Citations

ABSTRACT Glioblastoma (GBM) is universally lethal despite decades of research to find effective treatments. This highlights the need to identify druggable targets essential for sustaining the malignant phenotype but dispensable for normal tissue. We propose that the enzyme α-tubulin acetyl transferase ( ATAT1 ) meets these criteria. ATAT1 acetylates α-tubulin at lysine 40, which increases microtubule stability and promotes microtubule-based transport. While ATAT1 knockout mice have only a very mild phenotype, ATAT1 suppression in GBM has multiple therapeutic effects by reducing tumor invasion, proliferation, and therapeutic resistance. These translate not only into improved survival with ATAT1 targeting by itself, but also into synergy when ATAT1 deletion is combined with FDA approved therapies. This study strongly supports our conclusion that ATAT1 is a promising therapeutic target in GBM.

Introduction Recent transcriptome analysis has demonstrated increased expression of Vascular Endothelial Growth Factor receptor-1 (VEGFR-1/FLT1) and in AD brain. Increased expression of VEGFR1 and its ligand VEGFB were associated with a more rapid rate of cognitive decline, providing evidence of a potential link between increased VEGFR-1 expression in AD pathogenesis. In this study, we explored the potential role of VEGFR-1 expression in neurons on AD pathology. Methods To confirm VEGFR1 expression in AD brains, we first performed immunostaining in AD brain sections (AD - Braak stage V-VI, and normal controls - Braak 0-II). And to determine a potential detrimental role of neuronal VEGFR1 expression on AD associated pathologies, we exposed SH-SY5Y human neuroblastoma cells and mouse primary neurons to either hypoxia conditions (1%O 2 ) or 5 μ Aβ1-42 oligomers or fibrils for 24, 28 and 72hrs. Results In this study, we found preferential staining of VEGFR-1 in the neuropil and neuronal cell bodies both in AD and Control hippocampus and increased VEGFR-1 immunoreactivity in dystrophic neuritic processes in the vicinity of Thio-S positive amyloid plaques in AD brains. And treatment of SH-SY5Y human neuroblastoma cell line and mouse primary neurons, with either hypoxia conditions or Aβ1-42 oligomers, resulted in increased VEGFR-1 expression and cleaved caspase 3 activation, leading to neuronal toxicities/cell death. Similarly, treatment with Aβ1-42 fibrils also increased VEGFR-1 and cleaved caspase 3 protein levels in the SH-SY5Y cells whereas treatment with Aβ1-42 monomers had no effect on VEGFR-1 expression. In addition, we show that over-expression of VEGFR-1 intracellular domains in SH-SY5Y cells directly induced neuronal toxicities and importantly, siRNA-mediated knockdown of VEGFR-1 in neurons prevented the hypoxia, Aβ1-42 oligomer and Aβ1-42 fibril-induced toxicities and cell death phenotypes. Treatment with either hypoxia or Aβ1-42 oligomers also reduced expression of cell survival genes including VEGFR-2 and Hippo pathway YAP1 and siRNA-mediated VEGFR-1 knockdown in the neurons normalized expression of both VEGFR-2 and YAP1. Using differential gene expression analysis, we demonstrated upregulation of several inflammatory/interferon-stimulated genes (ISGs) as well as increased expression of genes involved in activation of oxidative stress and cell death pathways in response to Aβ1-42 oligomers treatment in mouse primary neurons. And siRNA-mediated VEGFR-1 knockdown in the mouse primary neurons, reduced gene expression of both the ISGs and oxidative stress/cell death pathways in response to Aβ1-42 oligomer treatment. Discussion In summary, these results show that siRNA-mediated knockdown of VEGFR-1 in neurons significantly prevented hypoxia, Aβ1-42 oligomer and Aβ1-42 fibril-induced cellular toxicities and cell death phenotypes, indicating a potential detrimental role of aberrant VEGFR-1 expression and signaling in response to AD associated pathologies.

Abstract Background While immune checkpoint inhibitors (ICIs) provide durable clinical benefit in a subset of patients, current biomarkers to predict response lack accuracy. Ex vivo cytokine profiling has shown promise for improved accuracy but has been mostly limited to ICIs targeting PD-1 [Voabil et al, 2021]. This poses the question of whether ICIs beyond αPD-1 – such as CTLA4 blockade, which has been reported to act within the draining lymph node – would be amenable to similar approaches. The Elephas platform characterizes response to ICIs in live tumor fragments (LTFs) from core needle biopsies (CNBs) and resections using longitudinal cytokine profiling. Using a panel of 7 clinically relevant ICI regimens, we demonstrate cytokine responses across diverse tumor types, characterize similarities and differences in cytokine response profiles across different ICIs, and explore specimen variability in responses across ICI treatments. Methods CNBs were collected through 3 observational clinical trials (NCT05478538, NCT05520099, NCT0634962) and a biobank collection study, while resections were procured from NCI CHTN. LTFs were generated using an automated cutting device and encapsulated in hydrogel. LTFs were treated with mono (αPD-1, αPD-L1, αCTLA4, αLAG-3) or combination therapy (αPD-1/αCTLA4, αPD-1/αLAG-3, αPD-L1/αCTLA4) using a sequential treatment strategy in which isotype control followed by ICI were added to the same tissue in a single well. This approach enabled the comparison of control and treatment conditions while mitigating the effects of tumor heterogeneity. Production rates for 46 cytokines were measured using a multiplex assay. Remaining T-cell reactivity was also evaluated at the end of the assay to better understand the presence of functional T cells in LTFs. Results Unsupervised hierarchical clustering of cytokine profiling data from CNB LTFs across 129 patients identified defined responder and non-responder clusters. Within the response cluster, 4 subclusters with distinct immune phenotypes were identified. While tight clustering of samples treated with the same ICI was not observed, some enrichment of samples treated with combination ICIs was observed within individual subclusters. Further, cytokine responses to αCTLA4 monotherapy were observed, suggesting that CTLA4 blockade may act beyond the draining lymph node. LTFs from resections enabled the characterization of cytokine response profiles across treatment conditions within individual specimens, including the magnitude of cytokine response and variability in cytokine response profiles. Evaluation of T-cell reactivity provided further insight into the presence of functional T-cells in LTFs and the extent of T-cell activation achieved by ICI treatment. Conclusions This study demonstrates the platform’s ability to recapitulate diverse mechanisms of immune activation ex vivo, enabling the characterization of cytokine responses to clinically relevant ICI mono or combination therapy in live tumor tissue and supporting future efforts to improve ICI response prediction beyond αPD-1. Citation Format: Pichet Adstamongkonkul, Pierre Cunin, Santanu Bhattacharya, Tanmay Kulkarni, Amreen Nasreen, Nathan Marhefke, Nina Chen, T S. Ramasubramanian, Laura Hrycyniak, Julie Zweng, Erika von Euw, Christina Vivelo, Sean Caenepeel, Debabrata Mukhopadhyay, Hinco Gierman, Hilario Ramos. Characterization of αPD1, αPD-L1, αCTLA4, and αLAG3 as mono and combination therapy in live tumor fragments using an ex vivo cytokine profiling platform [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B032.

Abstract Background Glioblastoma (GBM) is the most common high-grade primary malignant brain tumor, characterized by a notably poor prognosis. Current treatments for GBM have shown limited effectiveness in improving patient survival, highlighting the urgent need for novel therapeutic strategies. Combination therapy offers significant potential in overcoming resistance by targeting multiple signaling pathways; however, it often comes with increased toxicity compared to monotherapy. Co-encapsulating multiple therapeutic agents into a tumor-targeted drug delivery platform holds promise for overcoming these limitations and improving treatment outcomes. Methods We developed a tumor-targeted liposomal nanoformulation (TTL) using phospholipids, cholesterol, DSPE-(PEG)2000-OMe, and a proprietary tumor-targeting peptide (TTP). The TTL was loaded with everolimus (TTL-E), vinorelbine (TTL-V), rapamycin (TTL-R), a combination (TTL-EV), or (TTL-RV). These formulations were tested in vivo on orthotopic GBM mice, combined with temozolomide and radiation. RNA sequencing was performed to identify molecular and transcriptome changes post-treatment. Results TTL demonstrated tumor-specific uptake, effectively delivering drugs to GBM tumors. TTL-EV and TTL-RV outperformed single-drug formulations. Radiation combined with TTL-EV/RV improved tumor growth inhibition and survival, while temozolomide provided minimal benefit. Transcriptome analysis revealed differentially expressed genes (DEGs) linked to DNA damage repair, cell cycle, metabolism, and extracellular matrix pathways. Conclusion TTL crossed the blood-brain barrier, targeting tumors effectively. Radiation plus TTL-EV/RV enhanced tumor suppression and survival in GBM models. Gene expression analysis identified DEGs related to DNA damage and cell death. Mechanistic studies suggest TTL-EV plus radiation inhibits mTOR/MAPK pathways and sensitizes tumors to radiation. These findings offer a potential approach for improving GBM treatment.

Abstract Mesenchymal stem cell (MSC)-derived exosomes are recognized as critical mediators within the tumor microenvironment (TME), exerting both pro- and anti-tumorigenic effects depending on contextual factors. These vesicles are also gaining attention for their potential as therapeutic vehicles in regenerative medicine and targeted drug delivery. However, the influence of the TME on the physical characteristics of MSC-derived exosomes remains poorly understood. In this study, we utilized Atomic Force Microscopy (AFM) to investigate the morphological and nanomechanical properties of MSC-derived exosomes under standard and TME-like conditions. AFM imaging in fluid mode preserved the native structure of exosomes and enabled high-resolution analysis of their topography, surface roughness, stiffness, adhesion, and deformation. AFM offers unique advantages in exosome research by enabling label-free, nanoscale analysis of vesicle properties in near-physiological conditions. The ability to detect such subtle but functionally significant changes highlights the relevance of AFM in exosome characterization and quality assessment. Our results revealed that exposure to the TME induces marked changes in exosomal membrane morphology and mechanical behavior, including increased surface heterogeneity, higher stiffness, and altered adhesive interactions. These biophysical alterations may reflect changes in membrane composition and protein or lipid cargo, potentially affecting exosome function, uptake, and therapeutic efficacy. Overall, our findings provide new insights into how the TME modulates MSC exosome biophysics and underscore the utility of AFM-based techniques for advancing the development of exosome-based diagnostics and therapies. This can provide a conceptual framework for future mechanistic and translational studies in this area.

Tumor-Treating Fields (TTFields), a novel therapeutic avenue, is approved for therapy in Glioblastoma multiforme, malignant pleural mesothelioma, and metastatic non-small cell lung cancer (NSCLC). In pancreatic ductal adenocarcinoma (PDAC), several clinical trials are underway to improve outcomes, yet a significant knowledge gap prevails involving the cell-extracellular matrix (ECM) crosstalk. Herein, we hypothesized that treatment with TTFields influence this crosstalk, which is reflected by the dynamic alteration in nanomechanical properties (NMPs) of cells and the ECM in a co-culture system. We employed an ECM gel comprising collagen, fibronectin, and laminin mixed in 100:1:1 stoichiometry to co-culture of Panc1 and AsPC1 individually. This ECM mixture mimics the in vivo tumor microenvironment closely when compared to the individual ECM components studied before. A comprehensive frequency-dependent study revealed the optimal TTFields frequency to be 150 kHz. We also observed that irrespective of the ECM’s presence, TTFields increase cell membrane stiffness and decrease deformation several-folds in both Panc1 and AsPC1 cells at both 48 h and 72 h. Although adhesion for AsPC1 decreased at 48 h, at 72 h it was observed to increase irrespective of ECM’s presence. Moreover, it significantly alters the NMPs of ECM gels when co-cultured with PDAC cell lines. However, AsPC1 cells were observed to be more detrimental to these changes. Lastly, we attribute the stiffness changes in Panc1 cells to the membrane F-actin reorganization in the presence of TTFields. This study paves a path to study complex PDAC TME as well as the effect of various chemotherapeutic agents on such TME with TTFields in the future.

Tumor treating fields (TTFields), a noninvasive anticancer treatment modality, has received FDA approval for pleural mesothelioma, metastatic NSCLC following platinum-based therapy, newly diagnosed and recurrent glioblastoma multiforme. TTFields have also demonstrated potential benefits in other cancer types, including pancreatic ductal adenocarcinoma (PDAC). Although the PANOVA trial yielded positive results in PDAC patients, the mechanisms underlying these beneficial effects remain unclear. Severe desmoplasia is a hallmark characteristic of PDAC due to accumulation of excess extracellular matrix (ECM) caused by activated fibroblasts and cancer cells. This dense ECM contributes to an immunosuppressive tumor microenvironment (TME) and drug resistance, which ultimately leads to poor 5-year survival in PDAC patients. Our previous work with atomic force microscopy (AFM) showed that the nanomechanical properties of the ECM change dynamically based on its composition and the presence of PDAC cells, and vice versa. Herein, we prepared an ECM complex comprising of collagen, fibronectin and laminin at physiological concentrations. Following which, we investigated the alteration of stiffness of both PDAC cells and the ECM in presence of TTFields using an AFM. A frequency-dependent analysis of TTFields showed that a frequency of 150 KHz led to consistent decreases ECM stiffness at 48 and 72 hours. Next, we evaluated the influence of TTFields in PDAC cells alone. In Panc-1 cells exposed to TTFields for 48 and 72 hours, we observed a more than 6-fold increase in membrane stiffness compared to untreated controls. Additionally, AsPC-1 cells subjected to TTFields exhibited a greater than 2-fold increase in membrane stiffness at these time points. Furthermore, we have investigated influence of TTFields on PDAC cells and ECM components in a co-culture model. In this co-culture scenario for Panc-1 cells, TTFields increase membrane stiffness by 5-fold compared to untreated at these time points. Also, AsPC-1 cells demonstrated a 1.5-fold increase in membrane stiffness in the presence of TTFields compared to the untreated. Lastly, we also evaluated the influence of cells on the ECM stiffness in the presence of TTFields. It significantly increased the ECM stiffness in presence of Panc-1 co-culture by 1.3-folds compared to control. We also observed 3- and 4-fold increase in ECM matrix stiffness when co-cultured with AsPC-1 for 48 and 72 hours from TTFields, respectively compared to control. In conclusion, TTFields significantly modulate the stiffness of PDAC cells and the ECM, both individually and in co-culture. Citation Format: Tanmay Kulkarni, Hani Babiker, Debabrata Mukhopadhyay, Santanu Bhattacharya. Tumor treating fields (TTFields) alter nanomechanical attributes of cell-ECM complex in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5105.

Abstract The extracellular matrix (ECM) plays a critical role in cancer progression, with its biophysical properties influencing cancer behavior. ECM stiffness can induce long-lasting adaptations in cancer cells, promoting sustained invasion through a process known as “mechanical memory” (MM). Glioblastoma (GBM) exhibits spatial heterogeneity in stiffness, with stiffer ECM found at the tumor periphery where invasive cells reside and promote recurrence. We hypothesize that increased stiffness at the tumor periphery primes GBM cells for sustained invasion through MM. METHODS Cell lines were established from image-guided human GBM samples from tumor margin and core (n=10 patients, 20 cell lines). Cells from random locations were conditioned in physiologically relevant extracellular matrices for two weeks. Human organotypic assay (n=3), Migration Assays, Atomic Force Microscopy (AFM), Immunocytochemistry, Western Blot, and RNA-sequencing (n=6) were performed. RESULTS GBM cells at higher stiff surfaces displayed increased migration (p<0.01). Magnetic Resonance Elastography (n=9) confirmed GBM heterogeneity, with softer cores (0.2-0.5kPa) transitioning to stiffer margins (>2kPa). Organotypic scaffolds mimicking the tumor margin promoted enhanced migration of GFP-labeled GBM cells. Margin cells and cells conditioned on stiffer matrices demonstrated higher migratory potential compared to their core and soft-conditioned counterparts (p<0.05). These trends persist over time and across multiple passages. ECM stiffness exposure causes nanomechanical, cytoskeletal, and transcriptional adaptations. AFM shows that cells from the margin (n=6) are softer (p<0.01) and more deformable (p<0.01). RNAseq identified upregulation of MM-specific genes in GBM margin cells. The mechanosensitive transcription factor YAP1 emerged as a key driver of MM. Notably, YAP1 is targetable by the FDA-approved drug Verteporfin. YAP1 expression was elevated in margin and stiff-conditioned cells (p<0.05). Stiff-conditioned cells and margin cells displayed increased sensitivity to Verteporfin treatment (p<0.05), with treatment significantly reducing margin migration compared to the core (p<0.001). CONCLUSION Stiffer GBM margins promote the formation of aggressive phenotypes through MM. Targeting YAP1 with Verteporfin, a readily available drug, presents a promising strategy to halt GBM invasion and improve patient outcomes.

Abstract Radiation therapy (RT) is frequently used in the neoadjuvant setting in borderline resectable and some resectable pancreatic ductal adenocarcinoma (PDAC) and in the definitive setting in locally advanced PDACs which is third leading cause of cancer associated death. Higher doses of RT improve overall treatment outcomes but increasing the dose significantly above 50Gy in standard 2Gy fractions risks profound gastrointestinal (GI) toxicity. Additionally, genotoxic stress such as RT, can induce CXCL12 secretion by cancer associated fibroblast (CAFs) and overexpression of CXC motif chemokine receptor 4 (CXCR4) in cancer cells. CXCL12-CXCR4 downstream signaling results in immune suppression and consequent enhanced tumor growth and metastasis and poor prognosis in PDAC. To overcome these clinical challenges, we have developed multimodal gold nanoparticle constructs (GNPs) by functionalizing the CXCR4 antagonists to (i) bind to CXCR4 and mediate GNP internalization, (ii) abolish the feedback immune inhibitory signals mediated by CXCR4 overexpression, and (iii) enhance therapeutic benefit of radiation. To achieve these multimodal activities, we have covalently attached the CXCR4 antagonist, AMD070, to thiol-terminated polyethylene glycol (PEG) to synthesize AMD-PEG which was then conjugated with GNPs to produce AMD-PEG-GNP. In our experiment AMD-PEG-GNP has successfully inhibited CXCL12-CXCR4 downstream signalling such as reduction of ERK phosphorylation in PDAC cells. To analyze the radiosensitization role of GNP nanoconjugates, PDAC cells were incubated with AMD-PEG, PEG-GNP and AMD-PEG-GNPs for 4 hours and then treated with increasing doses of radiated, from 1-4Gy. AMD-PEG-GNP demonstrated a systematic enhancement on reactive oxygen species (ROS) production and downregulation of a colony formation. Collectively, the results from in vitro study with PDAC cells confirm that the nanoconjugate with AMD070 enhances radio-cytotoxicity and impair CXCL12-CXCR4 downstream signaling. Hence AMD-PEG-GNP has strong merit to enhance radiation dose locally and simultaneously restrict RT induced immunosuppression in PDAC. Citation Format: Tanmay Kulkarni, Shamit Dutta, Hazel Parent, Karington Perry, Yuri Mackeyev, Santanu Bhattacharya. Immune modulatory activity of radio-sensitizing gold nanoparticle [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3181.