Focused ultrasound for cellular mechanoactivation and nanomedicine delivery in cancer

Glycosylation is a complex post-translational modification essential for development, growth, and survival. Altered glycosylation is a hallmark of cancer, yet the roles of many glycoproteins remain unclear, hampering translation of glycoprotein-related vulnerabilities into therapeutic strategies. Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies, with a five-year survival rate under 13%. For decades, serum levels of CA19-9, a terminal tetra-saccharide glycan, conjugating many secreted and cell surface proteins, has been the single-most effective biomarker to track PDA progression in patients. Recent studies have identified Fibulin-3 (Fbln3) as a secreted, CA19-9–modified matricellular glycoprotein that drives EGFR hyperactivation in the pancreatic epithelium, which is essential for PDA development. However, the functional role of Fbln3 in PDA has remained largely unexplored. Here, we show that Fbln3 promotes PDA progression and tumor microenvironment (TME) remodeling. Fbln3 expression is significantly upregulated in both human and mouse PDA tissues. Overexpression and knockdown of Fbln3 modulate PDA growth rates both in vitro and in vivo. In CA19-9–expressing KRASG12D mutant PDA organoids, Fbln3 enhances activation of key oncogenic pathways, including EGFR, NFκB, and TGFβ signaling. Notably, Fbln3 regulates the expression of IL1A and TGF, cytokines known to impact both autocrine and paracrine signaling. In syngeneic orthotopic tumor models, Fbln3 promotes the expansion of antigen-presenting cancer associated fibroblasts and reduces infiltration of CD8+ T cells, contributing to an immunosuppressive microenvironment. Taken together, these findings demonstrate that CA19-9-modified Fbln3 drives pancreatic tumor progression and TME remodeling by regulating IL1A and TGFb production, highlighting the need to further investigate its potential as a therapeutic target. Citation Format: Hyemin Song, Jasper Hsu, Satoshi Ogawa, Kristina Peck, Kassidy Curtis, Chelsea Bottomely, McKenna Stamp, Dannielle D. Engle. Fibulin-3 drives tumor progression and microenvironment remodeling in CA19-9-induced pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr B009.

Mast cells are of paramount importance to allergies, pathogen immune responses during infections, and angiogenesis, as well as innate and adaptive immune regulations. Beyond all these roles, mast cells are now more and more being recognized as modulators of tumor microenvironment. Notwithstanding mounting evidences of mast cell accumulation in tumors, their exact role in tumor microenvironment is still incompletely understood. In this review, we discuss the significant role of mast cells in the remodeling of tumor microenvironment by either releasing various factors after activation or interacting with other cells within tumor and, as a result, the possible role of mast cell in cancer invasion and metastasis. We also discuss recent findings that mast cells actively release microparticles, which account for the transfer of membrane-type receptor signal and regulatory molecules such as microRNAs to tumor cells and immune cells. These findings on mast cells provide further insights into the complexity of tumor microenvironment remodeling.

Pancreatic cancer is one of most deadly cancers with a 5-year survival rate of 6%. Accumulation of hyaluronan (HA) is found in about 87% of human pancreatic adenocarcinomas, and removal of HA suppresses tumor growth in HA-rich preclinical models. In a transgenic pancreatic cancer mouse model (LSL-KrasG12D/+;LSLTrp53R172H/+;Pdx-1-Cre, KPC), removal of HA by pegylated human recombinant PH20 hyaluronidase (PEGPH20) inhibits tumor growth and increases survival in combination with gemcitabine compared to gemcitabine monotherapy. In this study, we explored the role of HA synthesizing (HAS) enzymes HAS2 and HAS3 and HA accumulation in pancreatic cancer tumor growth and remodeling of tumor microenvironment. HAS2 and HAS3 were overexpressed in BxPC3 human pancreatic cancer cells using lentiviral vectors. Stable HAS2 and HAS3 overexpressing pancreatic cancer cell lines secreted more HA to culture medium and produced larger pericellular HA matrices than parental BxPC3 cells. In vivo, overexpression of HAS2 or HAS3 led to an increase in BxPC3 xenograft tumor growth (peritibial i.m. tumor model) compared to parental cells. Interestingly, overexpression of HAS3 was more effective to enhance tumor growth than overexpression of HAS2. In addition, massive accumulation of extracellular HA was found in HAS3 overexpressing tumors while HAS2 overexpressing tumors contained both extracellular and intracellular HA. Treatment with PEGPH20 removed the majority of extracellular HA and induced a 87% reduction of tumor volume in BxPC3 HAS3 model (p<0.001) but had weaker effect on BxPC3 HAS2 (33%, p<0.001) and BxPC3 tumors (36%, p<0.01). Accumulation of extracellular HA was associated with enriched tumor stroma, loss of membranous E-cadherin and accumulation of cytoplasmic β-catenin in pancreatic cancer cells, suggesting HA-induced epithelial-mesenchymal transition (EMT). Removal of HA by PEGPH20 reversed the remodeling of the tumor stroma and induced translocation of E-cadherin and β-catenin to the plasma membrane.Translocation of E-cadherin was also observed in the KPC pancreatic tumors after PEGPH20 treatment. In conclusion, accumulation of extracellular HA by HAS3 overexpression favors tumor growth and leads to a strong response to PEGPH20 in a pancreatic cancer xenograft model. Deposition of extracellular HA is associated with optimization of the tumor microenvironment and EMT. Depletion of HA by PEGPH20 reverses changes in the tumor stroma and induces translocation of epithelial markers to the plasma membrane. Citation Format: Anne Kultti, Chunmei Zhao, Susan Zimmerman, Ryan J. Osgood, Yanling Chen, Rebecca Symons, Ping Jiang, Curtis B. Thompson, David A. Tuveson, Gregory I. Frost, H Michael Shepard, Zhongdong Huang. Extracellular hyaluronan accumulation by hyaluronan synthase 3 promotes pancreatic cancer growth and modulates tumor microenvironment via epithelial-mesenchymal transition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4844. doi:10.1158/1538-7445.AM2014-4844

Simple SummaryColon cancer is one of the most common types of cancer worldwide. Immune checkpoint inhibitors have promising effects on various types of cancers with limited efficacy in colon cancer. Midostaurin (PKC412) is currently used for the treatment of patients with acute myeloid leukemia harboring FLT3-mutation. The aim of this study was to assess the potential effect of midostaurin on the modulation of TME and the efficacy of anti-PD-1 against colon cancer. We showed midostaurin inhibited colorectal adenocarcinoma cell growth and induced multinucleation and micronuclei formation. Midostaurin inhibited colorectal adenocarcinoma cell growth associated with the formation of dsDNA and ssDNA; the up-regulation of mRNA expression of cGAS, STING, IRF3, and IFNAR1; the down-regulation of Trex-1, c-Kit, and Flt3 protein expression. The tumor-implanted model displayed a combination of midostaurin-enhanced efficacy of anti-PD-1 to suppress tumor growth. In TME, midostaurin diminished Treg cells and increased M1 macrophage. The expressions of STING and INFβ proteins were elevated in the tumor specimens. Our results suggest that midostaurin may have the potential to enhance immunotherapy in clinical practice.Immunotherapy modulating the tumor microenvironment (TME) immune function has a promising effect on various types of cancers, but it remains as a limited efficacy in colon cancer. Midostaurin (PKC412) has been used in the clinical treatment of fms-like tyrosine kinase 3 (FLT3)-mutant acute myeloid leukemia and has demonstrated immunomodulatory activity. We aimed to evaluate the effect of midostaurin on the modulation of TME and the efficacy of anti-programmed cell death protein 1 (PD-1) against colon cancer. Midostaurin inhibited the growth of murine CT26 and human HCT116 and SW480 cells with multinucleation and micronuclei formation in morphology examination. The cell cycle arrested in the G2/M phase and the formation of the polyploid phase was noted. The formation of cytosolic DNA, including double-strand and single-strand DNA, was increased. Midostaurin increased mRNA expressions of cGAS, IRF3, and IFNAR1 in colorectal adenocarcinoma cells and mouse spleen macrophages. The protein expressions of Trex-1, c-KIT, and Flt3, but not PKCα/β/γ and VEGFR1, were down-regulated in midostaurin-treated colorectal adenocarcinoma cells and macrophages. Trex-1 protein expression was abrogated after FLT3L activation. In vivo, the combination of midostaurin and anti-PD-1 exhibited the greatest growth inhibition on a CT26-implanted tumor without major toxicity. TME analysis demonstrated that midostaurin alone decreased Treg cells and increased neutrophils and inflammatory monocytes. NKG2D+ and PD-1 were suppressed and M1 macrophage was increased after combination therapy. When combined with anti-PD-1, STING and INFβ protein expression was elevated in the tumor. The oral administration of midostaurin may have the potential to enhance anti-PD-1 efficacy, accompanied by the modulation of cytosolic DNA-sensing signaling and tumor microenvironment.

BackgroundRenal cell carcinoma (RCC) is characterized by significant vascularization and immunogenicity, which contributes to drug resistance and immune escape. CD248, a pericytes marker in tumor vasculature, might help explain tumor microenvironment (TME) remodeling and serve as a novel therapeutic target.MethodsTranscriptome data and clinical information of RCC patients were obtained from The Cancer Genome Atlas (TCGA) database. ESTIMATE and microenvironment cell population (MCP)-counter algorithms were adopted to calculate immune and stromal contents. The prognostic value of TME was evaluated via Kaplan-Meier and Wilcoxon signed rank test. Pearson’s correlation coefficient was employed to explore the correlation between angiogenesis and TME, and the relationship between CD248 and TME or RCC progression. CD248 overexpression and vascular colocalization in RCC were confirmed via histology staining. The weighted gene coexpression network analysis (WGCNA) and enrichment analysis were performed to explore CD248-mediated regulatory mechanism in angiogenesis and TME remodeling. CD248-based drug response was predicted through CellMiner database.ResultsTumor angiogenesis contributed to deteriorated RCC progression, which might be involved with immunosuppression. More specifically, upregulated immune checkpoints exhausted infiltrated T cells. CD248 overexpressed in RCC vessels correlated with TME and predicted a bad survival outcome. CD248 and coexpressed genes participated in angiogenesis and TME remodeling. Several clinical approved drugs that might inhibit CD248-mediated tumor promoting effects were selected.ConclusionsCD248 appears to contribute to angiogenesis and immunosuppressive TME, and may thus be a promising prognostic and therapeutic target for RCC. CD248-based medication guidance might benefit RCC patients.

The tumor microenvironment (TME) plays a pivotal role in tumorigenesis, metastasis, and resistance to therapy. Although immunotherapeutic approaches such as CAR-T cells, immune checkpoint inhibitors, and tumor vaccines have demonstrated promising therapeutic effectiveness, tumor heterogeneity and immunosuppression continue to limit their efficacy in solid tumors, therefore, development of effective low-cost TME targeted therapy is necessary. Recent studies indicate, selective serotonin reuptake inhibitors display immune modulatory effects. Fluoxetine is an US Food and Drug Administration-approved selective serotonin reuptake inhibitor widely used in treatments of depression in patients with cancer, its effects toward immune responses in TME and effectiveness in management of solid tumor has not been explored previously. In this study, we investigated the immunoregulatory and antitumor effects of fluoxetine on solid tumor using syngeneic orthotropic B16-F10 melanoma and 4T1 breast carcinoma models in mice. Fluoxetine oral application significantly decreased the primary tumor burden and development of metastatic nodules. Histopathology, flowcytometry and confocal imaging revealed that fluoxetine significantly activates host antitumor immune system through increased cytotoxic interferon gamma secreting T cells and M1-macrophages accumulation at both primary and metastatic sites that results in elevated activated caspase-8 in cancer cells leading to immune mediated tumor cell death. Fluoxetine treatment led to decrease in immunosuppressive cells populations such as myeloid derived suppressor cells, M2-macrophages, regulatory T cells, while also reducing the level of cancer stemness markers and proteins associated with epithelia to mesenchymal transition and metastasis. Collectively, our study indicates the multifaceted role of fluoxetine in modulating the solid TME and highlight its potential in designing of novel anticancer treatment regimen. SIGNIFICANCE STATEMENT: Fluoxetine, an US Food and Drug Administration-approved selective serotonin reuptake inhibitor reduces solid tumor (breast and melanoma) load through immune mediated tumor cell death and restore antitumor immunity. It remodels tumor microenvironment, activates T cells, reduces cancer stemness and metastasis via regulation of epithelial mesenchymal transition factors, hypoxia Inducible factor 1α, and β-catenin.

Pickering emulsion with tumor vascular destruction and microenvironment modulation for transarterial embolization therapy.

Focused ultrasound (FUS) is a rapidly advancing noninvasive energy delivery technology with the capacity to precisely modulate the tumor microenvironment (TME) through acoustic waves. Glioblastoma (GBM) is characterized by profound TME immune suppression and treatment resistance and has emerged as a key subject to treatment with FUS therapy. This review examines the technical evolution of FUS and its expanded applications in GBM, including subtypes of low- and high-intensity FUS and their mechanistic contributions to therapeutic effect. A comprehensive literature review was conducted using PubMed, Scopus, and Google Scholar to identify preclinical and clinical studies utilizing FUS in the context of GBM. Articles were included if they discussed FUS mechanisms (thermal, mechanical), bioeffects (immunomodulation, barrier permeability, cell death), or combinatory approaches (e.g., drug delivery, CAR T cells, sonodynamic therapy). A literature search yielded 312 studies; 95 met inclusion criteria (67 preclinical, 14 clinical trials, 14 reviews) with defined FUS parameters and biological endpoints. FUS enables spatiotemporal control of thermal and mechanical effects in GBM. Modulation of duty cycle, acoustic pressure, and exposure time allows FUS to operate across therapeutic regimes. Preclinical data support using FUS for targeted drug delivery, immune cell repolarization, and synergistic effects with immunotherapies. Clinical trials demonstrate the safety and feasibility of several FUS platforms. FUS offers a tunable multimodal platform with the potential to overcome core resistance mechanisms in GBM. Recurrent glioblastoma could be effectively treated by integrating FUS as an adjunct therapy alongside emerging immunotherapies and targeted drug delivery systems.

E7386, a novel orally active CBP/beta-catenin modulator, has an impact on cancer cells with aberrant activation of Wnt/beta-catenin signaling pathway driven by adenomatous polyposis coli (APC) mutation or beta-catenin mutation and shows significant antitumor activity in xenograft models. CBP/beta-catenin transcriptional activation has an important role in not only malignancy of cancer cells but also regulation of tumor microenvironment such as fibroblast, pericyte, endothelial cells and immune cells. In this study, we investigated E7386 effect on tumor microenvironment and if it leads to enhance antitumor activity of lenvatinib. Lenvatinib is a potent anti-angiogenic inhibitor targeting vascular endothelial growth factor receptors, fibroblast growth factor receptors and other proangiogenic and oncogenic kinases. Firstly, we tested the combination of E7386 with lenvatinib in Wnt-1 tumor isogenic models, where tumors isolated from MMTV-Wnt1 transgenic mice were inoculated in mice. While E7386 and lenvatinib individually suppressed tumor growth and caused tumor dormancy, the combination resulted in approximately 75% tumor reduction. To clarify the effect on tumor microenvironment, we tested an isograft model of 4T1 murine breast cancer cells which was resistant to E7386 in vitro proliferation. E7386 and lenvatinib individual treatments showed a significant antitumor effect, but the antitumor effect of the combination was significantly superior to that of each mono-treatment. Therefore, we compared effects of E7386 and lenvatinib on tumor microenvironment in immunohistochemical analysis using CD31 Ab as an endothelial marker and alpha-SMA Ab as a pericyte and cancerous fibroblast marker. E7386 significantly decreased alpha-SMA positive cells and microvessel density in tumors. In addition, remaining tumor vessels were not covered with pericytes. In lenvatinib treated tumors, greater reduction of microvessel density was observed than in E7386 treated tumors, but tumor vessels covered with pericytes, which are known to be resistant against VEGF inhibitors, were remained. In combination treatment, most of the tumor vessels disappeared. The enhancement of antitumor activity in the combination was also observed in SEKI human melanoma xenograft model which is relatively resistant to lenvatinib. These data suggest E7386 sensitizes tumor to lenvatinib through the modulation of the tumor microenvironment that is resistant to VEGF inhibitors. Taken together, E7386 shows not only antitumor activity via the effect on cancer cells but also through the modulation of tumor microenvironment, and the treatment of E7386 with lenvatinib is a novel combination therapy to overcome resistance to VEGF inhibitors. Citation Format: Yoichi Ozawa, Yusaku Hori, Kazuhiko Yamada, Yasuhiro Funahashi, Junji Matsui, Tomohiro Matsushima, Takashi Owa. E7386, an orally active CBP/beta-catenin modulator, effects tumor microenvironment, resulting to the enhancement of antitumor activity of lenvatinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5176. doi:10.1158/1538-7445.AM2017-5176

Dorsal root ganglion (DRG) neurons have a wide range of functions, including touch, pain and itch. These neurons have recently emerged as promising targets for non-invasive focused ultrasound (FUS) neuromodulation. However, our understanding of the molecular and physical mechanisms underlying FUS-evoked responses in DRG neurons remains limited. Here, we explore the neuromodulatory effects of FUS on cultured DRG neurons using calcium imaging to track neural responses. We find that a 20-MHz FUS burst of 1-ms duration at an acoustic pressure of 5 MPa elicited calcium responses in 52% of DRG neurons. Single-cell RNA sequencing reveals that more than half of FUS-sensitive neurons belong to two subsets: the TH-expressing C low-threshold mechanoreceptors (C-LTMRs) and the MRGPRD-expressing C high-threshold mechanoreceptors (C-HTMRs), both of which express the Gαi-interacting protein (GINIP). This finding was further confirmed by using a ginip mouse model. We demonstrate that FUS excites both GINIP + and GINIP- neurons through membrane deformation, likely mediated by mechanosensitive ion channels. Our findings identify specific FUS parameters that activate distinct subsets of DRG neurons, opening new possibilities for using FUS to modulate DRG neuron activity.

Dorsal root ganglion (DRG) neurons have a wide range of functions, including touch, pain and itch. These neurons have recently emerged as promising targets for non-invasive focused ultrasound (FUS) neuromodulation. However, our understanding of the molecular and physical mechanisms underlying FUS-evoked responses in DRG neurons remains limited. Here, we explore the neuromodulatory effects of FUS on cultured DRG neurons using calcium imaging to track neural responses. We find that a 20-MHz FUS burst of 1-ms duration at an acoustic pressure of 5 MPa elicited calcium responses in 52% of DRG neurons. Single-cell RNA sequencing reveals that more than half of FUS-sensitive neurons belong to two subsets: the TH-expressing C low-threshold mechanoreceptors (C-LTMRs) and the MRGPRD-expressing C high-threshold mechanoreceptors (C-HTMRs), both of which express the Gαi-interacting protein (GINIP). This finding was further confirmed by using a ginip mouse model. We demonstrate that FUS excites both GINIP+ and GINIP- neurons through membrane deformation, likely mediated by mechanosensitive ion channels. Our findings identify specific FUS parameters that activate distinct subsets of DRG neurons, opening new possibilities for using FUS to modulate DRG neuron activity.

Oncolytic viruses (OVs) have emerged as a transformative approach in cancer therapy, offering tumor-specific lysis while sparing normal tissues. In addition to their direct cytolytic effects, OVs actively reshape the tumor microenvironment (TME) by enhancing immune infiltration, disrupting immunosuppressive signals, and promoting tumor antigen presentation. However, the complexity of the TME poses challenges, often necessitating combination therapies to improve OV efficacy and overcome tumor resistance. This review explores the evolution of oncolytic virotherapy, from the early use of naturally occurring viruses to the development of genetically engineered OVs. Among the most significant advancements, T-VEC, an FDA-approved herpesvirus, has been modified to express GM-CSF, enhancing immune activation in metastatic melanoma. Similarly, JX-594, a vaccinia virus, has been engineered for selective replication in tumor cells, demonstrating the potential of OVs to combine direct oncolysis with immune modulation. Other HSV-based OVs, such as HF10 and HSV1716, further highlight the ability of OVs to enhance immune cell infiltration and increase antigen presentation within the TME. Recent advances in tumor microenvironment remodeling have expanded OV therapeutic strategies. By converting immunologically “cold” tumors into “hot” tumors, OVs can overcome immune evasion through mechanisms such as enhanced antigen release, immune checkpoint inhibition, and metabolic reprogramming. To maximize therapeutic potential, researchers are developing genetically engineered OVs carrying immune-stimulatory transgenes, exploring synergistic combination therapies with immune checkpoint inhibitors, and utilizing nanoparticle-based delivery systems for improved precision. Additionally, novel OVs—including measles virus, Newcastle virus, Zika virus, and SARS-CoV-2—are being investigated for their unique ability to disrupt the TME and enhance anti-tumor immunity. Looking ahead, OV therapy will depend on optimizing TME-targeted strategies, improving viral delivery mechanisms, and identifying predictive biomarkers to personalize patient responses. Advances in viral engineering and immunomodulation hold the potential to revolutionize cancer treatment, offering more precise and effective therapeutic options. This review provides a comprehensive analysis of current progress in oncolytic virotherapy, emphasizing its potential to remodel the TME and improve clinical outcomes.

The unique physiology of tumors limits the efficacy of chemotherapeutics. In efforts to improve the effectiveness of the existing chemotherapy drugs, nanomedicine emerged as a new hope but proved inadequate due to the transport barriers present within the tumor tissues, which limits the potential of nanomedicine. Dense collagen networks in fibrotic tissues contribute to hindering the penetration of molecular- or nano-scale medicine through tumor interstitium. In the present study, human serum albumin (HSA)-based nanoparticles (NPs) were developed for gemcitabine (GEM) and losartan (LST), which could offer secreted protein acids rich in cysteine (SPARC) and enhanced permeability and retention effect (EPR)-mediated drug accumulation in tumors. Also, the tumor microenvironment (TME) modulation approach using LST was coupled to investigate the impact on antitumor efficacy. GEM-HSA NPs and LST-HSA NPs were prepared by the desolvation-cross-linking method and characterized for size, potential, morphology, drug loading, drug-polymer interactions, and hemocompatibility. For investigating the efficacy of prepared NPs, cytotoxicity and mechanisms of cell death were elucidated in vitro by using various assays. Intracellular uptake studies of prepared HSA NPs indicated their uptake and cytoplasmic localization. Furthermore, in vivo studies demonstrated significantly improved anticancer efficacy of GEM-HSA NPs in combination with LST pretreatment. Extended LST treatment further improved the anticancer potential. It was shown that the improved efficacy of the nanomedicine was correlated with the reduced thrombospondin-1 (TSP-1) and collagen level in tumor tissue upon LST pretreatment. Moreover, this approach exhibited augmented nanomedicine accumulation in the tumor, and hematological, biochemical, and tissue histology indicated the safety profile of this combination regimen. Concisely, the undertaken study demonstrated the potential of the triple targeting (SPARC, EPR, TME modulation) approach for augmented efficacy of chemotherapeutics.

Purpose of Study: Diffuse midline glioma (DMG) is an aggressive brain tumor and the leading cause of pediatric death caused by cancer. Despite great strides in the understanding of this disease, survival is still dismal. One of the objectives of our lab is to modulate the tumor microenvironment (TME) towards a proinflammatory phenotype to render these tumors amenable to immunotherapy. TIM-3 is a member of the TIM family of immunoregulatory proteins expressed on multiple immune cell types, including T-cells, NK, myeloid populations, and microglia, regulating adaptive and innate immunity. Therefore, the aim of this project is to study the antitumor effect of the anti-TIM-3 monoclonal antibody and its effect on the DMG tumor microenvironment. Experimental Procedures: TIM-3 expression in DMG patients was analyzed using total mRNA sequencing data and single-cell RNAseq data. To perform all the experiments, DIPG murine cell lines were used (NP53 and XFM). For in vivo experiments, cells were injected into the pons of mice using a screw-guided system. The antibody was administered intracranially (25µg) with the same system and two times intraperitoneally (10mg/kg) 3, 7, and 11 days after the cell implantation respectively. Tumor immune populations, chemokines, and cytokines were analyzed by flow cytometry. Results: In silico assessment of TIM-3 expression in DMG mRNA and single-cell datasets showed a robust expression of this gene mainly in microglia and macrophages uncovering this molecule as a potential target in DMGs. In vivo studies showed that TIM-3 blockade with an antibody significantly increased the overall survival of two DMG immunocompetent orthotopic models, led to long-term survivors (50%), and showed immune memory. TIM-3 inhibition resulted in significant increase in the number and proliferative state of microglia, NK, and CD8+ cells and higher levels of IFNγ, GrzB and TNFα corresponding to NK and T-cell activate phenotypes. Interestingly, there was a decrease in the Treg population, which caused an increase in the pro-inflammatory CD8/Treg ratio. Chemokine studies demonstrated an augmentation of CCL5, CCL2 chemotactic chemokines, and CXCL10, IL-1β and IFN-γ pro-inflammatory cytokines in the tumor microenvironment of treated mice. Additionally, DCs, CD4+, and CD8+ cells were increased in treated draining lymph nodes and of functional significance, expressed higher amounts of pro-inflammatory cytokines than in control mice. Interestingly, the depletion NK, CD4 and CD8 immune populations did not completely abrogate the treatment efficacy. However, microglia and macrophages depletion with an anti-CSF1R resulted in a total loss of efficacy indicating a critical role of these populations in the effect of TIM-3 blockade. Conclusions: These data uncover TIM-3 as a potential target for the treatment of DMG and its role as an immune regulator of the DMG tumor microenvironment. Citation Format: Iker Ausejo-Mauleon, Sara Labiano, Virginia Laspidea, Daniel de la Nava, Oren Becher, Mariella G Filbin, Fernando Pastor, Marta M Alonso. Modulation of tumor microenvironment with TIM-3 blockade improves survival in diffuse midline glioma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6369.

Acquired therapeutic resistance in glioblastoma multiforme (GBM) constitutes a major determinant of its refractory and tumor recurrence. Both tumor-intrinsic epigenetic regulation and tumor microenvironment (TME) remodeling are now understood to play pivotal roles in this resistance; however, the synergistic mechanisms and key molecular mediators underlying this interplay remain poorly defined. In this study, we demonstrated that temozolomide (TMZ) could activate the autophagy-dependent secretory pathway to promote extracellular secretion of Alpha-enolase (ENO1). Extracellular soluble ENO1 robustly enhanced GBM cell proliferation, migration, and invasion in vitro. Clinically, serum ENO1 levels were markedly elevated in GBM patients and strongly correlated with TMZ therapeutic response, suggesting its potential as a diagnostic biomarker for predicting TMZ efficacy. Mechanistically, secreted ENO1 could bind to the Toll-like receptor 4 (TLR4) receptor on GBM cells, enhancing the PI3K/Akt pathway to promote cell invasion and proliferation. Meanwhile, ENO1/TLR4 axis activated the downstream ERK/SPHK1 signaling cascade, inducing phosphorylation and membrane translocation of SPHK1 at Ser225, thereby promoting the biosynthesis of sphingosine-1-phosphate (S1P), a critical sphingolipid metabolite. Notably, extracellular ENO1 and its downstream metabolite S1P synergistically polarized tumor-associated macrophages (TAMs) toward an M2-like phenotype, fostering an immunosuppressive tumor microenvironment (TME) and conferring chemoresistance. Importantly, in vivo studies confirmed that combined therapy with the SPHK1 inhibitor PF-543, the TLR4 antagonist TAK-242, and TMZ synergistically suppressed tumor growth and significantly enhanced the efficacy of TMZ. Collectively, these findings reveal that ENO1 mediates intercellular crosstalk between GBM cells and M2-TAMs via autophagy-dependent secretion, thereby driving TMZ chemoresistance and functioning as an oncogene in GBM. Targeting the ENO1/TLR4 signaling axis reshapes the immune microenvironment and enhances the efficacy of TMZ, offering a promising therapeutic strategy and potential combinatorial targets for precision therapy in GBM.