Bhattacharya, Santanu

Tumor Treating Fields (TTFields) represent a novel, non-invasive therapeutic modality in oncology that employs low-intensity, intermediate-frequency alternating electric fields to disrupt mitotic processes and induce cancer cell death. This review integrates mechanistic, preclinical, and emerging clinical evidence supporting the integration of TTFields with immunotherapeutic strategies in pancreatic ductal adenocarcinoma (PDAC). Although immunotherapy has transformed the treatment landscape across multiple malignancies, its efficacy in PDAC remains limited due to the tumor’s dense stroma, immunosuppressive microenvironment, and low immunogenicity. Preclinical investigations suggest that TTFields may potentiate immune-based therapies by enhancing antigen presentation, modulating the tumor microenvironment (TME), and attenuating mechanisms of immune resistance. We highlight studies evaluating TTFields in combination with immune checkpoint inhibitors (ICI), adoptive cellular therapies, and cancer vaccines, emphasizing their potential synergistic effects in PDAC. Clinically, the phase II PANOVA-2 trial demonstrated feasibility and encouraging survival outcomes with TTFields in combination with gemcitabine and nab-paclitaxel, providing the rationale for the ongoing phase III PANOVA-3 trial and the phase II PANOVA-4 trial, which combines TTFields with chemotherapy and atezolizumab. Additional clinical experiences in glioblastoma (GBM) and non-small-cell lung cancer (NSCLC) further substantiate the broader applicability of TTFields as an immunomodulatory adjunct. Remaining challenges include optimizing treatment sequencing, identifying predictive biomarkers, and managing TTFields-associated toxicities. Collectively, current evidence positions TTFields as a promising strategy to augment immunotherapy in PDAC, warranting further translational and clinical investigation to establish its role in reshaping therapeutic paradigms.

Abstract RMC-6236 (daraxonrasib) is a novel multiselect RAS(ON) inhibitor under investigation for pancreatic ductal adenocarcinoma (PDAC) and other RAS-mutant cancers. This compound shares structural similarities with RMC-7977 and exhibits a conserved binding site and interaction pattern within the tri-complex formed between active RAS (RAS(ON)) proteins and cyclophilin A (CypA). Tumor Treating Fields (TTFields) are low-intensity, alternating electric fields that disrupt mitosis and have shown clinical benefit in solid tumors. The PANOVA-3 trial demonstrated that TTFields combined with gemcitabine plus nab-paclitaxel improved survival, pain control, and quality of life in locally advanced PDAC. Our previous work demonstrated that TTFields abrogate oncogenic KRAS pathway. We thus hypothesize a potential for increase in effectievness with the combination. In this study, the metastatic PDAC cell line AsPC-1 was treated with TTFields (150 kHz, 1.8 V/cm), RMC-6236 (15 nM; sub-IC50), or combination. A sub-IC50 concentration (15 nM) of RMC-6236 was selected based on MTS assay results, and subsequent experiments were performed at this dose. The MTS assay revealed that both TTFields and RMC-6236 independently reduced AsPC-1 cell viability, with a more pronounced reduction observed at 72 hours compared to 48 hours. RMC-6236 alone produced minimal effects at either 48 hours (85.82 ± 13.9%) or 72 hours (90.48 ± 15.4%) compared with control (100.0 ± 8.4%, 48 h; 100.0 ± 8.2%, 72 h). TTFields treatment significantly reduced viability to 74.42 ± 21.4% at 48 hours and 50.17 ± 26.5% at 72 hours. The combination treatment (TTFields + RMC-6236) further decreased viability to 63.8 ± 25.2% at 48 hours and 40.93 ± 18.1% at 72 hours, demonstrating synergistic cytotoxicity relative to monotherapy. Clonogenic assays were performed to evaluate cell survival fractions following treatment with RMC-6236, TTFields, and their combination. Cell migration was assessed using scratch assays, revealing that untreated cells exhibited a migration rate of 91.49 ± 0.85%, RMC-6236-treated cells showed 89.48 ± 1.3%, TTFields alone reduced migration to 61.33 ± 16.3%, and combination treatment markedly suppressed migration to 24.6 ± 22.13% . For every case we were compared with 72h cell migration rate with 0 h migration rate. These findings indicate that TTFields alone significantly impair cellular motility, while their combination with RMC-6236 results in a profound anti-migratory effect, substantially inhibiting wound closure. Additionally, western blot analysis was performed to assess changes in KRAS protein expression, a key PDAC biomarker. Overall, our results demonstrate that the combination of TTFields and RMC-6236 significantly inhibits PDAC cell growth, migration, and survival at sub-IC50 concentrations, suggesting a promising alternative therapeutic approach for PDAC management. Citation Format: Ishita Saha, Santanu Bhattacharya, Debabrata Mukhopadhyay, Hani M. Babiker. Synergistic antitumor activity of tumor-treating fields and multiselect-RAS (ON) inhibitor RMC-6236 in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4478.

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 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 Background: TTFields are non-invasive and novel treatment modality that utilizes low intensity, alternating, and intermediate frequency electrical fields to inhibit cancer cell proliferation through a multitude of mechanisms. These include inhibition of mitosis, disruption of cancer cell membranes, metabolomic dysregulation of cancer cells, and immunogenic activation of the tumor microenvironment (TME). TTFields are approved in glioblastoma multiforme, non-small cell lung cancer and malignant mesothelioma. The PANOVA2 trial demonstrated promising efficacy for TTFields when applied together with chemotherapy in patients with PDAC. Mechanisms of TTFields in PDAC need further delineation. We sought to investigate the efficacy of TTFields in vitro and in vivo in PDAC cell lines and mouse models. Preclinical studies were conducted in concert with an going clinical trial (NCT04605913). Experimental Procedures: Utilizing the inovitro apparatus we studied the effect of TTFields (150kHz, intensity 2.9+0.2 V cm RMS, 37ċ, over 72 hrs) in an array of PDAC (Panc1, KPC, Panc2, and AsPC1) and human pancreatic stellate cells (hPSC) cell lines, alone or together with cisplatin (Cis, 0.1μM) or gemcitabine (Gem, 0.01μM), compared to control. Cell viability analysis was conducted in each of the groups. In addition, cell lysates from Panc1 cells from concomitant treatment of TTFields plus Gem were probed for STAT3 and α-SMA to analyze stromal proteins and RAS pathway components (KRAS, P38, P42, and others) were performed via Westen Blot. We also performed RNA sequencing for the different groups to analyze gene expression levels. We further assessed the effect of TTFields (150kHz, intensity 1.8-2 V/cm RMS, over 2 weeks) using the inovivo system with and without chemotherapy (Cis, 0.25 mg/Kg per dose (i.p.) 3 times a week) compared to control in C57BL6 orthotopic murine models (6-7 weeks old C57BL6 mice). Summary of Results: Our analysis revealed statistically significant abrogation of PDAC cancer cell lines for the different groups and hPSC cell viability when compared to control for TTFields monotherapy (p <0.001) and TTFields concomitantly with Gem or Cis (p <0.0001). Western blot analysis demonstrated that TTFields alone and when combined with chemotherapy alters both the stroma and RAS pathways with confirmed downregulation of pSTAT3 and α-SMA and KRAS, P-P38, and P-P42. In vivo analysis revealed significant decrease in tumor volume and weight for the concomitant effect of TTFields + Cis. Conclusion: Our preclinical studies demonstrated the efficacy of TTFields as monotherapy and concomitantly with chemotherapy in PDAC and hPSC cell lines and mouse models. Mechanistic insights from the pre-clinical studies will inform the design of future TTFields studies in PDAC. Citation Format: Hani M. Babiker, Santanu Bhattacharya, Douaa Albelal, Shamit Kumar Dutta, Enfeng wang, Debabrata Mukhopadhyay. umor treating fields (TTFields) plus chemotherapy abrogates pancreatic ductal adenocarcinoma (PDAC) proliferation and alters the tumor microenvironment [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 1834.

Abstract Gold nanoparticles for therapeutic interventions have become a widely popular source due to the ease in surface chemistry available for modification. Several studies undertaken focusses on the size, shape of nanoparticles as well as the signaling mechanisms and yet, knowledge regarding the alteration in membrane dynamics such as nanomechanical properties and cytokines involved during their uptake has not been studies before. To overcome the knowledge deficit, we employed atomic force microscopy and qRT-PCR techniques to acquire nanomechanical attributes and cytokines expressions respectively, in Panc-1 and AsPC-1 cell lines treated with surface modified gold nanoparticles for 1- and 24-hours’ time point. In nanomechanics paradigm, impetus was given on both linear attributes such as membrane stiffness, deformation, and adhesion as well as non-linear attributes such as drained Poisson’s ratio, diffusion coefficient and pore size. On the other hand, qRT-PCR technique yielded alterations in relative mRNA expressions of pro-tumorigenic cytokines such as CCL2, CXCL1, CXCL2, CXCL3, IL8, IL11, IL18 and TGFβ-1. Further, using Pearson’s correlation, we deduced the dependency between nanomechanical properties and cytokines expressions. We evaluated several criteria such as receptor dependent vs independent, PEGylated vs non-PEGylated and 1 hr vs 24hrs, to deduce correlations between cytokines and nanomechanical attributes. In Panc-1 cell line, we observed that PEGylation vs non-PEGylation of gold nanoparticles demonstrated most significant correlations, whereas in AsPC-1 cell line, receptor independent mechanism played a pivotal role in establishing significant correlations. For instance, D_C and P_D were highly correlated with CXCL1, CXCL2 and TGFβ-1 when non-PEGylated gold nanoparticles were up taken by Panc-1 cells. Similarly, D_C and P_D were highly correlated consistently with our cytokine panel except for TGFβ-1. In AsPC-1 cell line, deformation was significantly correlated with CCL2, CXCL2, CXCL3 and IL-8 whereas, D_C and P_D were significantly correlated with CCL2, CXCL1, CXCL3, IL-18 and TGFβ-1 in receptor independent scenario. This study demonstrated that during the uptake of gold nanoparticles, non-linear nanomechanical attributes are detrimental over linear attributes. Moreover, Surface modified gold nanoparticles induce varying levels of alteration in pro-tumorigenic cytokines. Taken together, this study boosts our insights into correlations describing cytokines regulating nanomechanical attributes in endocytosis paradigm. Citation Format: Tanmay Kulkarni, Enfeng Wang, Ramcharan Singh Angom, Debabrata Mukhopadhyay, Santanu Bhattacharya. Insights into correlation between nanomechanical properties and cytokines expression during gold nanoparticle endocytosis [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 496.

Glioblastoma multiforme (GBM) is a primary malignant brain tumor characterized by a high grade of malignancy and an extremely unfavorable prognosis. The current efficacy of established treatments for GBM is insufficient, necessitating the prompt development of novel therapeutic approaches. The progress made in the fundamental scientific understanding of GBM is swiftly translated into more advanced stages of therapeutic studies. Despite extensive efforts to identify new therapeutic approaches, GBM exhibits a high mortality rate. The current efficacy of treatments for GBM patients is insufficient due to factors such as tumor heterogeneity, the blood–brain barrier, glioma stem cells, drug efflux pumps, and DNA damage repair mechanisms. Considering this, pharmacological cocktail therapy has demonstrated a growing efficacy in addressing these challenges. Towards this, various forms of immunotherapy, including the immune checkpoint blockade, chimeric antigen receptor T (CAR T) cell therapy, oncolytic virotherapy, and vaccine therapy have emerged as potential strategies for enhancing the prognosis of GBM. Current investigations are focused on exploring combination therapies to mitigate undesirable side effects and enhance immune responses against tumors. Furthermore, clinical trials are underway to evaluate the efficacy of several strategies to circumvent the blood–brain barrier (BBB) to achieve targeted delivery in patients suffering from recurrent GBM. In this review, we have described the biological and molecular targets for GBM therapy, pharmacologic therapy status, prominent resistance mechanisms, and new treatment approaches. We also discuss these promising therapeutic approaches to assess prospective innovative therapeutic agents and evaluated the present state of preclinical and clinical studies in GBM treatment. Overall, this review attempts to provide comprehensive information on the current status of GBM therapy.