Prolonged Survival Of Mice Research Articles

Nanomaterial-Mediated Delivery of MLKL Plasmids Sensitizes Tumors to Immunotherapy and Reduces Metastases.

Cancer immunotherapy has emerged as a promising approach for the induction of an antitumor response. While immunotherapy response rates are very high in some cancers, the efficacy against solid tumors remains limited caused by the presence of an immunosuppressive tumor microenvironment. Induction of immunogenic cell death (ICD) in the tumor can be used to boost immunotherapy response in solid cancers by eliciting the release of immune-stimulatory components. However, the delivery of components inducing ICD to tumor sites remains a challenge. Here, a novel delivery method is described for antitumor therapy based on MLKL (Mixed Lineage Kinase Domain-Like), a key mediator of necroptosis and inducer of ICD. A novel highly branched poly (β-amino ester)s (HPAEs) system is designed to efficiently deliver MLKL plasmid DNA to the tumor with consequent enhancement of immune antigen presentation for T cell responses in vitro, and improved antitumor response and prolonged survival in tumor-bearing mice. Combination of the therapy with anti-PD-1 treatment revealed significant changes in the composition of the tumor microenvironment, including increased infiltration of CD8+ T cells and tumor-associated lymphocytes. Overall, the HPAEs delivery system can enhance MLKL-based cancer immunotherapy and promote antitumor immune responses, providing a potential treatment to boost cancer immunotherapies.

Antibody targeting of anaerobic bacteria warms cold tumors and improves the abscopal effect of radiotherapy

BackgroundThe combination of immune checkpoint inhibitors with radiotherapy can enhance the immunomodulation by RT and reduce the growth of distant unirradiated tumors (abscopal effect); however, the results are still not very satisfactory. Therefore, new treatment options are needed to enhance this effect. Our previous study showed that the combination of Bifidobacterium (Bi) and its specific monoclonal antibody (mAb) could target and alleviate hypoxia at the tumor site and act as a radiosensitizer. In this study, we explored the anti-tumor efficacy of quadruple therapy (Bi + mAb and RT + αPD-1). The current study also aimed to probe into the complex immune mechanisms underlying this phenomenon.MethodsConstructed 4T1 breast and CT26 colon cancer tumor models. A comprehensive picture of the impact of constructed quadruple therapy was provided by tumor volume measurements, survival analysis, PET/CT imaging, immune cell infiltration analysis and cytokine expression levels.ResultsThe abscopal effect was further amplified in the “cold” tumor model and prolonged survival in tumor-bearing mice. Bi can colonized in primary and secondary tumors and direct the mAb to reach the tumor site, activate complement, enhance the ADCC effect and initiate the innate immune response. Then combined with αPD-1 and radiotherapy to stimulate adaptive immune response and synergize with cytokines to expand the immune efficacy and generate effective anti-tumor immune response.ConclusionsBi was used as an artificially implanted anaerobic target to cause a transient “infection” at the tumor, causing the tumor to become locally inflamed and “hot”, and at the same time, mAb was used to target Bi to enhance the local immune effect of the tumor, and then combined with radiotherapy and αPD-1 to amplify the abscopal effect in multiple dimensions. Therefore, the present study provided a new idea for the multipotent immune-activating function of antibody-targeted anaerobic bacteria for the RT treatment of extensively metastasized cancer patients.

Stem Cell Therapy Using Bone Marrow-Derived Muse Cells Repairs Radiation-Induced Intestinal Injury Through Their Intestine-Homing via Sphingosine Monophosphate-Sphingosine Monophosphate Receptor 2 Interaction

PurposeThere is still no effective treatment for the gastrointestinal side effects of radiation therapy. Multilineage-differentiating stress enduring (Muse) cells are tissue stem cells that have the ability to spontaneously homing in on injured tissues and repairing them. Several clinical trials have shown that stem cell therapy using human bone marrow-derived Muse (hBM-Muse) cells is effective in treating various diseases, but it is not known whether they are effective in treating radiation-induced intestinal injury. In this study, we investigated whether hBM-Muse cells homing to the radiation-damaged intestine and promote its repair. Methods and MaterialshBM-Muse cells were injected into the tail vein of mice 2 hours after high-dose total body irradiation. Then, homing analysis, crypt assay, BrdU assay, TUNEL assay, immunostaining, and survival time measurements were performed. In addition, we analyzed the expression of sphingosine monophosphate (S1P), a Muse cell inducing factor, in the mouse small intestine after irradiation. Finally, we investigated whether administration of JTE-013, an S1P receptor 2 (S1PR2)-specific antagonist, inhibits hBM-Muse cells homing to the injured intestine. ResultsS1P expression increased in mouse intestine after irradiation, with hBM-Muse cells homing in on the injured intestine. Injection of hBM-Muse cells after radiation exposure significantly increased the number of crypts, proliferating cells in the crypts, and small intestinal component cells such as intestinal stem cells, inhibited radiation-induced apoptosis, and prolonged mouse survival. Treatment with JTE-013 significantly inhibited intestinal homing and therapeutic effects of hBM-Muse cells. These findings indicate that hBM-Muse cells homed in on the injured intestine through the S1P-S1PR2 interaction to exert therapeutic effects on the radiation-induced intestinal injury. ConclusionThis study indicates that hBM-Muse cells are effective in treating radiation-induced intestinal injury, suggesting that hBM-Muse cell-based stem cell therapy has the potential to overcome the gastrointestinal side effects that limit the indications for radiation therapy.

Elevated SLC3A2 associated with poor prognosis and enhanced malignancy in gliomas

The role of SLC3A2, a gene implicated in disulfidptosis, has not been characterized in gliomas. This study aims to clarify the prognostic value of SLC3A2 and its influence on glioma. We evaluated the expression of SLC3A2 and its prognostic importance in gliomas using publicly accessible databases and our clinical glioma samples and with reliance on Meta and Cox regression analysis approaches. Functional enrichment analyses were performed to explore SLC3A2's function. Immune infiltration was evaluated using CIBERSORT, ssGSEA, and single-cell sequencing data. Additionally, Tumor immune dysfunction and exclusion (TIDE) and epithelial-mesenchymal transition scores were determined. CCK8, colony formation, migration, and invasion assays were utilized in vitro, and an orthotopic glioma xenograft model was employed in vivo, to investigate the role of SLC3A2 in gliomas. Bioinformatics analyses indicated high SLC3A2 expression correlates with adverse clinicopathological features and poor patient prognosis. Upregulated SLC3A2 influenced the tumor microenvironment by altering immune cell infiltration, particularly of macrophages, and tumor migration and invasion. SLC3A2 expression positively correlated with immune therapy indicators, including immune checkpoints and TIDE. Elevated SLC3A2 was revealed as an independent risk element for poor glioma prognosis through Cox regression analyses. In vitro experiments showed that reduced SLC3A2 expression decreased cell proliferation, migration, and invasion. In vivo, knockdown of SLC3A2 led to a reduction in tumor volume and prolonged survival in tumor-bearing mice. Therefore, SLC3A2 is a prognostic biomarker and associated with immune infiltration in gliomas.

Phage display-derived alpaca nanobodies as potential therapeutics for Naja atra snake envenomation.

Naja atra, the Chinese cobra, is a major cause of snake envenomation in Asia, causing hundreds of thousands of clinical incidents annually. The current treatment, horse serum-derived antivenom, has unpredictable side effects and presents manufacturing challenges. This study focused on developing new-generation snake venom antidotes by using microbial phage display technology to derive nanobodies from an alpaca immunized with attenuated N. atra venom. Following confirmation of the immune response in the alpaca, we amplified VHH genes from isolated peripheral blood mononuclear cells and constructed a phage display VHH library of 1.0 × 107 transformants. After four rounds of biopanning, the enriched phages exhibited increased binding activity to N. atra venom. Four nanobody clones with high binding affinities were selected: aNAH1, aNAH6, aNAH7, and aNAH9. Specificity testing against venom from various snake species, including two Southeast Asian cobra species, revealed nanobodies specific to the genus Naja. An in vivo mouse venom neutralization assay demonstrated that all nanobodies prolonged mouse survival and aNAH6 protected 66.6% of the mice from the lethal dosage. These findings highlight the potential of phage display-derived nanobodies as valuable antidotes for N. atra venom, laying the groundwork for future applications in snakebite treatment.IMPORTANCEChinese cobra venom bites present a formidable medical challenge, and current serum treatments face unresolved issues. Our research applied microbial phage display technology to obtain a new, effective, and cost-efficient treatment approach. Despite interest among scientists in utilizing this technology to screen alpaca antibodies against toxins, the available literature is limited. This study makes a significant contribution by introducing neutralizing antibodies that are specifically tailored to Chinese cobra venom. We provide a comprehensive and unbiased account of the antibody construction process, accompanied by thorough testing of various nanobodies and an assessment of cross-reactivity with diverse snake venoms. These nanobodies represent a promising avenue for targeted antivenom development that bridges microbiology and biotechnology to address critical health needs.

DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation.

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

Systemic delivery of oncolytic herpes virus using CAR-T cells enhances targeting of antitumor immuno-virotherapy

Recent studies have indicated that combining oncolytic viruses with CAR-T cells in therapy has shown superior anti-tumor effects, representing a promising approach. Nonetheless, the localized delivery method of intratumoral injection poses challenges for treating metastatic tumors or distal tumors that are difficult to reach. To address this obstacle, we employed HSV-1-infected CAR-T cells, which systemically delivery HSV into solid tumors. The biological function of CAR-T cells remained intact after loading them with HSV for a period of three days. In both immunocompromised and immunocompetent GBM orthotopic mouse models, B7-H3 CAR-T cells effectively delivered HSV to tumor lesions, resulting in enhanced T-cell infiltration and significantly prolonged survival in mice. We also employed a bilateral subcutaneous tumor model and observed that the group receiving intratumoral virus injection exhibited a significant reduction in tumor volume on the injected side, while the group receiving intravenous infusion of CAR-T cells carrying HSV displayed suppressed tumor growth on both sides. Hence, CAR-THSV cells offer notable advantages in the systemic delivery of HSV to distant tumors. In conclusion, our findings emphasize the potential of CAR-T cells as carriers for HSV, presenting significant advantages for oncolytic virotherapy targeting distant tumors.

Ultrasound -Induced Thermal Effect Enhances the Efficacy of Chemotherapy and Immunotherapy in Tumor Treatment.

The inadequate perfusion, frequently resulting from abnormal vascular configuration, gives rise to tumor hypoxia. The presence of this condition hinders the effective delivery of therapeutic drugs and the infiltration of immune cells into the tumor, thereby compromising the efficacy of treatments against tumors. The objective of this study is to exploit the thermal effect of ultrasound (US) in order to induce localized temperature elevation within the tumor, thereby facilitating vasodilation, augmenting drug delivery, and enhancing immune cell infiltration. The selection of US parameters was based on intratumor temperature elevation and their impact on cell viability. Vasodilation and hypoxia improvement were investigated using enzyme-linked immunosorbent assay (ELISA) and immunofluorescence examination. The distribution and accumulation of commercial pegylated liposomal doxorubicin (PLD) and PD-L1 antibody (anti-PD-L1) in the tumor were analyzed through frozen section analysis, ELISA, and in vivo fluorescence imaging. The evaluation of tumor immune microenvironment was conducted using flow cytometry (FCM). The efficacy of US-enhanced chemotherapy in combination with immunotherapy was investigated by monitoring tumor growth and survival rate after various treatments. The US irradiation condition of 0.8 W/cm2 for 10 min effectively elevated the tumor temperature to approximately 40 °C without causing any cellular or tissue damage, and sufficiently induced vasodilation, thereby enhancing the distribution and delivery of PLD and anti-PD-L1 in US-treated tumors. Moreover, it effectively mitigated tumor hypoxia while significantly increasing M1-phenotype tumor-associated macrophages (TAMs) and CD8+ T cells, as well as decreasing M2-phenotype TAMs. By incorporating US irradiation, the therapeutic efficacy of PLD and anti-PD-L1 was substantially boosted, leading to effective suppression of tumor growth and prolonged survival in mice. The application of US (0.8 W/cm2 for 10 min) can effectively induce vasodilation and enhance the delivery of PLD and anti-PD-L1 into tumors, thereby reshaping the immunosuppressive tumor microenvironment and optimizing therapeutic outcomes.

Intermittent fasting boosts antitumor immunity by restricting CD11b+Ly6ClowLy6Glow cell viability through glucose metabolism in murine breast tumor model

Intermittent fasting can benefit breast cancer patients undergoing chemotherapy or immunotherapy. However, it is still uncertain how to select immunotherapy drugs to combine with intermittent fasting. Herein we observed that two cycles of fasting treatment significantly inhibited breast tumor growth and lung tissue metastasis, as well as prolonged overall survival in mice bearing 4T1 and 4T07 breast cancer. During this process, both the immunosuppressive monocytic- (M-) and granulocytic- (G-) myeloid-derived suppressor cell (MDSC) decreased, accompanied by an increase in interleukin (IL) 7R+ and granzyme B+ T cells in the tumor microenvironment. Interestingly, we observed that Ly6Glow G-MDSC sharply decreased after fasting treatment, and the cell surface markers and protein mass spectrometry data showed potential therapeutic targets. Mechanistic investigation revealed that glucose metabolism restriction suppressed the splenic granulocyte-monocyte progenitor and the generation of colony-stimulating factors and IL-6, which both contributed to the accumulation of G-MDSC. On the other hand, glucose metabolism restriction can directly induce the apoptosis of Ly6Glow G-MDSC, but not Ly6Ghigh subsets. In summary, these results suggest that glucose metabolism restriction induced by fasting treatment attenuates the immune-suppressive milieu and enhances the activation of CD3+ T cells, providing potential solutions for enhancing immune-based cancer interventions.

ATRT-07. PRC1/BMI1 ACTIVITY ALTERS CHROMATIN ACCESSIBILITY TO REGULATE DIFFERENTIATION IN ATYPICAL TERATOID RHABDOID TUMORS

Abstract BACKGROUND SMARCB1 is a member of the SWI/SNF chromatin remodeling complex that is responsible for determining cellular pluripotency and lineage commitment. Atypical Teratoid Rhabdoid Tumor (ATRT) is a highly aggressive pediatric brain tumor and characterized by SMARCB1 deletion. METHODS Genome wide RNAi screen, RNA-seq and ChIP-Seq analysis, colony formation assay, cell cycle, western blotting, immunoprecipitation, mass spectrometry analysis, mouse xenograft model, IVIS and MRI imaging. RESULTS We performed an RNAi-based functional genomic screen to identify key epigenetic regulators that co-operate with SMARCB1 loss and contribute to tumorigenesis in ATRT. 406 genes associated with epigenetic regulation in patient-derived ATRT cell lines were targeted. This screen identified BMI1, a component of the Polycomb Repressive Complex 1 (PRC1), as one of the top targets essential for ATRT cell growth. Using RNA-seq and ChIP-Seq analysis we demonstrated that BMI1 co-operates with SMARCB1 deletion to suppress transcription of pro-differentiation pathways and promote self-renewal of tumor stem cells. In SMARCB1 deficient cells BMI1 forms a partial PRC1 complex devoid of DNA binding components. We found that genetic inactivation of BMI1 suppresses ATRT cell clonogenicity and neurosphere formation, leads to S-phase arrest and induces ATRT cell differentiation in vitro. In vivo BMI1 inhibition induces apoptosis, leading to significantly increasing survival of treated mice. By Doxycycline-inducible SMARCB1 re-expression system we revealed activation of proapoptotic proteins p16 and p21. With immunoprecipitation for BMI1, followed by mass spectrometry analysis we demonstrated that re-expression of SMARCB1 activates two PRC1 chromatin localizing components CBX4 and CBX8. Moreover, SMARCB1 re-expression significantly prolongs mice survival and decreases tumor size as evaluated by IVIS imaging system and T2-MRI. CONCLUSION Thus, we demonstrated that SMARCB1 deletion results in reprograming of BMI1 chromatin occupancy away from lineage specification by altering the components of the PRC1 complex and that PRC1/BMI1 inhibition is a novel therapeutic approach in ATRT.

MDB-01. A POSITIVE FEEDBACK LOOP BETWEENLNC-HLX-2-7, HLX, AND MYC STRONGLY PROMOTES GROUP 3 MEDULLOBLASTOMA

Abstract Recent studies suggest that long non-coding RNAs (lncRNAs) contribute to medulloblastoma formation and progression. We have identified a lncRNA, lnc-HLX-2-7, as a potential therapeutic target in group 3 (G3) medulloblastomas. lnc-HLX-2-7 RNA specifically accumulates in the promoter region of HLX, its host gene, which activates HLX expression by recruiting multiple factors, including enhancer elements. RNA sequencing and chromatin immunoprecipitation reveal that HLX binds to and activates the promoters of several oncogenes, including TBX2, LIN9, HOXM1, and MYC. Intravenous treatment with cerium oxide nanoparticle–coated antisense oligonucleotides targeting lnc-HLX-2-7 (CNP-lnc-HLX-2-7) inhibits tumor growth by 40-50% in an intracranial medulloblastoma xenograft mouse model (p<0.01). Combining CNP-lnc-HLX-2-7 with standard-of-care cisplatin further inhibits tumor growth and significantly prolongs mouse survival compared with CNP-lnc-HLX-2-7 monotherapy (p<0.01). Thus, the lnc-HLX-2-7–HLX–MYC axis is important for regulating G3 medulloblastoma progression, providing a strong rationale for using lnc-HLX-2-7 as a therapeutic target for G3 MBs in children.

Biomimetic Hydrogel-Mediated Mechano-Immunometabolic Therapy for Inhibition of ccRCC Recurrence After Surgery.

The unique physical tumor microenvironment (TME) and aberrant immune metabolic status are two obstacles that must be overcome in cancer immunotherapy to improve clinical outcomes. Here, an in situ mechano-immunometabolic therapy involving the injection of a biomimetic hydrogel is presented with sequential release of the anti-fibrotic agent pirfenidone, which softens the stiff extracellular matrix, and small interfering RNA IDO1, which disrupts kynurenine-mediated immunosuppressive metabolic pathways, together with the multi-kinase inhibitor sorafenib, which induces immunogenic cell death. This combination synergistically augmented tumor immunogenicity and induced anti-tumor immunity. In mouse models of clear cell renal cell carcinoma, a single-dose peritumoral injection of a biomimetic hydrogel facilitated the perioperative TME toward a more immunostimulatory landscape, which prevented tumor relapse post-surgery and prolonged mouse survival. Additionally, the systemic anti-tumor surveillance effect induced by local treatment decreased lung metastasis by inhibiting epithelial-mesenchymal transition conversion. The versatile localized mechano-immunometabolic therapy can serve as a universal strategy for conferring efficient tumoricidal immunity in "cold" tumor postoperative interventions.

CXCR4-Mediated Codelivery of FLT3 and BCL-2 Inhibitors for Enhanced Targeted Combination Therapy of FLT3-ITD Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is often associated with poor prognosis and survival. Small molecule inhibitors, though widening the treatment landscape, have limited monotherapy efficacy. The combination therapy, however, shows suboptimal clinical outcomes due to low bioavailability, overlapping systemic toxicity and drug resistance. Here, we report that CXCR4-mediated codelivery of the BCL-2 inhibitor venetoclax (VEN) and the FLT3 inhibitor sorafenib (SOR) via T22 peptide-tagged disulfide cross-linked polymeric micelles (TM) achieves synergistic treatment of FLT3-ITD AML. TM-VS with a VEN/SOR weight ratio of 1/4 and T22 peptide density of 20% exhibited an extraordinary inhibitory effect on CXCR4-overexpressing MV4-11 AML cells. TM-VS at a VEN/SOR dosage of 2.5/10 mg/kg remarkably reduced leukemia burden, prolonged mouse survival, and impeded bone loss in orthotopic MV4-11-bearing mice, outperforming the nontargeted M-VS and oral administration of free VEN/SOR. CXCR4-mediated codelivery of BCL-2 and FLT3 inhibitors has emerged as a prospective clinical treatment for FLT3-ITD AML.

Metabolic determinants of leukemia onset variability in genetically homogeneous AKR mice

Leukemia is a complex disease shaped by the intricate interplay of genetic and environmental factors. Given our preliminary data showing different leukemia incidence in genetically homogenous AKR mice harboring the spontaneous leukemia-inducing mutation Rmcfs, we sought to unravel the role of metabolites and gut microbiota in the leukemia penetrance. Our metabolomic analysis revealed distinct serum metabolite profiles between mice that developed leukemia and those that did not. We discovered that linoleic acid (LA), an essential ω-6 polyunsaturated fatty acid, was significantly decreased in the leukemia group, with the lower levels observed starting from 25 weeks before the onset. A predictive model based on LA levels demonstrated high accuracy in predicting leukemia development (area under curve 0.82). In vitro experiment confirmed LA's cytotoxic effects against leukemia cells, and in vivo study showed that a diet enriched with LA prolonged survival in AKR mice. Furthermore, gut microbiome analysis identified specific Lachnospiraceae species, that affect host lipid metabolism, are exclusively present in the leukemia group, suggesting their potential influence on LA metabolism and leukemia development. These findings shed light on the complex relationship between metabolites, gut microbiota, and leukemia development, providing valuable insights into the role of non-genetic factors in leukemia penetrance and potential strategies for leukemia prevention.

Comparison of seven CD19 CAR designs in engineering NK cells for enhancing anti-tumour activity.

Chimeric antigen receptor-natural killer (CAR-NK) cell therapy is emerging as a promising cancer treatment, with notable safety and source diversity benefits over CAR-T cells. This study focused on optimizing CAR constructs for NK cells to maximize their therapeutic potential. We designed seven CD19 CAR constructs and expressed them in NK cells using a retroviral system, assessing their tumour-killing efficacy and persistence. Results showed all constructs enhanced tumour-killing and prolonged survival in tumour-bearing mice. In particular, CAR1 (CD8 TMD-CD3ζ SD)-NK cells showed superior efficacy in treating tumour-bearing animals and exhibited enhanced persistence when combined with OX40 co-stimulatory domain. Of note, CAR1-NK cells were most effective at lower effector-to-target ratios, while CAR4 (CD8 TMD-OX40 CD- FcεRIγ SD) compromised NK cell expansion ability. Superior survival rates were noted in mice treated with CAR1-, CAR2 (CD8 TMD- FcεRIγ SD)-, CAR3 (CD8 TMD-OX40 CD- CD3ζ SD)- and CAR4-NK cells over those treated with CAR5 (CD28 TMD- FcεRIγ SD)-, CAR6 (CD8 TMD-4-1BB CD-CD3ζ 1-ITAM SD)- and CAR7 (CD8 TMD-OX40 CD-CD3ζ 1-ITAM SD)-NK cells, with CAR5-NK cells showing the weakest anti-tumour activity. Increased expression of exhaustion markers, especially in CAR7-NK cells, suggests that combining CAR-NK cells with immune checkpoint inhibitors might improve anti-tumour outcomes. These findings provide crucial insights for developing CAR-NK cell products for clinical applications.

Magnesium microspheres to enhance lipiodol-mediated transarterial chemo-embolization (TACE) of hepatocellular carcinoma: From bench to a pilot clinical study.

e16204 Background: Transarterial chemoembolization (TACE) has been extensively used in clinic to treat unresectable hepatocellular carcinoma (HCC). However, arterial embolization would worsen the hostile physicochemical features in the tumor microenvironment (TME) by further reducing its pH and increasing hypoxia, leading to immunosuppressive TME and drug resistance. Methods: Magnesium microspheres (Mg MSs), acting as H2 donors and pH modulators, were prepared via gas atomization pulverization. Characterization included inverted optical microscopy, SEM, and XRD analysis. Their H2 generation and acid neutralization capabilities were confirmed. The Lip-Mg dispersion (Mg MSs dispersed in lipiodol) was tested for therapeutic efficacy in H22 murine liver tumor-bearing mice, 4T1, and CT26 subcutaneous tumor models, as well as in vivo interventional TAE therapy for rabbit orthotopic liver tumors. The modulated capacities of Lip-Mg in H2 gas generation, acid neutralization, and promotion of antitumor immune response were investigated in vivo. Finally, a single-center, single-arm, open-label trial (ChiCTR2200064999) evaluated the safety and efficacy of TACE treatment with Lip-Mg (M-TACE) in Chinese HCC patients. Results: Inverted optical microscopy and SEM images confirmed uniform, smooth Mg microspheres (~60 μm) with a grayish-white color. In acidic buffer (pH 6.4), Mg microspheres rapidly increased pH, as measured by pH meter or fluorescent probe. Lip-Mg treatment significantly inhibited tumor growth compared to controls, prolonging mouse survival by ~2-fold, validated in 4T1 and CT26 models. Ultrasonic and multispectral fluorescence imaging demonstrated Lip-Mg's ability to release H2 and neutralize pH in vivo, reversing the immunosuppressive tumor microenvironment. Clinical study showed manageable treatment-related adverse events, with no unexpected toxicities and negligible impact on serum magnesium levels, confirming the safety of Mg microspheres for M-TACE. According to mRECIST, M-TACE exhibited high CR (61.5%), PR (38.5%), and ORR (100%) rates, with an overall response rate and CR rate of 90% and 60%, respectively. Conclusions: Metallic Mg microspheres (~60 μm) were developed to enhance lipiodol-mediated TAE/TACE for HCC. When combined with lipiodol and injected, they neutralized the acidic TME, triggered hydrogen therapy, and reversed immunosuppression, effectively inhibiting tumor growth. Lip-Mg showed superior outcomes to lipiodol alone for TAE therapy, with M-TACE treatment exhibiting higher ORR and CR rates than cTACE. Post-M-TACE, adverse events were managed well without treatment discontinuations. Our study introduces Mg microspheres as a promising embolic device for M-TACE therapy, potentially improving TACE benefits. Clinical trial information: ChiCTR2100050327.

NXL-004: A novel trans-differentiation therapy for glioblastoma treatment.

e14020 Background: Gliomas, tumors originating from glial cells in the brain and spinal cord, constitute 80% of all malignant primary tumors affecting the central nervous system, with glioblastomas (GBM) as the most prevalent and aggressive subtype accounting for approximately 60% of cases. Current treatment options for GBM, including surgery, radiation therapy, and chemotherapy, have limited effectiveness in improving patient outcomes. Therefore, there is a critical need for more effective therapeutic approaches to treat GBM. Methods: We present NXL-004, an adeno-associated virus (AAV) vector delivering the neural transcription factor NeuroD1 directly into GBM tumor cells, as a novel therapy for GBM. Various GBM models were utilized to investigate NXL-004 treatment for GBM, including U87 intracranial and subcutaneous models in mice, PDX model and GBM organoid. Results: The results demonstrated that NXL-004 significantly reduced tumor size compared to the control group; (D) NXL-004 exhibited a significant effect in prolonging the survival of the GBM mouse model. The median survival of mice treated with NXL-004 was 34 days post tumor implantation, whereas the control group had a median survival of only 16 days. Conclusions: Preclinical studies of NXL-004 have demonstrated that this intervention effectively reprograms tumor cells, downregulating glial cell- and tumor-related gene expression, while concurrently upregulating neuron-related gene expression. The induced differentiation towards non-dividing neurons results in significant inhibition of tumor cell proliferation, substantial reductions in tumor volume and prolonged survival in GBM-bearing mice. Additionally, NXL-004 has exhibited a favorable safety profile in toxicity studies in rats and non-human primates. These preclinical outcomes paved the way for subsequent clinical evaluation, representing a potential breakthrough in GBM treatment.

LC3-associated phagocytosis of neutrophils triggers tumor ferroptotic cell death in glioblastoma

Necrosis in solid tumors is commonly associated with poor prognostic but how these lesions expand remains unclear. Studies have found that neutrophils associate with and contribute to necrosis development in glioblastoma by inducing tumor cell ferroptosis through transferring myeloperoxidase-containing granules. However, the mechanism of neutrophilic granule transfer remains elusive. We performed an unbiased small molecule screen and found that statins inhibit neutrophil-induced tumor cell death by blocking the neutrophilic granule transfer. Further, we identified a novel process wherein neutrophils are engulfed by tumor cells before releasing myeloperoxidase-containing contents into tumor cells. This neutrophil engulfment is initiated by integrin-mediated adhesion, and further mediated by LC3-associated phagocytosis (LAP), which can be blocked by inhibiting the Vps34-UVRAG-RUBCN-containing PI3K complex. Myeloperoxidase inhibition or Vps34 depletion resulted in reduced necrosis formation and prolonged mouse survival in an orthotopic glioblastoma mouse model. Thus, our study unveils a critical role for LAP-mediated neutrophil internalization in facilitating the transfer of neutrophilic granules, which in turn triggers tumor cell death and necrosis expansion. Targeting this process holds promise for improving glioblastoma prognosis.

Combined cryoablation and PD-1 inhibitor synergistically enhance antitumor immune responses in Lewis lung adenocarcinoma mice via the PI3K/AKT/mTOR pathway

Cryoablation is a therapeutic modality for lung adenocarcinoma that destroys target tumors using lethal levels of cold, resulting in the release of large amounts of specific antigens that activate immune responses. However, tumor immune checkpoint escape mechanisms prevent these released self-antigens from inducing effective anti-tumor immune responses. To overcome this challenge, we propose the use of immune checkpoint inhibitors to relieve T cell inhibition by immune checkpoints and enhance the anti-tumor immune response mediated by cryoablation. We used bilateral tumor-bearing mouse models and a specific cryoablation instrument to study the efficacy of cryoablation combined with PD-1 inhibitors in Lewis lung adenocarcinoma model mice. We found that cryoablation combined with PD-1 inhibitors significantly inhibited the growth of mouse lung adenocarcinoma, prolonged mouse survival, and enhanced the anti-tumor immune response. Moreover, this combined regimen could synergistically promote the activation and proliferation of T cells via the PI3K/AKT/mTOR pathway. The present study provides a strong theoretical basis for the clinical combination of cryoablation and PD-1 inhibitors.