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Metastases are often the direct cause of death from pancreatic ductal adenocarcinoma (PDAC). The role of genomic alterations (GA) in mediating tropism and metastasis formation by PDAC cells is currently unknown. We aimed to identify GAs predisposing colonization of PDAC cells to the liver and decipher mechanisms enabling this process. In order to reveal specific genes, we studied the frequency of GA in 8,880 local and 7,983 metastatic PDAC samples. We observed differential pattern of GA in the local tumor and specific metastatic sites, with liver metastases characterized by deletion of CDKN2A/B (encoding p16/p15, respectively). The role of CDKN2A/B in promoting liver metastasis was evidenced by enhanced tumorigenic phenotype of p15/p16-deleted PDAC cells when exposed to hepatocytes conditioned media. The liver is characterized by high-ammonia low-glutamine environment and transcriptomic assays indicated unique adaptation of PDAC cells to these conditions, including regulation of genes leading to reduced glutaminolysis, like overexpression of GLUL and reduction in GLS2. Furthermore, metabolic assays indicated an increase in glutamate derived from [U

Simple Summary We aimed to study the role of the anti-aging protein klotho and its secreted isoform, sKL, in pancreatic cancer. Three in vivo models, including a novel genetic mouse model and bioinformatics analyses, indicated klotho as a tumor suppressor in pancreatic ductal adenocarcinoma, and unveiled a unique klotho DNA hypermethylation pattern in pancreatic tumors. These results possess significant prognostic value, and further suggest that sKL may serve as a therapeutic agent for pancreatic ductal adenocarcinoma. Abstract Klotho is an anti-aging transmembrane protein, which can be shed and can function as a hormone. Accumulating data indicate that klotho is a tumor suppressor in a wide array of malignancies, and designate the subdomain KL1 as the active region of the protein towards this activity. We aimed to study the role of klotho as a tumor suppressor in pancreatic ductal adenocarcinoma (PDAC). Bioinformatics analyses of The Cancer Genome Atlas (TCGA) datasets revealed a correlation between the survival of PDAC patients, levels of klotho expression, and DNA methylation, and demonstrated a unique hypermethylation pattern of klotho in pancreatic tumors. The in vivo effects of klotho and KL1 were examined using three mouse models. Employing a novel genetic model, combining pancreatic klotho knockdown with a mutation in Kras, the lack of klotho contributed to PDAC generation and decreased mousece survival. In a xenograft model, administration of viral particles carrying sKL, a spliced klotho isoform containing the KL1 domain, inhibited pancreatic tumors. Lastly, treatment with soluble sKL prolonged survival of Pdx1-Cre; KrasG12D/+;Trp53R172H/+ (KPC) mice, a model known to recapitulate human PDAC. In conclusion, this study provides evidence that klotho is a tumor suppressor in PDAC. Furthermore, these data suggest that the levels of klotho expression and DNA methylation could have prognostic value in PDAC patients, and that administration of exogenous sKL may serve as a novel therapeutic strategy to treat PDAC.

Colorectal cancer (CRC) is currently the fourth leading etiology of brain metastasis (BM). Yet, mechanisms supporting the formation of CRC BM are mostly unknown. In order to identify drivers that lead to tropism and adaptation of CRC cells to the brain environment, we analyzed an extensive genomic database, consisting of over 36,000 human CRC primary and metastasis samples. Several genomic alterations specific for BM were noted, among them increased prevalence of insulin receptor substrate 2 (IRS2) amplification, observed in 7.6% of BM, compared to only 2.9% of primary tumors or other metastatic sites (pAccession no GSE203017 and here is the link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE203017

Introduction: Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression, ultimately leading to cell death. TTFields therapy is approved for treatment of adult patients with glioblastoma (GBM) or unresectable pleural mesothelioma. Clinical trials are underway in other solid tumors, including ovarian cancer and non-small cell lung carcinoma (NSCLC). The objective of this study is to identify possible mechanisms involved in reduced sensitivity of cancer cell to TTFields, and explore strategies to circumvent them. Methods: Ovarian cancer A2780, GBM U-87 MG, and NSCLC H1299 cells with reduced sensitivity to TTFields were generated by continuous long-term application of TTFields. Luminex multiplex assay was employed to examine changes in signaling pathways in these cells, and specific pathway markers were validated by Western blot. In vivo validation was performed by immunohistochemistry of ovarian cancer, hepatocellular carcinoma, and NSCLC tumor sections from animals treated with TTFields. Next, TTFields concomitant with alpelisib, an isoform specific PI3K inhibitor, was evaluated both in vitro and in an in-vivo model of ovarian cancer. Results: TTFields inflicted a continuous cytotoxic effect on the different cancer cells albeit sensitivity to treatment was reduced following prolonged duration of application. Luminex analysis revealed activation of the PI3K/AKT signaling pathway in treated cells, and kinetics experiments showed that amplitude of AKT signaling increased over time, with significant increases in phosphorylation levels of AKT and focal adhesion kinase (FAK). AKT phosphorylation was also demonstrated in tumor sections of animals treated with TTFields. Experiments performed with concomitant alpelisib sensitized the cells to TTFields and enhanced cytotoxicity in vitro and treatment efficacy in vivo. Conclusions: The current study demonstrates that the PI3K/AKT signaling pathway is involved in reduced cancer cell sensitivity to TTFields, and that PI3K inhibition can further sensitize cancer cells to TTFields. Citation Format: Anat Klein-Goldberg, Tali Voloshin, Efrat Zemer-Tov, Rom Paz, Lilach Koren, Kerem Wainer-Katsir, Alexandra Volodin, Bella Koltun, Boris Brant, Yiftah Barsheshet, Tal Kan, Cfir David, Tharwat Haj Khalil, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. PI3K inhibition sensitize cancer cells to tumor treating fields (TTFields). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4860.

Background: Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression, ultimately leading to cell death. In addition, application of TTFields to glioblastoma cells has been shown to increase cell membrane permeability. The aim of the current study was to examine whether this mechanism is relevant in other tumor types, and may be leveraged to facilitate cellular internalization of the anticancer agent doxorubicin (DOX). Methods: Lung fibroblast (MRC-5), brain endothelial (HBMVEC), and several cancer cell lines - breast mammary carcinoma (4T1), breast adenocarcinoma (MCF-7), and uterine sarcoma (MES-SA) - were treated with TTFields (100-400 kHz, 1.7 V/cm RMS) using the inovitroTM system. Intracellular accumulation of 7-aminoactinomycin D (7-AAD) was measured to determine membrane permeability, and cell counts were examined to evaluate cytotoxicity. To examine the kinetics and reversibility of the phenomenon, 7-AAD was added at different time points following TTFields application initiation or termination. TTFields were also applied together with DOX to DOX-sensitive and matched DOX-resistant 4T1 cells, followed by flow cytometry examination of DOX accumulation and cytotoxicity measurements. Mice orthotopically inoculated with 4T1 cells were treated with TTFields for 72 h and concomitant DOX injected 24 h before treatment cessation. DOX florescence was measured by flow cytometry in single-cell tumor suspension and by whole tumor in vivo imaging system (IVIS). Results: TTFields increased intracellular accumulation of 7-AAD specifically in the cancer cell lines, with no such effect seen on the non-cancer MRC-5 and HBMVEC cells. In 4T1 cells, maximal TTFields-induced cellular permeability was recorded with 300 kHz TTFields, whereas highest TTFields-induced cytotoxicity was observed at 150 kHz. TTFields application allowed for DOX accumulation to the same extent in both DOX-resistant and DOX-sensitive cells, and sensitized both cell types to DOX cytotoxicity. In vivo, a 2- to 3-fold higher DOX accumulation was seen in tumors isolated from mice treated with TTFields relative to control mice. Conclusions: TTFields elevated cancer cell permeability, resulting in enhanced cell accumulation of DOX and improved drug efficacy, even in DOX resistant cells. TTFields-induced accumulation of DOX was also demonstrated in vivo. Citation Format: Bella Koltun, Tali Voloshin, Tal Kan, Cfir David, Lilach Koren, Yaara Porat, Alexandra Volodin, Noa Kaynan, Anat Klein-Goldberg, Rom Paz, Boris Brant, Yiftah Barsheshet, Efrat Zemer-Tov, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Sensitizing cancer cell to doxorubicin by tumor treating fields (TTFields)-induced, elevated membrane permeability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1738.

BackgroundTumor Treating Fields (TTFields) are low intensity (1–3 V/cm), intermediate frequency (100–500 kHz), alternating electric fields, with demonstrated anti-mitotic effects on cancerous cells. TTFields are clinically approved for treatment of patients with glioblastoma and mesothelioma in the US and Europe. The current study aimed to examine the potential of TTFields to polarize unstimulated M0 macrophages and to regulate the phenotypes of M1 and M2 macrophages.MethodsBone marrow–derived macrophages (BMDMs) were generated from bone marrow cells flushed from the femurs and tibias of 5–8-week-old Balb\C mice. Unstimulated (M0 phenotype) BMDMs and BMDMs stimulated with LPS+IFN-γ (M1 polarization) or IL-4 (M2 polarization) were treated with TTFields (150 kHz) for 24 or 48 hours. Surface expression of the macrophage biomarker F4/80 and the activation markers CD80, major histocompatibility complex class II (MHC II), and inducible nitric oxide synthase (iNOS) were examined by flow cytometry. The heterogeneity of the stimulated macrophages was examined by a multiplexed secretion assay, capturing 13 different proteins: CXCL1 (KC), IL-18, IL-23, IL-12p70, IL6, TNF-α, IL-12p40, free active TGF-β1, CCL22 (MDC), IL-10, IL-6, G-CSF, CCL17 (TARC) and IL-1β.ResultsApplication of TTFields to polarized (M1 or M2) or unpolarized BMDMs significantly increase in the percentage of CD80+/MHC IIhigh cells. M1 polarized BMDMs treated with TTFields also displayed elevation of intracellular iNOS levels. Cell supernatants of M1 and M2 stimulated BMDMs, as well as of unstimulated M0 BMDMs, displayed a pro-inflammatory secretion pattern following delivery of TTFields, with increased levels of CXCL1, IL-18, IL-23, IL-12p70, TNF-α, IL-12p40, CCL22, G-CSF, CCL17 and IL-1β.ConclusionsThis research showed that TTFields polarized unstimulated BMDMs to the M1 phenotype, elevated the pro-inflammatory phenotype of M1 polarized BMDMs, and induced phenotype skewing of M2 polarized BMDMs to the M1 phenotype. These results elucidate a novel immunoregulatory role of TTFields on macrophage polarization. Future studies will aim to focus on the mechanism governing this phenotypic skewing.

Tumor Treating Fields (TTFields) are alternating electric fields, which disrupt cellular process critical for cancer cell survival and tumor progression. TTFields therapy is approved for the treatment of glioblastoma (GBM) and unresectable malignant pleural mesothelioma, and is being tested in clinical studies for the treatment of other solid tumors, including ovarian cancer, non-small cell lung carcinoma (NSCLC), and hepatocellular carcinoma (HCC). The current study aimed to detect potential mechanisms that may reduce cellular sensitivity to TTFields, and target these pathways in order to re-sensitize the cells to TTFields. Cancer cells (Ovarian A2780, GBM U-87 MG, and NSCLC H1299) that display reduced sensitivity to TTFields were generated by continuous long-term TTFields application (7 or 13 days, depending on the cell line). A Luminex multiplex assay revealed activation of the PI3K/AKT/mTOR signaling pathway in these cells, with significant increases in phosphorylation levels of AKT and RPS6. This elevation was also observed by immunohistochemistry in tumor sections from N1S1 HCC tumor-bearing rats treated with TTFields relative to sham. Treatment of cells with PI3K inhibitors re-sensitized them to TTFields and downregulated the phosphorylation of AKT. Concomitant application of TTFields with the PI3K inhibitor alpelisib in mice orthotopically implanted with MOSE-L firefly luciferase (FFL) ovarian cancer cells resulted in enhanced efficacy, as determined by In Vivo Imaging System (IVIS) measurements of tumor volume. Overall, this study demonstrated that the PI3K/AKT/mTOR signaling pathway is involved in reduced cancer cell sensitivity to long-term application of TTFields, and that re-sensitization may be achieved with relevant inhibitors. The results provide a rationale for further examining the potential benefit of TTFields concomitant with PI3K inhibitors.

Background Tumor Treating Fields (TTFields) are alternating electric fields disrupting cellular processes critical for cancer cell replication and tumor progression. TTFields therapy is approved for treatment of newly diagnosed glioblastoma (GBM), recurrent GBM, and unresectable malignant pleural mesothelioma, and is currently under clinical investigation for treating other solid tumors, such as ovarian cancer, non-small cell lung carcinoma (NSCLC), and hepatocellular carcinoma (HCC). The research herein aimed to identify potential treatment escape mechanisms and explore the possibility of targeted inhibition of these pathways for re-sensitizing the cells to TTFields. Material and Methods GBM U-87 MG, ovarian A2780, and NSCLC H1299 cells were treated with TTFields (1.7 V/cm RMS, 200 or 150 kHz) for continuous long-term application (7 or 13 days, specific conditions depending on the cell line). Changes in signaling pathways in these cells relative to cells exposed to short term TTFields application (3 or 7 days, depending on cell line) were examined by Luminex multiplex assay. Specific pathway markers were examined by immunohistochemistry of tumor sections from sham or TTFields-treated rats bearing N1S1 HCC tumors. TTFields were then co-applied with relevant pathway inhibitors, followed by cell count measurements and western blot examinations for specific pathway markers. The concomitant application of TTFields with a selected inhibitor was tested in mice inoculated orthotopically with MOSE-L firefly luciferase (FFL) ovarian cancer cells. Tumor volume was measured at study end by luciferin signal detection using the In Vivo Imaging System (IVIS). Results Cancer cells exposed to long-term application of TTFields displayed decreased sensitivity to TTFields. The PI3K/AKT/mTOR pathway was activated in these cells, with significant increases in AKT and RPS6 phosphorylation levels also observed in HCC tumors from rats treated with TTFields. PI3K inhibitors re-sensitized the cells to TTFields cytotoxicity, with associated down regulation of AKT phosphorylation. Application of TTFields concomitant with the PI3K inhibitor alpelisib resulted in enhanced efficacy in the ovarian cancer mouse model. Conclusions The current research demonstrated that PI3K/AKT/mTOR signaling pathway activation was involved in the response to long-term application of TTFields, with increased phosphorylation of key proteins observed both in vitro and in vivo. Concomitant treatment with PI3K inhibitors and TTFields re-sensitized the cells to TTFields, as it alleviated TTFields-induced AKT activation.

Objective: Tumor Treating Fields (TTFields), encompassing intermediate frequency, alternating electric fields, are an anticancer treatment delivered loco-regionally and non-invasively, through transducer arrays placed on the skin, to the tumor region. TTFields therapy has demonstrated efficacy and safety[for full text, please go to the a.m. URL], 72. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), Joint Meeting mit der Polnischen Gesellschaft für Neurochirurgie

Introduction: Tumor Treating Fields (TTFields) are low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields that are delivered non-invasively to tumors. TTFields therapy is approved in the US and Europe for the treatment of patients with glioblastoma or mesothelioma. In addition to an established anti-mitotic mechanism of action, previous studies have shown that TTFields-induced cell death stimulates anti-tumor immunity and promotes the maturation of dendritic cells. In the current research, the effect of TTFields on the polarization of unstimulated macrophages and the phenotypic regulation of M1 and M2 macrophages was investigated. Methods: Bone marrow-derived macrophages (BMDMs) were generated from the femurs and tibias of 5-8-week-old Balb\C mice. TTFields (150 kHz) were applied for 24 h to unstimulated (M0 phenotype) BMDMs and BMDMs stimulated with LPS+IFN-γ (M1 polarization) or IL-4 (M2 polarization). Flow cytometry was used to identify surface expression of the macrophage biomarker F4/80 and activation markers CD80, major histocompatibility complex class II (MHC II), inducible nitric oxide synthase (iNOS), CD206, and ARG-1. Multiplexed secretion assays were conducted to quantify CXCL1 (KC), IL-18, IL-23, IL-12p70, IL6, TNF-α, IL-12p40, free active TGF-β1, CCL22 (MDC), IL-10, IL-6, G-CSF, CCL17 (TARC), and IL-1β as to examine the heterogeneity of the stimulated macrophages. Results: The percentage of cells expressing the pro-inflammatory M1 markers CD80+ and MHC IIhigh was significantly increased following application of TTFields to polarized (M1 or M2) or unpolarized BMDMs, while expression of the M2 markers CD206 and ARG-1 was significantly decreased. A pro-inflammatory secretion pattern, with increased levels of CXCL1, IL-18, IL-23, IL-12p70, TNF-α, IL-12p40, CCL22, G-CSF, CCL17 and IL-1β was observed in cell supernatants of M1 and M2 stimulated BMDMs, and unstimulated M0 BMDMs, following delivery of TTFields. Taken together, TTFields polarized unstimulated BMDMs to the M1 phenotype, and induced phenotype skewing of M2 polarized BMDMs to the M1 phenotype. Conclusions: TTFields therapy displays a novel immunoregulatory role in macrophage polarization and promotes a pro-inflammatory phenotype. Future investigations will focus on defining the underlying mechanism of this phenotypic skewing. Citation Format: Yiftah Barsheshet, Boris Brant, Tali Voloshin, Alexandra Volodin, Lilach Koren, Bella Koltun, Anat Klein-Goldberg, Efrat Zemer-Tov, Tal Kan, Rom Paz, Moshe Giladi, Uri Weinberg, Yoram Palti. Tumor Treating Fields (TTFields) promote a pro-inflammatory phenotype in macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1305.

INTRODUCTION: Tumor Treating Fields (TTFields) are alternating electric fields at intermediate frequencies that exert anti-mitotic effects on cancerous cells. TTFields therapy is approved in several territories for treatment of glioblastoma (GBM) and unresectable malignant pleural mesothelioma. Recently, membrane permeability of GBM cells has been found to be increased in response to TTFields application. The current study aimed to further explore this effect, testing the potential of TTFields to facilitate cellular accumulation of the anticancer agent doxorubicin (DOX) in breast carcinoma cells. METHODS: 4T1 breast mammary carcinoma cells were treated with TTFields (1.7 V/cm RMS) for 72 h across a frequency range (50-500 kHz). Cytotoxicity was examined by cell counts, and permeability determined by 7-aminoactinomycin D (7-AAD) intracellular accumulation, both measured by flow cytometry. Next, TTFields at the frequency inducing highest permeability was applied to chemotherapy-sensitive and matched chemotherapy-resistant cells. Intracellular accumulation of DOX and drug-induced cytotoxicity were measured by flow cytometry. In vivo validation was performed by 72 h delivery of TTFields at the frequency of maximal permeability to mice orthotopically inoculated with 4T1 cells and injected with DOX 24 h before treatment cessation. DOX florescence was measured using in vivo imaging system (IVIS) for whole tumor assessment and flow cytometry for detection at the single-cell level. RESULTS: While highest TTFields-induced cytotoxicity was observed at 150 kHz, 7-AAD intracellular accumulation was maximal at 300 kHz. When TTFields were delivered concomitant with DOX, the drug accumulated to the same extent in chemotherapy-resistant cells as in chemotherapy-sensitive cells. Application of TTFields also sensitized both cell types to DOX, with cytotoxicity observed at low drug concentrations. Furthermore, 2- to 3-fold higher DOX accumulation was seen in tumors isolated from mice treated with TTFields relative to control. CONCLUSIONS: Permeability of 4T1 breast cancer cells was elevated by TTFields, allowing enhanced intracellular accumulation of DOX and improving drug efficacy, even in chemotherapy-resistant cells. Increased cellular accumulation of DOX was also demonstrated in vivo. Citation Format: Bella Koltun, Tali Voloshin, Tal Kan, Lilach Koren, Yaara Porat, Alexandra Volodin, Noa Kaynan, Anat Klein-Goldberg, Rom Paz, Boris Brant, Yiftah Barsheshet, Efrat Zemer-Tov, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Application of Tumor Treating Fields (TTFields) to cancer cells enhances their membrane permeability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1801.

INTRODUCTION Tumor Treating Fields (TTFields), encompassing alternating electric fields within the intermediate frequency range, is an anticancer treatment delivered to the tumor region through transducer arrays placed non-invasively on the skin. This novel loco-regional treatment has demonstrated efficacy and safety and is FDA-approved in patients with glioblastoma (GBM) and malignant pleural mesothelioma. TTFields are currently being investigated in other solid tumors in ongoing trials. Recently, TTFields were reported to alter the cellular membrane structure of GBM cells, rendering them more permeable. The objective of this study was to characterize TTFields-induced cellular permeability in cancerous cell lines. METHODS TTFields were applied to uterine sarcoma, glioblastoma, and breast adenocarcinoma cell lines across a range of frequencies (50–500 kHz). Cellular permeability was assessed by quantifying the percentages of cells with accumulated 7-aminoactinomycin D (7-AAD) using flow cytometry and cytotoxicity was assessed based on cell counts. Kinetics were determined using different 7-AAD exposure times relative to TTFields treatment end. Changes in intracellular accumulation of anthracycline chemotherapeutics were evaluated in chemotherapy-sensitive and chemotherapy-resistant cells. RESULTS TTFields induced cellular permeability to 7-AAD in all 4 cancer cell lines tested. The optimal frequency for TTFields-induced cellular permeability was different from the optimal cytotoxic frequency. Kinetics measurements demonstrated that TTFields-induced permeability is transient and is effective only during application of TTFields. In combination experiments, TTFields improved intracellular accumulation of chemotherapeutic agents. Furthermore, combining chemotherapy with TTFields treatment facilitated accumulation of chemotherapeutics in chemotherapy-resistant cells to levels comparable with accumulation in chemotherapy-sensitive cancer cells. CONCLUSIONS This study demonstrates that TTFields can transiently increase cancer cell permeability in vitro with an optimal frequency that is variable from the frequency that is used to induce cancer cell cytotoxicity. Moreover, this effect is reversible and cellular permeability is restored to a normal state upon TTFields treatment cessation.

INTRODUCTION Tumor Treating Fields (TTFields) therapy is an approved anti-cancer treatment modality applied non-invasively and loco-regionally to the tumor region. TTFields have been demonstrated to extend life, however, most patients do eventually progress. The current study aimed to identify potential molecular mechanisms involved in reduced cellular sensitivity to TTFields. METHODS Cancer cells that exhibit reduced sensitivity to TTFields were generated by continuous long duration application of TTFields (7 or 13 days, depending on the cell line). Changes in cellular signaling pathways were evaluated in ovarian A2780 and glioblastoma U-87 MG cancer cells exposed to long-term relative to short-term (3 or 7 days, depending on the cell line) treatment with TTFields using Luminex multiplex assay followed by Western blot validation. The relevance of the affected pathways was confirmed through evaluation of the response to long-term application of TTFields in combination with pharmacological pathway inhibitors by measuring cell counts, apoptosis, and clonogenicity. Relevant pathway markers in tumor sections from tumor-bearing rats treated with TTFields were examined using immunohistochemistry. RESULTS Continuous long-term application of TTFields reduced cellular sensitivity to TTFields and was accompanied by increased levels of phosphorylated AKT, mTOR and additional proteins from the PI3K/AKT/mTOR signaling pathway. Increased phosphorylation of AKT was also evident in tumor sections from rats treated with TTFields. Concomitant use of TTFields with PI3K inhibitors or PI3K/mTOR dual inhibitors sensitized A2780 cells to long-term TTFields application. CONCLUSION Our study demonstrates that decreased cancer cell sensitivity to long-term TTFields application is mediated by activation of the PI3K/AKT/mTOR signaling pathway and provides a rationale for further examining the potential benefit of combining TTFields with PI3K or PI3K/mTOR dual inhibitors.

Purpose/Objective(s) Tumor Treating Fields (TTFields) therapy is a clinically applied loco-regional, non-invasive, anti-mitotic treatment modality delivering to the tumor low intensity (1-3 V/cm) alternating electric fields (100-300 kHz). While TTFields therapy has demonstrated proven efficacy in management of glioblastoma and malignant pleural mesothelioma, tumor recurrence still occurs in some patients. In order to anticipate potential molecular mechanisms of resistance to TTFields, we developed TTFields-resistant cancer cell lines, and analyzed the changes in their signaling pathways. We also examined the in vitro efficacy of TTFields when combined with agents targeting the molecular candidates that possibly confer resistance to TTFields. Materials/Methods To evaluate the mechanisms of cellular resistance to TTFields, we developed resistant ovarian (A2780), glioma (U-87-MG), and non-small cell lung carcinoma (H1299) cancer cell lines through continued long-term application of TTFields (one or two weeks, depending on the cell line), without treatment breaks. We then assessed the concomitant changes in cellular signaling pathways using an immunoassay followed by Western blot analysis. Finally, we characterized the mechanism of resistance by examining the in vitro efficacy of continued long-term application of TTFields in combination with pharmacological inhibitors including a PI3K/mTOR dual inhibitor (BGT226), and Pan- and isoform-specific inhibitors of PI3K (alpelisib, pictilisib, and BKM120), using cell count, apoptosis, and colony formation assays. Results Continued long-term application of TTFields resulted in reduced sensitivity to TTFields in all tested cell lines. Immunoassay and Western blot analysis showed significant activation of the PI3K/AKT/mTOR signaling pathway in response to TTFields treatment. The combination therapy of TTFields with either alpelisib, pictilisib, BKM120 or BGT226, inhibited AKT S473 phosphorylation and sensitized cancer cells to “long-term” TTFields application. Conclusion Our study demonstrates that the PI3K/ AKT/ mTOR signaling pathway confers resistance to TTFields. This provides a rationale for the combined targeting of PI3K in patients treated with TTFields.