Abstract Glioblastoma (GB) is the most lethal primary brain cancer in adults, characterized by a profoundly immunosuppressive microenvironment dominated by tumor-associated macrophages and microglia (TAMs). We previously identified Interleukin-33 (IL-33), a dual-function cytokine with nuclear and extracellular roles, as a key modulator of this innate immune landscape. While full-length IL-33 accelerates GB progression by recruiting immunosuppressive myeloid cells, a nuclear-deficient variant lacking the nuclear localization sequence (ND-IL-33) potently halts tumor growth and prolongs survival. This growth-restrictive state is marked by the emergence of a distinct pro-inflammatory, anti-tumor myeloid population. Secretome profiling reveals that ND-IL-33-expressing glioma cells produce a unique repertoire of immune-stimulatory factors, consistent with enhanced innate immune activation, suggesting that loss of IL-33 nuclear activity reshapes tumor-intrinsic signaling to drive myeloid reprogramming. The clinical relevance of this biology is supported in patient-derived GB specimens, where high IL-33 expression is associated with increased immunosuppressive TAM infiltration and reduced overall survival. Ongoing bulk and single-cell RNA sequencing, integrated with functional co-culture assays, aims to define the transcriptional programs that distinguish tumor-inhibiting from tumor-promoting myeloid populations and to uncover therapeutic pathways capable of inducing this anti-tumor state. Together, these findings position IL-33 activity as a targetable regulator of myeloid plasticity in GB and offers a promising avenue to leverage innate immunity for improved therapeutic outcomes in glioblastoma. Citation Format: Tala-Maria Mouannes, Shyam V. Menon, Peipei Zeng, Jianbo Zhang, Isabelle Carrier, Eduardo Diez, Stephen M. Robbins, Donna L. Senger, . IL-33-mediated control of myeloid immunity in glioblastoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7457.

Despite intensive basic and clinical research over the past 30 years, there has been minimal improvement in outcomes for patients with glioblastoma, the deadliest form of adult brain cancer. Several factors contribute to this therapeutic challenge, including a complex interaction between the tumor and the innate immune compartment of the brain microenvironment, that is thought to support glioma proliferation and treatment resistance. Previously, we established that glioma cells can communicate with microglia/macrophage by producing the dual-function (secreted and nuclear) cytokine interleukin 33 (IL33). We found that IL33 supports the recruitment and reprogramming of pro-tumorigenic macrophage to fuel rapid and fatal tumor growth. However, in contrast, when IL33 is prevented from entering the nucleus, by loss of its nuclear localization signal (ND-IL33), tumor growth is dramatically suppressed. Using multiplex immunohistochemistry across different stages of tumor progression, we uncovered a population of macrophages unique to this growth restrictive environment, which we term glioma-inhibitory macrophages (GIMs). To resolve the molecular features of GIMs, we performed spatial transcriptomics and a computational workflow based on unsupervised deconvolution to identify cell types and activities enriched within the ND-IL33 environment. This strategy revealed that GIMs upregulate phagocytosis and antigen processing/presentation pathways and exhibit features of granulocytic cells. When examining xenografts established from patient-derived brain tumor-initiating cells, we observed a notable increase in the presence of GIMs in xenografts that exhibited long-term survival (>300 days) compared to those with short-term survival (<100 days). The tumor-suppressive nature of GIMs was further supported by experiments in which tumors formed by a combination of ND-IL33-expressing cancer cells and highly tumorigenic cells led to a growth-restrictive environment that significantly extended survival. Further functional characterization of this macrophage phenotype and development of strategies to deliver ND-IL33 to brain tumors is necessary to determine whether the recruitment and activation of GIMs could be an effective therapeutic approach for patients with glioblastoma. Shyam V. Menon, Xueqing Lun, Tala-Maria Mouannes, Peipei Zeng, Varsha T. Manoharan, Jianbo Zhang, Isabelle Carrier, Eduardo Diez, Alisha Poole, Ngoc Ha Dang, Bo Young Ahn, Katalin Osz, Sorana A. Morrissy, Jennifer A. Chan, Daniela F. Quail, Stephen M. Robbins, Donna L. Senger. Harnessing glioma-inhibitory macrophages for sustained control of glioblastoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5264.

Abstract Glioblastoma is the most common and deadly form of brain cancer. Even with aggressive treatment including surgery, chemotherapy, and radiotherapy, survival outcomes for newly diagnosed glioblastoma patients remains less than two years. Novel high throughput omics technologies have expanded our understanding of the role of innate immune system in brain tumors, that are generally believed to drive glioma progression and enable evasion of the adaptive immune system. However, targeting of this axis in the clinic remains an unmet opportunity. Previously, we discovered that the dual-function (secreted and nuclear) cytokine IL-33 is a crucial regulator of the inflammatory microenvironment that promotes glioma tumorigenesis through phenotypic and functional changes in the innate immune cell repertoire. Strikingly, when IL-33 is prevented from entering the nucleus, by deletion of its nuclear localization sequence (ΔNLS IL-33), but is still secreted, in vivo tumor growth is dramatically inhibited resulting in prolonged long-term survival. Using multiplex immunohistochemistry and spatial transcriptomics with temporal resolution across different stages of tumor progression, we identified a population of glioma-inhibitory macrophages (GIMs) unique to this suppressive environment. Assessment of GIMs in xenografts generated from patient brain tumor initiating cells found an enriched presence of these cells in xenografts with long-term survival (greater than 300 days) versus short-term survival (less than 100 days). The ability of GIMs to inhibit glioma progression was demonstrated when tumors established using a combination of ΔNLS IL-33 expressing cancer cells together with highly tumorigenic cells resulted in a growth inhibitory environment that significantly extended survival. A deeper molecular characterization of this phenotype and development of clinical strategies are currently underway. Citation Format: Shyam V. Menon, Xueqing Lun, Peipei Zeng, Jianbo Zhang, Bo Young Ahn, Henry Yu, Alisha Poole, Ngoc Ha Dang, Katalin Osz, Jennifer A. Chan, Daniela F. Quail, Stephen M. Robbins, Donna L. Senger. Durable control of brain tumors by glioma inhibitory macrophages and IL33 [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 6812.

Objective Dipeptidase-1 (DPEP-1) is a recently discovered leucocyte adhesion receptor for neutrophils and monocytes in the lungs and kidneys and serves as a potential therapeutic target to attenuate inflammation in moderate-to-severe COVID-19. We aimed to evaluate the safety and efficacy of the DPEP-1 inhibitor, LSALT peptide, to prevent specific organ dysfunction in patients hospitalised with COVID-19.Design Phase 2a randomised, placebo-controlled, double-blinded, trial.Setting Hospitals in Canada, Turkey and the USA.Participants A total of 61 subjects with moderate-to-severe COVID-19.Interventions Randomisation to LSALT peptide 5 mg intravenously daily or placebo for up to 14 days.Primary and secondary outcome measures The primary endpoint was the proportion of subjects alive and free of respiratory failure and/or the need for renal replacement therapy (RRT). Numerous secondary and exploratory endpoints were assessed including ventilation-free days, and changes in kidney function or serum biomarkers.Results At 28 days, 27 (90.3%) and 28 (93.3%) of subjects in the placebo and LSALT groups were free of respiratory failure and the need for RRT (p=0.86). On days 14 and 28, the number of patients still requiring more intensive respiratory support (O2 ≥6 L/minute, non-invasive or invasive mechanical ventilation or extracorporeal membrane oxygenation) was 6 (19.4%) and 3 (9.7%) in the placebo group versus 2 (6.7%) and 2 (6.7%) in the LSALT group, respectively (p=0.14; p=0.67). Unadjusted analysis of ventilation-free days demonstrated 22.8 days for the LSALT group compared with 20.9 in the placebo group (p=0.4). LSALT-treated subjects had a significant reduction in the fold expression from baseline to end of treatment of serum CXCL10 compared with placebo (p=0.02). Treatment-emergent adverse events were similar between groups.Conclusion In a Phase 2 study, LSALT peptide was demonstrated to be safe and tolerated in patients hospitalised with moderate-to-severe COVID-19.Trial registration number NCT04402957.

Supplementary Figure S1:TNC expression in mice implanted with human BTIC. Supplementary Figure S2: TNC expression, BTIC growth and invasiveness in orthotopic BT53M xenograft. Supplementary Figure S3: Tumor growth in orthotopic BTIC 53M xenograft implanted in mice - correspondence of TNC with nucleolin+ areas. Supplementary Figure S4: TNC increases BTIC growth in a concentration dependent manner over time. Supplementary Figure S5: Integrin expression in BTICs and elucidation of the role of alpha2beta1 integrin in TNC promoted BTIC growth. Supplementary Figure S6: Data extraction from Oncomine show elevated JAG1 and NOTCH1 expression in human brain tumors. Supplementary Figure S7: Expression of JAG1 and NOTCH1 is negatively correlated with longevity of patients. Supplementary Figure S8: Correlation between TNC, JAG1, NOTCH1 and alpha2beta1 integrin as determined through the TCGA database. Supplementary Figure S9: Inhibitors, Notch activity and JAG1 expression. Supplementary Figure S10: shRNA to reduce TNC expression in BTICs.

Abstract Despite a sophisticated treatment regimen, including surgery, chemotherapy, and radiotherapy, survival outcomes for glioblastoma remain at a dismal 14.6 months. Multi-omics profiling have established that myeloid phagocytes drive glioma progression and contribute to therapeutic resistance. However, effective targeting of this axis remains an unmet clinical opportunity. Previously, we found that the dual-function (secreted and nuclear) cytokine IL-33 is a key regulator of the inflammatory microenvironment that aids glioma tumorigenesis through phenotypic and functional changes in the innate immune cell repertoire. Strikingly, when IL-33 is prevented from entering the nucleus, by deletion of its nuclear localization signal (ΔNLS IL-33), but is still secreted, in vivo tumor growth is dramatically suppressed resulting in extended long-term survival. Using spatial transcriptomics and multiplex immunohistochemistry with temporal resolution at different stages of tumor progression, we identified a population of glioma-inhibitory macrophages (GIMs) unique to this suppressive environment. Assessment of xenografts generated from patient brain tumor initiating cells found an enrichment of GIMs in xenografts with long-term survival (>300 days) compared to short-term survivors (<100 days). The ability of GIMs to inhibit glioma progression was further highlighted when tumors established using a combination of ΔNLS IL-33 expressing cancer cells together with highly tumorigenic cells resulted in a growth inhibitory environment that significantly prolonged survival through the polarization and activation of GIMs. Additional characterization of this phenotype and development of clinical strategies to deliver ΔNLS IL-33 to brain tumors is warranted to determine if recruitment and activation of GIMs is a translatable therapeutic strategy for glioma patients.

The nervous system regulates cancer progression, and the importance of neuron–glioma communication in tumor growth is evident in glioblastoma. This tumor-promoting communication presents a potential therapeutic axis, a concept reinforced by a study that identifies a specific potassium channel complex as a therapeutic target.

This supplementary data of microarray information contains results that support the tenascin C to notch pathway highlighted in this manuscript

Supplementary data of microarray information contains results that support the tenascin C to notch pathway highlighted in this manuscript

Figure S1: Preclinical assessment of clioquinol. Figure S2: Preclinical assessment of montelukast. Figure S3: Secondary validation of compounds identified via HTS. Figure S4: Sensitivity of BTICs to TMZ. Figure S5: Copper is required for Disulfiram-mediated cell killing. Figure S6: Cell Viability and Apoptosis in the presence of DSF-Cu. Figure S7: Efficacy to DSF-Cu is independent of MGMT expression. Figure S8: Inhibition of proteasome activity by DSF-Cu. Figure S9: DSF-Cu in combination with TMZ prolongs survival in vivo. Figure S10: DSF-Cu mediated apoptosis in vivo. Table S1: Summary of BTIC Molecular Characterization. Table S2: Summary of Compound Priortization. Table S3: Down regulation of DNA repair genes by DSF-Cu.

Abstract Approximately 3000 Canadians a year are diagnosed with the most aggressive and fatal form of brain cancer called glioma. Even with surgical resection, chemotherapy, and radiotherapy these patients have an average survival of less than 20 months. Research in the past has primarily focused on identifying and targeting specific genetic mutations in the tumor. However, it is apparent that the environment surrounding the tumor can influence tumor growth, tumor spread into the brain, and drug resistance. The brain presents many challenges to glioma treatment, one of which is the unique environment in which gliomas grow, which contributes to poor patient outcomes. The interaction between tumor cells, surrounding normal brain tissue, and immune cells supports the aggressiveness of glioma. Using a model of chemosensitive and chemoresistant tumors, we have identified an increase in the GABA transporter GAT1 in the chemosensitive tumor treated with the standard chemotherapy temozolomide. GAT1 is primarily expressed on neurons, but has also been shown to be expressed on astrocytes and immune cells such as macrophages. Our lab has identified that macrophages tend to concentrate in the same region as GAT1 expressing cells within the tumor environment. In addition, multiple papers have shown that an increase in GABA promotes a glioma-promoting immune microenvironment. Thus, we believe that treatment with temozolomide causes an increase in environmental GAT1 expression, resulting in a reuptake of GABA, and promoting a glioma-inhibitory macrophage phenotype. We believe that understanding how the glioma-inhibitory macrophage phenotype occurs will help improve patient outcomes.

The mechanisms that drive leukocyte recruitment to the kidney are incompletely understood. Dipeptidase-1 (DPEP1) is a major neutrophil adhesion receptor highly expressed on proximal tubular cells and peritubular capillaries of the kidney. Renal ischemia reperfusion injury (IRI) induces robust neutrophil and monocyte recruitment and causes acute kidney injury (AKI). Renal inflammation and the AKI phenotype were attenuated in Dpep1−/− mice or mice pretreated with DPEP1 antagonists, including the LSALT peptide, a nonenzymatic DPEP1 inhibitor. DPEP1 deficiency or inhibition primarily blocked neutrophil adhesion to peritubular capillaries and reduced inflammatory monocyte recruitment to the kidney after IRI. CD44 but not ICAM-1 blockade also decreased neutrophil recruitment to the kidney during IRI and was additive to DPEP1 effects. DPEP1, CD44, and ICAM-1 all contributed to the recruitment of monocyte/macrophages to the kidney following IRI. These results identify DPEP1 as a major leukocyte adhesion receptor in the kidney and potential therapeutic target for AKI.

Alveolar soft part sarcoma (ASPS) is a rare pediatric malignancy which has characteristically poor clinical outcomes due to a propensity to metastasize along with a complete lack of chemotherapeutic options. One additional difficulty of this rare tumor is an absence of established in vivo models. Here, we describe the establishment of a patient derived xenograft model (PDX), and corresponding cell line, from the lung metastases of a 14-year-old female diagnosed with ASPS. Using this model, we performed a drug screen, from which the proteasome inhibitor carfilzomib inhibited tumor cell viability and growth in vitro and in vivo. To further elucidate factors implicated in tumor progression and chemotherapy resistance, we characterized the microenvironment and secretome of the ASPS PDX. In keeping with recent literature showing large numbers of tumor associated macrophage (TAMs) in ASPS, we found that 96% of the myeloid cells present in the PDX were TAMS, with 35% taking on a pro-tumor phenotype. Further, factors secreted by the tumor microenvironment largely support immune cell recruitment and pro-tumor phenotypes. Interestingly, in vivo assessment demonstrates that Iba1+ macrophage populations decrease by 60% with carfilzomib treatment. This prompted investigation into the potential impact of TAMs on ASPS growth and progression, and if they are implicated in the therapeutic response to carfilzomib. Based on preliminary findings that carfilzomib targets the pro-tumor phenotypic states of bone marrow derived macrophage in vitro, we hypothesize that carfilzomib alters the recruitment and phenotype of TAMs, decreasing tumor cell viability in vitro, and thus tumor burden in vivo. Citation Format: Alexis M. Philippot, Ngoc Ha Dang, Shyam V. Menon, Jennifer Bourdage, Xueqing Lun, Bo Young Ahn, Stephen M. Robbins, Donna L. Senger. Defining the microenvironment of alveolar soft part sarcoma & it’s role in therapeutic outcomes [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A006.

Sarcomas are a heterogeneous group of cancers occurring in tissues derived from the mesenchyme. Despite improved treatment strategies that include surgery and broad-based chemotherapeutics, survival of patients with sarcoma remains unchanged in the last 40 years with 5-year overall survival less than 25%. In part, this high rate of mortality can be attributed to the development of pulmonary metastases, a process that occurs in up to 50% of patients. We propose that preventing the occurrence or growth of lung metastases can improve the outcome for many of these patients. Our growing understanding of the dynamic relationship between inflammation and cancer has led to the investigation of anti-inflammatory approaches to treat cancer, including metastasis. Specifically, recent studies suggest a role for neutrophils in cancer metastasis, identifying a promising target for therapeutic intervention. The aim of the present study is to investigate the role of myeloid cells in the metastatic process, and their potential to act as therapeutic targets during the development of lung metastases. Herein we used human and syngeneic osteosarcoma lung metastatic models together with in vitro assays to assess the role of neutrophils in this process. Our results demonstrate that neutrophils are essential to facilitate the development of osteosarcoma pulmonary metastases and that treatment with the novel anti-inflammatory agent GM1-targeted linoleate-containing TLR2 ligand (GML), known to inhibit neutrophil recruitment, diminishes their occurrence. Overall, our results suggest that neutrophils play a role in mediating osteosarcoma lung metastasis and reveal GML as a potential anti-metastatic drug therapy. Citation Format: Liane Babes, Lauren A. Wierenga, Ngoc-Ha Dan, Xueqing Lun, Kimberly-Ann R. Goring, Stephen M. Robbins, Donna L. Senger. The novel anti-inflammatory agent GML (GM1-targeted linoleate-containing TLR2 ligand) inhibits sarcoma metastasis to the lung [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A014.

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.

Glioblastoma remains one of the most fatal cancers in humans. Despite the advent of optimal surgical resection, guided radiotherapy, and the addition of chemotherapy (temozolomide; TMZ) average survival remains at a dismal 14.6 months with very few long-term survivors. In this chapter we will discuss the various mouse and human glioma model systems that are used and the generation of in vivo selected models that recapitulate TMZ resistance together with the promise of repurposing disulfiram in the setting of relapsed glioblastoma. In addition, we will move beyond the sole targeting of the glioma cells and discuss the impact of the tumor microenvironment both at the level of the normal cellular repertoire that includes resident and recruited immune cells and the molecular and biophysical properties that contribute to gliomagenesis and therapeutic resistance where exciting new and novel therapeutic options are emerging.

Despite extensive molecular characterization, human glioblastoma remains a fatal disease with survival rates measured in months. Little improvement is seen with standard surgery, radiotherapy and chemotherapy. Clinical progress is hampered by the inability to detect and target glioblastoma disease reservoirs based on a diffuse invasive pattern and the presence of molecular and phenotypic heterogeneity. The goal of this study was to target the invasive and stem-like glioblastoma cells that evade first-line treatments using agents capable of delivering imaging enhancers or biotherapeutic cargo. To accomplish this, a combinatorial phage display library was biopanned against glioblastoma cell model systems that accurately recapitulate the intra- and inter-tumor heterogeneity and infiltrative nature of the disease. Candidate peptides were screened for specificity and ability to target glioblastoma cells in vivo. Cargo-conjugated peptides delivered contrast-enhancing agents to highly infiltrative tumor populations in intracranial xenograft models without the obvious need for blood brain barrier disruption. Simultaneous use of five independent targeting peptides provided greater coverage of this complex tumor and selected peptides have the capacity to deliver a therapeutic cargo (oncolytic virus VSVΔM51) to the tumor cells in vivo. Herein, we have identified a series of peptides with utility as an innovative platform to assist in targeting glioblastoma for the purpose of diagnostic or prognostic imaging, image-guided surgery, and/or improved delivery of therapeutic agents to glioblastoma cells implicated in disease relapse.

IL-33, a member of the IL-1 cytokine family has been shown to play a dual role within the body. First IL-33, similar to other IL-1 family members, is a secreted cytokine that binds to the cell surface receptor ST2 to induce a number of cell signaling pathways. Second, IL-33 enters the nucleus where it binds chromatin and directs transcriptional control of an array of growth factors and cytokines. Consistent with its complex cellular regulation, IL-33 mediates an array of biological functions by acting on a wide range of innate and adaptive immune cells. Recently, we found that IL-33 is expressed in a large number of human glioma patient specimens where its expression within the tumor correlates with the increased presence of Iba+ cells that include both resident microglia and recruited monocyte and macrophages. Strikingly, glioma derived expression of IL-33 correlates with a dramatic decrease in overall survival of tumor-bearing animals and thus supports its role as an influential factor in gliomagenesis. Notably however, when the nuclear localization function of IL-33 is crippled, the tumor microenvironment is programmed to be anti-tumorigenic and results in prolonged overall survival suggesting that when educated appropriately this could represent a novel therapeutic strategy for glioma (De Boeck et al. (2020), Nat Commun, doi: 10.1038/s41467-020-18569-4).

Despite a deeper molecular understanding, human glioblastoma remains one of the most treatment refractory and fatal cancers. It is known that the presence of macrophages and microglia impact glioblastoma tumorigenesis and prevent durable response. Herein we identify the dual function cytokine IL-33 as an orchestrator of the glioblastoma microenvironment that contributes to tumorigenesis. We find that IL-33 expression in a large subset of human glioma specimens and murine models correlates with increased tumor-associated macrophages/monocytes/microglia. In addition, nuclear and secreted functions of IL-33 regulate chemokines that collectively recruit and activate circulating and resident innate immune cells creating a pro-tumorigenic environment. Conversely, loss of nuclear IL-33 cripples recruitment, dramatically suppresses glioma growth, and increases survival. Our data supports the paradigm that recruitment and activation of immune cells, when instructed appropriately, offer a therapeutic strategy that switches the focus from the cancer cell alone to one that includes the normal host environment.