CD3+ cells from baseline, particularly within the effector CD8+ subpopulation (characterized by CD27+CD28+ expression), during CAR T cell therapy in patients with low COL1A1 expression. Furthermore, FACS analysis demonstrated a significant increase in the proportion of CD8+ T cells relative to CD4+ T cells in these patients. Conclusions Ongoing experiments are currently investigating the distribution of CAF subpopulations producing COL1A1 and their correlation with the response to CAR T cell therapy.

PDF file - 386K, A, Cell viability relative to day 0 is shown following the indicated treatments in the indicated cell lines. Error bars represent the mean plus-minus SD of three replicates per condition. B, qPCR assessment of MYC mRNA expression in three MYC-amplified patient derived medulloblastoma cell lines treated with JQ1R or JQ1S at doses shown. Values represent mean plus-minus SD of six replicate measurements.

PDF file - 291K, Densitometry quantification of Western Immunoblots shown in Figures 4 and 5.

PDF file - 66K, A, Primers used for qPCR assays B, MYC gene sets in GSEA.

PDF file - 72K, Nanostring CNV of MB002 and MB004.

PDF file - 63K, MYC gene sets enriched in medulloblastoma samples with high expression of MYC isoforms.

PDF file - 79K, C2 Gene Sets down-regulated following treatment with JQ1.

PDF file - 567K, A, Gene expression sub-group profiling of MB002 and MB004 patient-derived medulloblastoma cell lines. B, Western blot (left) and densitometry of western blot (right) depicting MYC expression relative to β actin loading control in patient derived medulloblastoma cell lines. ** depict cell lines which are known to harbor amplification of MYC.

PDF file - 71K, Differentially expressed genes following treatment with JQ1.

Abstract Purpose:MYC-amplified medulloblastomas are highly lethal tumors. Bromodomain and extraterminal (BET) bromodomain inhibition has recently been shown to suppress MYC-associated transcriptional activity in other cancers. The compound JQ1 inhibits BET bromodomain-containing proteins, including BRD4. Here, we investigate BET bromodomain targeting for the treatment of MYC-amplified medulloblastoma. Experimental Design: We evaluated the effects of genetic and pharmacologic inhibition of BET bromodomains on proliferation, cell cycle, and apoptosis in established and newly generated patient- and genetically engineered mouse model (GEMM)-derived medulloblastoma cell lines and xenografts that harbored amplifications of MYC or MYCN. We also assessed the effect of JQ1 on MYC expression and global MYC-associated transcriptional activity. We assessed the in vivo efficacy of JQ1 in orthotopic xenografts established in immunocompromised mice. Results: Treatment of MYC-amplified medulloblastoma cells with JQ1 decreased cell viability associated with arrest at G1 and apoptosis. We observed downregulation of MYC expression and confirmed the inhibition of MYC-associated transcriptional targets. The exogenous expression of MYC from a retroviral promoter reduced the effect of JQ1 on cell viability, suggesting that attenuated levels of MYC contribute to the functional effects of JQ1. JQ1 significantly prolonged the survival of orthotopic xenograft models of MYC-amplified medulloblastoma (P < 0.001). Xenografts harvested from mice after five doses of JQ1 had reduced the expression of MYC mRNA and a reduced proliferative index. Conclusion: JQ1 suppresses MYC expression and MYC-associated transcriptional activity in medulloblastomas, resulting in an overall decrease in medulloblastoma cell viability. These preclinical findings highlight the promise of BET bromodomain inhibitors as novel agents for MYC-amplified medulloblastoma. Clin Cancer Res; 20(4); 912–25. ©2013 AACR.

IntroductionGlioblastoma Multiforme (GBM) is one of the fatal cancers of the Central Nervous System (CNS). A variety of reasons exist for why previous immunotherapy strategies, especially Immune Checkpoint Blockers (ICBs), did not work in treating GBM patients. The cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a key immune checkpoint receptor. Its overexpression in cancer and immune cells causes tumor cell progression. CTLA-4 suppresses anti-tumor responses inside the GBM tumor-immune microenvironment.Areas coveredIt has been attempted to explain the immunobiology of CTLA-4 as well as its interaction with different immune cells and cancer cells that lead to GBM progression. Additionally, CTLA-4 targeting studies have been reviewed and CTLA-4 combination therapy, as a promising therapeutic target and strategy for GBM immunotherapy, is recommended.Expert opinionCTLA-4 could be a possible supplement for future cancer immunotherapies of GBM. However, many challenges remain such as the high toxicity of CTLA-4 blockers, and the unresponsiveness of most patients to immunotherapy. For the future clinical success of CTLA-4 blocker therapy, combination approaches with other targeted treatments would be a potentially effective strategy. Going forward, predictive biomarkers can be used to reduce trial timelines and increase the chance of success.

IL13Rα2 is an attractive target due to its overexpression in a variety of cancers and rare expression in healthy tissue, motivating expansion of interleukin 13 (IL13)–based chimeric antigen receptor (CAR) T cell therapy from glioblastoma into systemic malignancies. IL13Rα1, the other binding partner of IL13, is ubiquitously expressed in healthy tissue, raising concerns about the therapeutic window of systemic administration. IL13 mutants with diminished binding affinity to IL13Rα1 were previously generated by structure-guided protein engineering. In this study, two such variants, termed C4 and D7, are characterized for their ability to mediate IL13Rα2-specific response as binding domains for CAR T cells. Despite IL13Rα1 and IL13Rα2 sharing similar binding interfaces on IL13, mutations to IL13 that decrease binding affinity for IL13Rα1 did not drastically change binding affinity for IL13Rα2. Micromolar affinity to IL13Rα1 was sufficient to pacify IL13-mutein CAR T cells in the presence of IL13Rα1-overexpressing cells in vitro. Interestingly, effector activity of D7 CAR T cells, but not C4 CAR T cells, was demonstrated when cocultured with IL13Rα1/IL4Rα-coexpressing cancer cells. While low-affinity interactions with IL13Rα1 did not result in observable toxicities in mice, in vivo biodistribution studies demonstrated that C4 and D7 CAR T cells were better able to traffic away from IL13Rα1+ lung tissue than were wild-type (WT) CAR T cells. These results demonstrate the utility of structure-guided engineering of ligand-based binding domains with appropriate selectivity while validating IL13-mutein CARs with improved selectivity for application to systemic IL13Rα2-expressing malignancies.

Emergence of the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak identified in late 2019 in Wuhan, China, was declared a pandemic in March 2020. High fatality rate in afflicted patients prompted scientists and physicians to develop various vaccines against the virus. While administration of millions of doses of the adenoviral vector vaccines (e.g., Oxford-AstraZeneca (ChAdOx1 nCoV-19) and Janssen/Johnson & Johnson (Ad26.COV2. S)) has helped control the disease, numerous cases of cerebral venous sinus thrombosis (CVST) with thrombocytopenia have been reported in vaccinated individuals. In this article, we aim to review the cases reported thus far and further discuss the association between the vaccine administration and subsequent cerebral thromboembolic events. Our study was performed and reported based on the guidelines outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). PubMed, Google Scholar and Norris Medical Library databases were searched using the following terms: coronavirus disease 2019 (COVID-19) vaccines (“AstraZeneca” or “AZD1222 COVID vaccine” or “ChAdOx1 nCoV-19 COVID-19 vaccine” or “Janssen” or “Johnson & Johnson COVID vaccine” or “Ad26.COV2 COVID vaccine”), coagulopathy (“cerebral venous sinus thrombosis (CVST)” and “vaccine-induced immune thrombotic thrombocytopenia (VITT)” or “cerebral venous thrombosis (CVT)”) and thrombocytopenia. All the relevant studies within the English literature up to August 1, 2021, were included. Fourteen most recent articles reporting on 66 patients with CVST and VITT after adenoviral vector vaccination were reviewed by two independent authors. Age of the patients ranged from 18 to 60 years. The majority of cases were women (43 females versus 14 males). Platelet count was between 5 and 127 × 10 9 /L. Above-normal D-dimer was found in 86% of the patients. A total of 68% of the patients had positive platelet factor 4 IgG assay in the absence of prior exposure to heparin. Among CVST cases following COVID vaccination, 44% succumbed to death. Early diagnosis and treatment of CVST plays a fundamental role in decreasing morbidity and mortality. Health care professional should be familiar with this rare complication post vaccination against COVID-19. Given the rarity of CVST after the COVID-19 vaccine, the benefit of vaccination outweighs the potential harm. J Neurol Res. 2021;11(5):69-76 doi: https://doi.org/10.14740/jnr700

Abstract Chimeric antigen receptor (CAR) T cells mediate potent antigen-specific antitumor activity; however, their indirect effects on the endogenous immune system are not well characterized. Remarkably, we demonstrate that CAR T-cell treatment of mouse syngeneic glioblastoma (GBM) activates intratumoral myeloid cells and induces endogenous T-cell memory responses coupled with feed-forward propagation of CAR T-cell responses. IFNγ production by CAR T cells and IFNγ responsiveness of host immune cells are critical for tumor immune landscape remodeling to promote a more activated and less suppressive tumor microenvironment. The clinical relevance of these observations is supported by studies showing that human IL13Rα2–CAR T cells activate patient-derived endogenous T cells and monocytes/macrophages through IFNγ signaling and induce the generation of tumor-specific T-cell responses in a responding patient with GBM. These studies establish that CAR T-cell therapy has the potential to shape the tumor microenvironment, creating a context permissible for eliciting endogenous antitumor immunity. Significance: Our findings highlight the critical role of IFNγ signaling for a productive CAR T-cell therapy in GBM. We establish that CAR T cells can activate resident myeloid populations and promote endogenous T-cell immunity, emphasizing the importance of host innate and adaptive immunity for CAR T-cell therapy of solid tumors. This article is highlighted in the In This Issue feature, p. 2113