Increased Tumor Cell Research Articles

High α-lipoic acid-loaded hollow mesoporous prussian blue nanozymes for targeted therapy of nasopharyngeal carcinoma in mice.

This study successfully constructs a tumor-targeting α-lipoic acid-loaded hollow mesoporous prussian blue nanozyme (AHPRzyme) for targeted therapy of nasopharyngeal carcinoma in mice. In these nanozymes, Arg-Gly-Asp (RGD) acts as a targeting ligand, enabling effective targeting of tumor cells. Additionally, AHPRzyme exhibits multiple anti-tumor mechanisms: ① The prussian blue nanozymes in AHPRzyme have catalase (CAT) activity, which decomposes H2O2 in human nasopharyngeal carcinoma CEN2 cells into non-toxic H2O, reducing H2O2 levels and minimizing damage to normal cells. The released O2 helps alleviate the hypoxic environment of the tumor, inhibiting lactate production due to hypoxia and consequently suppressing tumor growth. ② The prussian blue nanozymes also have peroxidase (POD) activity, which catalyzes H2O2 in tumor cells to generate ·OH, a reactive oxygen species, leading to tumor cell apoptosis. ③ The α-lipoic acid structure in AHPRzyme contains disulfide bonds that react with GSH, depleting excess glutathione (GSH) in tumor cells, disrupting the oxidative stress balance within the cells, and making them more sensitive to reactive oxygen species, thereby increasing tumor cell apoptosis. In summary, AHPRzyme can inhibit tumor cell growth and promote tumor cell apoptosis by improving the tumor microenvironment, achieving the goal of anti-nasopharyngeal carcinoma therapy.

Nanobody-Mediated Cellular Uptake Maximizes the Potency of Polylysine Dendrimers While Preserving Solid Tumor Penetration.

Dendrimers are branched macromolecular structures that are useful nanocarriers for small-molecule drugs, such as cancer therapeutics. Their small size permits penetration into solid tumors, coupled with functionalization with a low-fouling PEG coating that minimizes transient cellular interactions and enhances plasma circulation time. While PEGylated dendrimers show significant promise as anticancer therapeutics, there is potential to increase tumor cell specificity and drive uptake of drugs into cells by conjugating cell-targeting ligands onto the dendrimers. To achieve this, we used an expanded genetic code and bio-orthogonal click chemistry to functionalize monomethyl auristatin E (MMAE)-loaded PEGylated dendrimers with a single tumor cell-targeting nanobody per dendrimer. The uniform addition of a single nanobody ligand facilitated greater intracellular uptake of the drug payload into HER2-positive target cells, while preserving the desirable circulatory characteristics of dendrimers. While the nanobody-dendrimer conjugates show similar levels of tumor infiltration over 24 h compared to unmodified dendrimers, the targeted dendrimers had significantly greater inhibition of tumor growth and long-term retention in the tumors. Our results highlight that biodistribution studies alone are poor predictors of therapeutic performance. The controlled conjugation strategy presented here preserves the size advantage and tissue penetration of dendrimers while maximizing targeted cellular uptake and potency in difficult-to-access solid tumor tissue.

Patient-Derived Cancer-Associated Fibroblasts Support the Colonization of Tumor Cells in Head and Neck Squamous Cell Carcinoma

Background: The crosstalk between cancer-associated fibroblasts (CAFs) and tumor cells promotes proliferation, tumor relapse, and the acquisition of a partial epithelial-to-mesenchymal (pEMT) phenotype in tumor cells. The aim of this study was to investigate the effects of patient-derived CAFs on tumor cell growth and radioresistance in head and neck squamous cell carcinoma (HNSCC). Methods: CAFs were isolated and cultured in a three-dimensional spheroid formation. SCC-25 tumor cells educated by the CAFs (SCC25-E cells) were subjected to irradiation, and the response of the CAF-stimulated tumor cells to radiotherapy was determined using an MTT assay, a clonogenic assay, and Western blotting. Tumor cell morphological changes and growth dynamics were assessed using 3D holotomographic microscopy and a live video microscope. Results: Patient-derived CAFs significantly increased the growth rate of SCC-25 cells. CAFs drove fibrosis in the tumor microenvironment (TME), functioned as a physical barrier, temporarily stopped tumor growth, and induced the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Viability after irradiation at 4–8 Gy was significantly higher in SCC25-E cells than in the controls (p = 8 × 10–4 or lower). Furthermore, irradiation triggered the pEMT profile in HNSCC cells. Conclusions: CAFs’ education of tumor cells and the induced p38 phosphorylation had no influence on irradiation sensitivity. SCC25-E cultures demonstrated increased tumor cell growth, viability, and stress-induced phospho-p38 activation.

PEGylated Granulocyte Colony-Stimulating Factor and Plerixafor Enhance Autologous Stem and Progenitor Cell Mobilization and Transplantation in Pediatric Patients.

Autologous hematopoietic stem cell transplantation is used to restore bone marrow function after high-dose chemotherapy. For apheresis, granulocyte colony-stimulating factor (G-CSF) is standard of care, but obtaining sufficient stem cells can be challenging. Other mobilization agents include plerixafor and PEGylated G-CSF (PEG-G-CSF). While efficacy of these is established in adults, limited data for their use in pediatric patients are available. Here, we compare Good versus Poor Mobilizers and study success of different mobilization regimens in regard to CD34+cell-collection, -quality, -phenotype and hematologic reconstitution in pediatric patients. In this multi-center retrospective study, we analyzed data of 278 patients with solid tumors and lymphoma, mobilized with either G-CSF (n = 224), PEG-G-CSF (n = 34), or G-CSF/PEG-G-CSF with additional plerixafor (n = 20). In Poor Mobilizers (13.7% of all patients), addition of plerixafor to G-CSF augmented CD34+cell collection, without adverse effects on hematologic reconstitution and CD34+cell quality. PEG-G-CSF-aided mobilization was successful as first-line treatment in two-thirds of patients and did not impair hematological reconstitution, compared to G-CSF-only. Within the Poor Mobilizer group, G-CSF+plerixafor increased primitive (CD45RA-CD38-CD90+CD49f+) and CXCR4-expressing CD34+cells in apheresis products compared to G-CSF-only, without exceeding levels of Good Mobilizers. No plerixafor-related increase in tumor cells was observed in apheresis products. In conclusion, our comprehensive study supports the use of plerixafor and furthermore demonstrates the potential of patient-friendly PEG-G-CSF for mobilization of pediatric patients.

Liposomes-mediated enhanced antitumor effect of docetaxel with BRD4-PROTAC as synergist for breast cancer chemotherapy/immunotherapy.

It has been reported that proteolysis-targeting chimeras (PROTACs) can effectively degrade intracellular oncogenic proteins, providing an ideal strategy for cancer treatment. ARV825, a bromodomain-containing protein 4 (BRD4)-PROTAC, has demonstrated the capacity to enhance the antitumor effect of the classic chemotherapeutic agent docetaxel (DTX). However, there are three major challenges to the broader in vivo application of ARV825: poor solubility, poor permeability, and off-target effects. Additionally, the efficient co-delivery of ARV825 and DTX to tumor tissues for a synergistic therapeutic effect remains unresolved. In this study, liposomes were utilized as co-delivery vehicles for ARV825 and DTX to effectively address these issues. The well-established liposomes significantly improved the solubility of both ARV825 and DTX while maintaining a sustained release profile in blood-mimetic conditions. The co-loaded liposomes accumulated in tumor tissues via the enhanced permeability and retention (EPR) effect. After internalization, ARV825 effectively degraded intracellular BRD4 proteins and downregulated the expression of both Bcl-2 and PD-L1 proteins, thereby increasing tumor cell apoptosis and enhancing the tumor immune response. This, in turn, augmented the antitumor effect of DTX in vivo without undesired side effects. In conclusion, BRD4-PROTAC may serve as a promising synergistic agent alongside the conventional chemotherapeutic agent DTX, with liposomes functioning as effective co-delivery vehicles.

Automated Cancer Detection Using CNNs: Innovations and Applications

Large-scale datasets and complicated cell architectures provide challenges for traditional tumor identification approaches, which often rely on human analysis or traditional machine-learning techniques. This may result in errors and inefficiencies. The inconsistent shape of tumor cells may be a challenge for these methods, making border detection and classification inconsistent. This paper suggests an automated tumor detection technique based on convolutional neural networks (CNNs) to increase tumor cell border detection and classification accuracy and efficiency. Several experiments were carried out to confirm the suggested strategy's efficacy. This entails developing and refining the CNN architecture in addition to merging deep learning and data augmentation methods. According to the findings, the model has performed well in a variety of experimental settings, especially when it comes to classifying complicated cell samples. Furthermore, the feasibility of using this model for real-time border detection was explored, indicating its suitability for use in a medical setting. Subsequent investigations will concentrate on enhancing the capabilities of models, expanding their range, and tackling problems like disparities in data. This discovery is significant because it has the potential to transform tumor identification by providing a more accurate, scalable, and efficient approach that might be used extensively in clinical settings and eventually lead to better patient outcomes.

Exploring tumor microenvironment interactions and apoptosis pathways in NSCLC through spatial transcriptomics and machine learning.

The most common type of lung cancer is non-small cell lung cancer (NSCLC), accounting for 85% of all cases. Programmed cell death (PCD), an important regulatory mechanism for cell survival and homeostasis, has become increasingly prominent in cancer research in recent years. As such, exploring the role of PCD in NSCLC may help uncover new mechanisms for therapeutic targets. We utilized the GEO database and TCGA NSCLC gene data to screen for co-expressed genes. To delve deeper, single-cell sequencing combined with spatial transcriptomics was employed to study the intrinsic mechanisms of programmed cell death in cells and their interaction with the tumor microenvironment. Furthermore, Mendelian randomization was applied to screen for causally related genes. Prognostic models were constructed using various machine learning algorithms, and multi-cohort multi-omics analyses were conducted to screen for genes. In vitro experiments were then carried out to reveal the biological functions of the genes and their relationship with apoptosis. Cells with high programmed cell death activity primarily activate pathways related to apoptosis, cell migration, and hypoxia, while also exhibiting strong interactions with smooth muscle cells in the tumor microenvironment. Based on a set of programmed cell death genes, the prognostic model NSCLCPCD demonstrates strong predictive capabilities. Moreover, laboratory experiments confirm that SLC7A5 promotes the proliferation of NSCLC cells, and the knockout of SLC7A5 significantly increases tumor cell apoptosis. Our data indicate that programmed cell death is predominantly associated with pathways related to apoptosis, tumor metastasis, and hypoxia. Additionally, it suggests that SLC7A5 is a significant risk indicator for the prognosis of non-small cell lung cancer (NSCLC) and may serve as an effective target for enhancing apoptosis in NSCLC tumor cells.

Epstein-Barr Virus BRRF1 Induces Butyrophilin 2A1 in Nasopharyngeal Carcinoma NPC43 Cells via the IL-22/JAK3-STAT3 Pathway.

Epstein-Barr virus is highly associated with nasopharyngeal carcinoma (NPC) with genes expressed for tumor transformation or maintenance of viral latency, but there are certain genes that can modulate immune molecules. Butyrophilin 2A1 (BTN2A1) is an important activating protein for presenting phosphoantigens for recognition by Vγ9Vδ2 T cells to achieve antitumor activities. We have previously shown that Vγ9Vδ2 T cells achieve efficacy against NPC when BTN2A1 and BTN3A1 are upregulated by stimulating EBV gene expression, particularly LMP1. While BTN3A1 can be induced by the LMP1-mediated IFN-γ/JNK/NLRC5 pathway, the viral gene that can regulate BTN2A1 remains elusive. We showed that BTN2A1 expression is directly mediated by EBV BRRF1, which can trigger the BTN2A1 promoter and downstream JAK3-STAT3 pathway in NPC43 cells, as shown by RNA-seq data and verified via inhibitor experiments. Furthermore, BRRF1 downregulated IL-22 binding protein (IL-22RA2) to complement the EBNA1-targeting probe (P4)-induced IL-22 expression. Therefore, this study elucidated a new mechanism of stimulating BTN2A1 expression in NPC cells via the EBV gene BRRF1. The JAK3-STAT3 pathway could act in concordance with IL-22 to enhance the expression of BTN2A1, which likely leads to increased tumor cell killing by Vγ9Vδ2 T cells for enhanced potential as immunotherapy against the cancer.

Identification and Validation of Molecular Features of the Anoikis Gene-Related Hub Genes in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from nasopharyngeal mucosa. Anoikis, a form of programmed cell death induced by detachment from the extracellular matrix, normally prevents metastasis. Resistance to anoikis in cancer cells can enhance their metastatic potential. This study identifies anoikis-related genes (ARGs) associated with NPC to elucidate tumorigenesis mechanisms. Analysis of the GSE12452 dataset from GEO revealed 77 differentially expressed ARGs in NPC tissues. GO and KEGG analyses highlighted significant enrichment in apoptosis-related pathways. A PPI network identified MYC, FN1, BRCA1, and FGF2 as Hub genes. Correlation analysis showed MYC positively correlated with activated dendritic cells (p < 0.01) but negatively with naive CD4 T cells (p < 0.001). FN1 was positively correlated with activated dendritic cells (p < 0.01) and negatively with M1 macrophages (p < 0.05). FGF2 negatively correlated with naive CD4 T cells (p < 0.001), while BRCA1 was positively correlated with eosinophils (p < 0.01). GSVA and GSEA indicated that MYC, FN1, BRCA1, and FGF2 were significantly enriched in cell cycle and DNA replication pathways. Immunohistochemistry and qPCR of 50 NPC samples confirmed the overexpression of these genes. Knockdown of MYC, FN1, BRCA1, and FGF2 led to increased tumor cell malignancy, with statistical significance (p < 0.05). This study identifies MYC, FN1, BRCA1, and FGF2 as anoikis-related genes (ARGs) with significant regulatory roles in nasopharyngeal carcinoma (NPC). These ARGs are found to be involved in the development and progression of NPC, suggesting their potential as therapeutic targets for this cancer.

A High-Throughput Immune-Oncology Screen Identifies Immunostimulatory Properties of Cytotoxic Chemotherapy Agents in TNBC.

Background: Triple-negative breast cancers (TNBCs) typically have a greater immune cell infiltrate and are more likely to respond to immune checkpoint inhibition (ICI) than ER+ or HER2+ breast cancers. However, there is a crucial need to optimize combining chemotherapy strategies with ICI to enhance overall survival in TNBC. Methods: Therefore, we developed a high-throughput co-culture screening assay to identify compounds that enhance CD8+ T-cell-mediated tumor cell cytotoxicity. Over 400 FDA-approved compounds or agents under investigation for oncology indications were included in the screening library. Results: Four chemotherapy agents were chosen as priority hits for mechanistic follow-up due to their ability to enhance T-cell-mediated cytotoxicity at multiple doses and multiple time points: paclitaxel, bleomycin sulfate, ispinesib, and etoposide. Lead compounds affected the expression of MHCI, MHCII, and PD-L1 and induced markers of immunogenic cell death (extracellular ATP or HMGB1). Conclusions: Based on the ability to increase tumor cell susceptibility to T-cell-mediated cytotoxicity while minimizing T-cell toxicity, bleomycin was identified as the most promising lead candidate. Overall, the results of these studies provide mechanistic insight into potential new chemotherapy partners to enhance anti-PD-1 efficacy in TNBC patients.

Reprogramming the Tumor Immune Microenvironment Through Activatable Photothermal Therapy and GSH depletion Using Liposomal Gold Nanocages to Potentiate Anti-Metastatic Immunotherapy.

Cancer immunotherapy offers significant clinical benefits for patients with advanced or metastatic tumors. However, immunotherapeutic efficacy is often hindered by the tumor microenvironment's high redox levels, leading to variable patient outcomes. Herein, a therapeutic liposomal gold nanocage (MGL) is innovatively developed based on photo-triggered hyperthermia and a releasable strategy by combining a glutathione (GSH) depletion to remodel the tumor immune microenvironment, fostering a more robust anti-tumor immune response. MGL comprises a thermosensitive liposome shell and a gold nanocage core loaded with maleimide. The flexible shell promotes efficient uptake by cancer cells, enabling targeted destruction through photothermal therapy while triggering immunogenic cell death and the maturation of antigen-presenting cells. The photoactivated release of maleimide depletes intracellular GSH, increasing tumor cell sensitivity to oxidative stress and thermal damage. Conversely, GSH reduction also diminishes immunosuppressive cell activity, enhances antigen presentation, and activates T cells. Moreover, photothermal immunotherapy decreases elevated levels of heat shock proteins in tumor cells, further increasing their sensitivity to hyperthermia. In summary, MGL elicited a robust systemic antitumor immune response through GSH depletion, facilitating an effective photothermal immunotherapeutic strategy that reprograms the tumor microenvironment and significantly inhibits primary and metastatic tumors. This approach demonstrates considerable translational potential and clinical applicability.

Uncovering the protective role of lipid droplet accumulation against acid-induced oxidative stress and cell death in osteosarcoma

Extracellular acidosis stemming from altered tumor metabolism promotes cancer progression by enabling tumor cell adaptation to the hostile microenvironment. In osteosarcoma, we have previously shown that acidosis increases tumor cell survival alongside substantial lipid droplet accumulation. In this study, we explored the role of lipid droplet formation in mitigating cellular stress induced by extracellular acidosis in osteosarcoma cells, thereby enhancing tumor survival during progression. Specifically, we examined how lipid droplets shield against reactive oxygen species induced by extracellular acidosis. We demonstrated that lipid droplet biogenesis is critical for acid-exposed tumor cell survival, as it starts shortly after acid exposure (24 h) and inversely correlates with ROS levels (DCFH-DA assay), lipid peroxidation (Bodipy assay), and the antioxidant response, as also revealed by NRF2 transcript. Additionally, extracellular metabolites, such as lactate, and interaction with mesenchymal stromal cells within the tumor microenvironment intensify lipid droplet build-up in osteosarcoma cells. Critically, upon targeting two key proteins implicated in LD formation - PLIN2 and DGAT1 - cell viability significantly declined while ROS production escalated. In summary, our findings underscore the vital reliance of acid-exposed tumor cells on lipid droplet formation to scavenge oxidative stress. We conclude that the rewiring of lipid metabolism driven by microenvironmental cues is of paramount importance for the survival of metabolically altered osteosarcoma cells in acidic condition. Overall, we suggest that targeting key members of lipid droplet biogenesis may eradicate more aggressive and resistant tumor cells, uncovering potential new treatment strategies for osteosarcoma.

Lactate reprograms glioblastoma immunity through CBX3-regulated histone lactylation.

Glioblastoma (GBM), an aggressive brain malignancy with a cellular hierarchy dominated by GBM stem cells (GSCs), evades antitumor immunity through mechanisms that remain incompletely understood. Like most cancers, GBMs undergo metabolic reprogramming toward glycolysis to generate lactate. Here, we show that lactate production by patient-derived GSCs and microglia/macrophages induces tumor cell epigenetic reprogramming through histone lactylation, an activating modification that leads to immunosuppressive transcriptional programs and suppression of phagocytosis via transcriptional upregulation of CD47, a "don't eat me" signal, in GBM cells. Leveraging these findings, pharmacologic targeting of lactate production augments efficacy of anti-CD47 therapy. Mechanistically, lactylated histone interacts with the heterochromatin component chromobox protein homolog 3 (CBX3). Although CBX3 does not possess direct lactyltransferase activity, CBX3 binds histone acetyltransferase (HAT) EP300 to induce increased EP300 substrate specificity toward lactyl-CoA and a transcriptional shift toward an immunosuppressive cytokine profile. Targeting CBX3 inhibits tumor growth by both tumor cell-intrinsic mechanisms and increased tumor cell phagocytosis. Collectively, these results suggest that lactate mediates metabolism-induced epigenetic reprogramming in GBM that contributes to CD47-dependent immune evasion, which can be leveraged to augment efficacy of immuno-oncology therapies.

Actin Dysregulation Induces Neuroendocrine Plasticity and Immune Evasion: A Vulnerability of Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is aggressive with limited therapeutic options. Despite recent advances in targeted therapies and immunotherapies, therapy resistance is a recurring issue, which might be partly due to tumor cell plasticity, a change in cell fate. Nonetheless, the mechanisms underlying tumor cell plasticity and immune evasion in SCLC remain elusive. CRACD, a capping protein inhibitor that promotes actin polymerization, is frequently inactivated in SCLC. Cracd knockout (KO) transforms preneoplastic cells into SCLC tumor-like cells and promotes in vivo SCLC development driven by Rb1, Trp53, and Rbl2 triple KO. Cracd KO induces neuroendocrine (NE) plasticity and increases tumor cell heterogeneity of SCLC tumor cells via dysregulated NOTCH1 signaling by actin cytoskeleton disruption. CRACD depletion also reduces nuclear actin and induces EZH2-mediated H3K27 methylation. This nuclear event suppresses the MHC-I genes and thereby depletes intratumoral CD8+ T cells for accelerated SCLC tumorigenesis. Pharmacological blockade of EZH2 inhibits CRACD-negative SCLC tumorigenesis by restoring MHC-I expression and immune surveillance. Unsupervised single-cell transcriptomics identifies SCLC patient tumors with concomitant inactivation of CRACD and downregulated MHC-I pathway. This study defines CRACD, an actin regulator, as a tumor suppressor that limits cell plasticity and immune evasion and proposes EZH2 blockade as a viable therapeutic option for CRACD-negative SCLC.

TMIC-58. LEVERAGING VIRO-IMMUNOTHERAPY BY TARGETING IGF2-IGF1R SIGNALING

Abstract While an FDA-approved oncolytic herpes simplex-1 virus (oHSV) has shown therapeutic promise with superior safety, accumulating clinical data revealed that its therapeutic efficacy is observed in a small subset of patients. Here, we show that NFκB-mediated Insulin-like Growth Factor 2 (IGF2)/Insulin-like Growth Factor-1 Receptor (IGF1R) signaling pathway as a key modulator for oHSV therapy-mediated tumor resistance. RNA sequencing on the oHSV-infected primary glioblastoma (GBM) and breast cancer (BC) cells identified IGF2 as one of the top 10 upregulated secretomes. Transcriptomic analysis also revealed significant upregulation/enrichment of genes related to IGF2-IGF1R-MAPK pathway in oHSV-treated tumor cells. Infection with oHSV in GBM and BC cells up-regulated IGF2 transcription via direct binding of NFκB in the IGF2 promoter 3. Compromising the IGF2-IGF1R signaling pathway using IGF2 neutralizing antibody significantly increased oHSV-mediated tumor cell killing in vitro and mice survival in vivo. To circumvent this and promptly translate our findings in clinical settings, we have developed a novel oHSV, oHSV-D11mt, by incorporating modified IGF2R domain 11 into the parental oHSV genome to function as IGF2 decoy receptor as a single-agent modality. oHSV-D11mt specifically bind to IGF2 not IGF1, blocks oHSV-induced IGF2-IGF1R signaling, increases tumor cell killing, decreases oHSV-induced neutrophils/PMN-MDSCs infiltration, immune suppressive/proangiogenic cytokine secretion, and increases CD8+ cytotoxic T lymphocytes (CTLs) activation, resulting in increased virus propagation and mice survival. These findings suggest that IGF2-IGF1R signaling is a novel target to overcome oHSV therapy resistance and oHSV-D11mt could be used for improved viro-immunotherapy.

EXTH-69. DEVELOPMENT OF A NOVEL, HUMAN ANTIBODY TO IMPROVE ONCOLYTIC VIRUS THERAPY FOR GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most aggressive, malignant, primary brain tumor with abysmal overall survival. GBM is regarded as an “immune cold” tumor due to poor immune cell infiltration and immunosuppressive signaling in the tumor microenvironment (TME). Oncolytic herpes virus (oHSV) therapy has emerged as a possible therapeutic avenue for solid tumors such as GBM. We have observed that oHSV-treated tumor cells release extracellular ATP (eATP) which functions as immunostimulatory signaling molecule. In the TME, eATP is rapidly hydrolyzed into immunosuppressive adenosine by ectoenzymes CD39 and CD73. Using global CD73 knock out (CD73KO) mice or a function-blocking murine antibody, we found that blocking CD73 activity improves oHSV therapy in vivo. Intracranial tumor-bearing CD73KO mice respond significantly better to oHSV therapy than wild type (WT). These long-term survivors also rejected a subsequent tumor challenge, demonstrating development of a memory response. scRNA-seq revealed significantly increased immune cell infiltration in CD73KO mice compared to WT. We evaluated cellular crosstalk using CellChat and uncovered a major CCL-directed signaling pathway in the CD73KO mice. Furthermore, we identified a novel, human CD73 antibody from panning an scFv phage display library derived from human tonsils. This antibody, Ab6, demonstrated functional inhibition of purified CD73 and very high binding affinity for CD73. Co-culture of human GBM cells treated with OV and Ab6 and donor PBMCs resulted in increased tumor cell death. Additionally, human DCs exposed to Ab6 and OV-treated conditioned media were better able to activate T cells. To evaluate in vivo efficacy of this novel antibody, we have generated custom humanized CD73 knock-in mice wherein human CD73 has replaced murine CD73. We are currently evaluating the efficacy of Ab6 and oHSV in these mice against intracranial glioma. These results will support the translation of Ab6 as a potential immune therapeutic to add to the arsenal against brain tumors.

DDDR-37. OHSV AND RADIATION THERAPIES SENSITIZE GLIOBLASTOMA TO IGF1R-TARGETED THERAPY AND SYNERGIZE IN TRIPLE COMBINATION THERAPY

Abstract Glioblastoma (GBM) continues to have a dismal prognosis despite remarkable advances in the field due to both tumor-intrinsic factors (i.e., IDH1 or EGFRvIII mutations) and extrinsic factors (i.e., relating to the tumor microenvironment (TME)), necessitating therapeutic innovation. Oncolytic herpes simplex virus-1 (oHSV) is an FDA-approved therapy that has shown promising results in clinical trials; however, therapeutic efficacy is limited to a small subset of patients. Thus, elucidating both the tumor-TME interplay and feedback/feedforward tumor-cell signaling in the context of viro-immunotherapy is essential in augmenting therapeutic outcomes. We recently discovered a novel mechanism of resistance to oHSVs, in which infection increases secretion of Insulin-like growth factor 2 (IGF2), activating the Insulin-like Growth Factor-1 Receptor (IGF1R). Upregulation of the IGF1R signaling pathway in GBM is correlated with poor prognosis and has been implicated in resistance to conventional therapies including radiotherapy (RTx). Herein, we further elucidate the impact of oHSVs on IGF1R activation, demonstrating a dose-dependent enhancement of downstream signaling. While IGF1R targeting therapies (e.g., OSI-966, Picropodophyllin (PPP)) have displayed limited efficacy in clinical trials, they significantly increased oHSV-induced cytotoxicity in all tested primary GBM cells compared to either agent alone in vitro and in vivo, resulting in a dose-dependent increase in PARP and caspase 3 activity by western blot and immunohistochemistry. We further found that RTx-induced IGF1 secretion and subsequent IGF1R activation were enhanced when combined with oHSV treatment. Triple combination therapy with oHSV, IGF1R inhibition, and RTx significantly increased tumor cell killing in vitro and improved survival of orthotopic GBM tumor-bearing mice. Collectively, our study provides preclinical evidence of enhanced therapeutic efficacy of oHSVs and RTx in combination with IGF1R blockade, supporting future use of IGF1R inhibitors in combination with the standard of care and FDA-approved oHSV therapies in patients with GBM.

PX-478 induces apoptosis in acute myeloid leukemia under hypoxia by inhibiting the PI3K/AKT/mTOR pathway through downregulation of GBE1

Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy characterized by limited therapeutic options and a pronounced tendency for relapse. PX-478, a novel inhibitor of hypoxia-inducible factor 1-alpha (HIF-1α), has demonstrated antitumor activity across various cancer models, but its specific role in AML remains unexplored. This study aimed to explore the potential target and mechanism of PX-478-induced AML cell apoptosis. First, PX-478 induced AML cell apoptosis in vitro under hypoxia via modulation of the Bcl-2 family and activation of the mitochondria-mediated caspase cascade, exhibiting a concentration-dependent effect. Additionally, in vivo administration of PX-478 led to notable inhibition of subcutaneous AML xenograft growth in mice, coupled with increased tumor cell apoptosis. RNA sequencing and cellular studies revealed downregulation of the PI3K/AKT/mTOR signaling pathway in PX-478-treated cells. Consistently, cellular studies also implicated PI3K/AKT/mTOR pathway in PX-478-induced AML cell apoptosis. Furthermore, by screening for RNA sequencing differential genes and subsequent experimental verification, Glycogen branching enzyme 1 (GBE1) may be involved in PX-478-induced apoptosis in AML cells. We found that inhibiting GBE1 expression in AML cells (siGBE1) led to downregulation of the PI3K/AKT/mTOR pathway and induced apoptosis. In experiments using AML cells with reduced GBE1 expression (shGBE1), PX-478 treatment did not further downregulate the pathway or enhance apoptosis. Re-expression of GBE1 in shGBE1 cells alleviated apoptosis and reduced PX-478- induced apoptosis and pathway downregulation. In conclusion, our findings provide convincing evidence that PX-478 induces apoptosis by inhibiting the PI3K/AKT/mTOR pathway through downregulation of GBE1 in AML cells.

Hsa_circ_0109320 Serves as a Novel Circular RNA Biomarker in Non-small Cell Lung Cancer by Promoting Metastasis.

Non-small cell lung cancer (NSCLC), including squamous cell carcinoma and adenocarcinoma, ranks among the top 10 cancers worldwide in terms of prevalence and mortality. NSCLC, a highly malignant tumor, exhibits distant invasion and migration as well as an unfavorable prognosis. As an innovative circular RNA, hsa _circ_0109320 (circ_0109320) has been recognized as a promising cancer modulator. However, our understanding of the influence of circ_0109320 in NSCLC remains insufficient. Our research explored the clinical significance and effects of circ_0109320 on oncogenic non-small cell lung cancer (NSCLC) phenotypes. Microarray analysis and qPCR indicated that circ_0109320 expression in NSCLC specimens increased relative to that in adjacent normal tissues and was further elevated in metastatic lymph nodes. The specimens acquired from 25 patients confirmed these findings. Additionally, circ_0109320 indicated a good score (AUC = 0.688, P = 0.013) on the ROC curves, which suggests its suitability as a promising biomarker for lung cancer. Meanwhile, circ_0109320 was noticeably upregulated in lung cancer (LC) cell lines compared to human bronchial epithelial cells. Next, we performed loss- and gain-of-function experiments to examine the role of circ_0109320 in the tumor phenotypes of the cell lines. We observed that depletion or overexpression of circ_0109320 did not alter cell viability. However, the ectopic removal of circ_0109320 repressed the migration and invasion of A549 and SK-MES-1 cells, whereas circ_0109320 overexpression promoted cell migration and invasion. Furthermore, the examination of epithelial-mesenchymal transition (EMT) markers indicated that circ_0109320 elevates cell EMT activity. In conclusion, circ_0109320 level was highly associated with increased tumor cell proliferation and metastasis. circ_0109320 could be a promising predictor of clinical outcomes and a reliable target to treat NSCLC by inhibiting metastasis.

TGF-β Signaling Loop in Pancreatic Ductal Adenocarcinoma Activates Fibroblasts and Increases Tumor Cell Aggressiveness.

The interaction between cancer cells and cancer-associated fibroblasts (CAFs) is a key determinant of the rapid progression, high invasiveness, and chemoresistance of aggressive desmoplastic cancers such as pancreatic ductal adenocarcinoma (PDAC). Tumor cells are known to reprogram fibroblasts into CAFs by secreting transforming growth factor beta (TGF-β), amongst other cytokines. In turn, CAFs produce soluble factors that promote tumor-cell invasiveness and chemoresistance, including TGF-β itself, which has a major role in myofibroblastic CAFs. Such a high level of complexity has hampered progress toward a clear view of the TGFβ signaling loop between stromal fibroblasts and PDAC cells. Here, we tackled this issue by using co-culture settings that allow paracrine signaling alone (transwell systems) or paracrine and contact-mediated signaling (3D spheroids). We found that TGF-β is critically involved in the activation of normal human fibroblasts into alpha-smooth muscle actin (α-SMA)-positive CAFs. The TGF-β released by CAFs accounted for the enhanced proliferation and resistance to gemcitabine of PDAC cells. This was accompanied by a partial epithelial-to-mesenchymal transition in PDAC cells, with no increase in their migratory abilities. Nevertheless, 3D heterospheroids comprising PDAC cells and fibroblasts allowed monitoring the pro-invasive effects of CAFs on cancer cells, possibly due to combined paracrine and physical contact-mediated signals. We conclude that TGF-β is only one of the players that mediates the communication between PDAC cells and fibroblasts and controls the acquisition of aggressive phenotypes. Hence, these advanced in vitro models may be exploited to further investigate these events and to design innovative anti-PDAC therapies.