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Tumor Treating Fields (TTFields) are electric fields that exert physical forces to disrupt cellular processes critical for cancer cell viability and tumor progression. TTFields induce anti-mitotic effects through the disruption of the mitotic spindle and abnormal chromosome segregation, which trigger several forms of cell death, including immunogenic cell death (ICD). The efficacy of TTFields concomitant with anti-programmed death-1 (anti-PD-1) treatment was previously shown in vivo and is currently under clinical investigation. Here, the potential of TTFields concomitant with anti- PD-1/anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) or anti-programmed death-ligand 1 (anti-PD-L1) immune checkpoint inhibitors (ICI) to improve therapeutic efficacy was examined in lung tumor-bearing mice. Increased circulating levels of high mobility group box 1 protein (HMGB1) and elevated intratumoral levels of phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α) were found in the TTFields-treated mice, indicative of ICD induction. The concomitant application of TTFields and ICI led to a significant decrease in tumor volume as compared to all other groups. In addition, significant increases in the number of tumor-infiltrating immune cells, specifically cytotoxic T-cells, were observed in the TTFields plus anti-PD-1/anti-CTLA-4 or anti-PD-L1 groups. Correspondingly, cytotoxic T-cells isolated from these tumors showed higher levels of IFN-γ production. Collectively, these results suggest that TTFields have an immunoactivating role that may be leveraged for concomitant treatment with ICI to achieve better tumor control by enhancing antitumor immunity.

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.

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.

Throughout the female menstrual cycle, physiological changes occur that affect the biodistribution of nanoparticles within the reproductive system. We demonstrate a 2-fold increase in nanoparticle accumulation in murine ovaries and uterus during ovulation, compared to the non-ovulatory stage, following intravenous administration. This biodistribution pattern had positive or negative effects when drug loaded nanoparticles, sized 100-nm or smaller, were used to treat different cancers. For example, treating ovarian cancer with nanomedicines during mouse ovulation resulted in higher drug accumulation in the ovaries, improving therapeutic efficacy. Conversely, treating breast cancer during ovulation, led to reduced therapeutic efficacy, due to enhanced nanoparticle accumulation in the reproductive system rather than at the tumor site. Moreover, chemotherapeutic nanoparticles administered during ovulation increased ovarian toxicity and decreased fertility compared to the free drug. The menstrual cycle should be accounted for when designing and implementing nanomedicines for females.

Acute Respiratory Distress Syndrome (ARDS), associated with Covid-19 infections, is characterized by diffuse lung damage, inflammation and alveolar collapse that impairs gas exchange, leading to hypoxemia and patient' mortality rates above 40%. Here, we describe the development and assessment of 100-nm liposomes that are tailored for pulmonary delivery for treating ARDS, as a model for lung diseases. The liposomal lipid composition (primarily DPPC) was optimized to mimic the lung surfactant composition, and the drug loading process of both methylprednisolone (MPS), a steroid, and N-acetyl cysteine (NAC), a mucolytic agent, reached an encapsulation efficiency of 98% and 92%, respectively. In vitro, treating lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages with the liposomes decreased TNFα and nitric oxide (NO) secretion, while NAC increased the penetration of nanoparticles through the mucus. In vivo, we used LPS-induced lung inflammation model to assess the accumulation and therapeutic efficacy of the liposomes in C57BL/6 mice, either by intravenous (IV), endotracheal (ET) or IV plus ET nanoparticles administrations. Using both administration methods, liposomes exhibited an increased accumulation profile in the inflamed lungs over 48 h. Interestingly, while IV-administrated liposomes distributed widely throughout the lung, ET liposomes were present in lungs parenchyma but were not detected at some distal regions of the lungs, possibly due to imperfect airflow regimes. Twenty hours after the different treatments, lungs were assessed for markers of inflammation. We found that the nanoparticle treatment had a superior therapeutic effect compared to free drugs in treating ARDS, reducing inflammation and TNFα, IL-6 and IL-1β cytokine secretion in bronchoalveolar lavage (BAL), and that the combined treatment, delivering nanoparticles IV and ET simultaneously, had the best outcome of all treatments. Interestingly, also the DPPC lipid component alone played a therapeutic role in reducing inflammatory markers in the lungs. Collectively, we show that therapeutic nanoparticles accumulate in inflamed lungs holding potential for treating lung disorders. SIGNIFICANCE: In this study we compare intravenous versus intratracheal delivery of nanoparticles for treating lung disorders, specifically, acute respiratory distress syndrome (ARDS). By co-loading two medications into lipid nanoparticles, we were able to reduce both inflammation and mucus secretion in the inflamed lungs. Both modes of delivery resulted in high nanoparticle accumulation in the lungs, intravenously administered nanoparticles reached lung endothelial while endotracheal delivery reached lung epithelial. Combining both delivery approaches simultaneously provided the best ARDS treatment outcome.

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.

This chapter explores tumoral barriers for the successful implementation of nanomedicines. Nanotechnologies hold great clinical promise to improve the efficacy of diagnostic and therapeutic anticancer agents. In cancer therapy, many nanotechnologies target solid tumors through the leaky tumor vasculature and/or using surface ligands with molecular specificity to biomarkers in the tumor tissue. Traditional approaches of loading nanoparticles solely with drugs aimed at killing the cancer cells are being replaced by multiple agents for targeting the various components of the tumor microenvironment. The demand for nutrients and oxygen by a developing tumor is accommodated by angiogenesis— the formation of new blood vessels. The extracellular matrix (ECM) is a woven net between tissues and organs composed of water, minerals, proteins, proteoglycans and glycoproteins such as collagen, fibronectin, hyaluronic acid, and many others. The dynamic nature of the ECM is particularly apparent during development, wound healing, cancer and other diseases.

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) are alternating electric fields, delivered noninvasively to the tumor site. TTFields therapy is currently approved for treatment of patients with newly diagnosed glioblastoma (GBM), recurrent GBM, or unresectable malignant pleural mesothelioma. Investigations are ongoing in additional tumor types, including non-small cell lung carcinoma (NSCLC), ovarian cancer, and hepatocellular carcinoma (HCC). Although TTFields have been demonstrated to extend life, most patients will eventually progress. The current research aimed to identify molecular mechanisms involved in reduced cancer cellular sensitivity to TTFields, and the potential of targeting these pathways to re-sensitize the cells to TTFields. Methods: Continuous long-term application of TTFields (7 or 13 days, depending on the cell line) generated cancer cells with reduced sensitivity to TTFields. Luminex multiplex assay was used to detect changes in signaling pathways in ovarian A2780 and GBM U-87 MG cells, and relevant pathway markers were validated by Western blot. Further validation was performed by immunohistochemistry of tumor sections from N1S1 HCC tumor-bearing rats treated with sham or TTFields. The significance of the identified pathways in reducing cancer cell sensitivity to TTFields was evaluated through in vitro combination treatment with PI3K inhibitors, followed by cell count measurements. Finally, the concomitant application of TTFields and the PI3K inhibitor Alpelisib was evaluated in mice orthotopically implanted with MOSE-L firefly luciferase (FFL) ovarian cancer cells. Tumor volume was measured using the In Vivo Imaging System (IVIS) to detect the luciferin signal, before and after treatment. Results: Cancer cell sensitivity to TTFields was reduced following continuous long-term application of TTFields. This was accompanied by activation of the PI3K/AKT/mTOR signaling pathway, with significant increases in the levels of phosphorylated AKT and RPS6 observed in cell cultures and in rat tumor sections following application of TTFields. Application of PI3K inhibitors re-sensitized the cells to TTFields in vitro. In vivo, concomitant application of TTFields with Alpelisib resulted in enhanced efficacy. Conclusions: The current study demonstrated that reduced cancer cell sensitivity to long-term application of TTFields is mediated by activation of the PI3K/AKT/mTOR signaling pathway. Furthermore, PI3K inhibitors were shown to re-sensitize the cells to TTFields, providing a rationale for further examining the potential benefit of TTFields concomitant with PI3K inhibitors. 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, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Inhibition of PI3K sensitized cancer cells to Tumor Treating Fields (TTFields) [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 2659.

Throughout the female menstrual cycle, physiological changes occur that affect the biodistribution of nanoparticles within the reproductive system. This can have positive or negative effects. We demonstrate a 2-fold increase in nanoparticle accumulation in the ovaries during female mouse ovulation compared to the non-ovulatory stage following intravenous administration. Accumulation in the reproductive system is favored by nanoparticles smaller than 100 nm. Chemotherapeutic nanoparticles administered during ovulation increased ovarian toxicity and decreased short-term and long-term fertility when compared to the free drug. Breast cancer treated with nanomedicines during ovulation results in higher drug accumulation in the reproductive system rather than at the site of the tumor, reducing treatment efficacy. Conversely, ovarian cancer treatment was improved by enhanced nanoparticle accumulation in the ovaries during ovulation. Our findings suggest that the menstrual cycle should be considered when designing and implementing nanotherapeutics for females.

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.

Introduction: Tumor Treating Fields (TTFields) therapy is a clinically applied anti-neoplastic treatment modality delivered via noninvasive application of low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields. In this study we investigated the potential of TTFields for regulating macrophage phenotype skewing, with or without lipopolysaccharide (LPS) and interferon-γ (IFN-γ) or IL-4 activation. Methods: Bone marrow cells were flushed from the femurs and tibias of 5-8 week old Balb\C mice to generate bone marrow-derived macrophages (BMDMs). To study individual macrophage responses to TTFields, we profiled macrophages that were stimulated with LPS+IFN-γ (M1 polarization) or IL-4 (M2 polarization), followed by treatment with TTFields (150 kHz) for 24 hours. Flow cytometry was used to assess surface expression of F4/80 (a macrophage biomarker) and the activation markers that included major histocompatibility complex class II (MHC II), CD80, and inducible nitric oxide synthase (iNOS). A multiplexed secretion assay was used to measure the heterogeneity of the stimulated macrophages. We captured the secretion of 13 different proteins including 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. Results: TTFields treatment polarized IL-4 stimulated M2 macrophages to the M1 phenotype, as indicated by a significant increase in the percentage of CD80+MHC IIhigh cells. Treatment with TTFields of BMDMs that were either stimulated with IFN-γ and LPS (M1 phenotype) or had remained unstimulated increased the percentage of CD80+MHC IIhigh cells to a lesser degree. Moreover, treatment with TTFields significantly increased intracellular iNOS levels in M1 polarized BMDMs. In unstimulated BMDMs, application of TTFields resulted in significant increases in secretion of the pro-inflammatory cytokines CXCL1, IL-18, IL-23, IL-12p70, TNF-α, IL-12p40, CCL22, G-CSF, CCL17 and IL-1β. Also, a similar pattern was observed in cell supernatants of M1 and M2 stimulated BMDMs after TTFields application. Conclusions: These results elucidate a novel immunoregulatory role of TTFields on macrophage polarization. Future studies will aim to focus on the mechanism governing phenotypic skewing of macrophages treated with TTFields and to explore the effect of TTFields on macrophage in the tumor microenvironment. Citation Format: Boris Brant, Tali Voloshin, Alexandra Volodin, Lilach Koren, Anat Klein-Goldberg, Efrat Zemer-Tov, Rom Paz, Moshe Giladi, Uri Weinberg, Yoram Palti. A novel immunoregulatory role of tumor treating fields (TTFields) on macrophage polarization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1692.

Introduction Tumor Treating Fields (TTFields) therapy is a clinically applied anti-neoplastic treatment modality, approved for the treatment of glioblastoma and malignant pleural mesothelioma. TTFields are low intensity (1-3 V/cm) alternating electric fields within the intermediate frequency range (100-300 kHz), that are applied loco-regionally and non-invasively to the tumor site. Although TTFields therapy has demonstrated improved progression free survival and overall survival, tumor recurrence still occurs in some patients. To evaluate the cellular mechanisms of treatment resistance to TTFields, we analyzed changes in signaling pathway mediators in treated human glioma (U-87 MG) and ovarian (A2780) cancer cell lines. Also, we examined the in vitro efficacy of TTFields in combination with therapies that target molecular candidates that may confer resistance to TTFields. Methods To establish TTFields resistant glioma and ovarian cancer cell lines in vitro, 2 approaches were tested: 1) cyclic repetitions, where TTFields treatment was repeatedly applied for 72 hours with 24 hours breaks between treatment cycles; and 2) continuous, long-duration application of TTFields (1 or 2 weeks, depending on the cancer cell line type) without treatment breaks. Luminex assay followed by Western blot analysis were used to study changes in signaling pathways following TTFields treatment. Finally, we examined the in vitro efficacy of continuous, long-duration application of TTFields in combination with BGT226 (a phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dual inhibitor). Results Application of TTFields using cyclic repetition significantly decreased proliferation of human glioma and ovarian cancer cells, whereas cells treated using continuous, long-duration application of TTFields showed reduced sensitivity to TTFields. Luminex analysis clearly showed activation of the PI3K/mTOR/AKT signaling pathway in response to TTFields treatment. To clarify the importance of this signaling pathway on regulating treatment resistance to TTFields, we examined the effect of TTFields, alone and in combination, with a PI3K/mTOR inhibitor (BGT226) on cancer cell proliferation, apoptosis, and activation of AKT. While long-duration application of TTFields led to Akt activation, TTFields in combination with BGT226 resulted in reduction of p-AKT S473, with a corresponding blockade of proliferation, induction of apoptosis, and decrease in the clonogenic potential. Conclusions We propose here a new mechanism of resistance to prolonged TTFields treatment mediated by the PI3K/mTOR/AKT signaling pathway in glioma and ovarian cancer cells. We demonstrate that combination therapy of TTFields and a targeted PI3K/mTOR dual inhibitor (BGT226) inhibited AKT S473 phosphorylation and sensitized cancer cells to “long-duration” TTFields application. Citation Format: Anat Klein-Goldberg, Tali Voloshin, Efrat Zemer-Tov, Rom Paz, Lilach Koren, Alexandra Volodin, Boris Brant, Moshe Giladi, Uri Weinberg, Yoram Palti. Targeting Akt signaling pathway potentiates the antitumor effect of Tumor Treating Fields (TTFields) in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1382.

Rational Pressure overload induces adaptive and maladaptive cardiac remodeling processes in the heart. Part of the maladaptive process is the cross-talk between cardiomyocytes and macrophages which is dependent on the function of the Activating Transcription Factor 3, ATF3. Yet, the molecular mechanism involved in cardiomyocytes-macrophages communication leading to macrophages recruitment to the heart and cardiac maladaptive remodeling is currently unknown. Methods and results Isolated peritoneal macrophages from either wild type or ATF3-KO mice were cultured in serum free medium to collect conditioned medium (CM). CM was used to probe an antibody cytokine/chemokine array. The interferon γ induced protein 10kDa, CXCL10, was found to be enriched in wild type macrophages CM. Wild type cardiomyocytes treated with CXCL10 in vitro, resulted in significant increase in cell volume as compared to ATF3-KO cardiomyocytes. In vivo, pressure overload was induced by phenylephrine (PE) infusion using micro-osmotic pumps. Consistently, CXCL11 (CXCL10 competitive agonist) and CXCL10 receptor antagonist (AMG487) attenuated PE-dependent maladaptive cardiac remodeling. Significantly, we show that the expression of the CXCL10 receptor, CXCR3, is suppressed in cardiomyocytes and macrophages derived from ATF3-KO mice. CXCR3 is positively regulated by ATF3 through an ATF3 transcription response element found in its proximal promoter. Finally, mice lacking CXCR3 display a significant reduction of cardiac remodeling processes following PE infusion. Conclusions Chronic PE infusion results in a unique cardiomyocytes-macrophages cross-talk that is mediated by IFNγ. Subsequently, macrophages that are recruited to the heart secrete CXCL10 resulting in maladaptive cardiac remodeling mediated by the CXCR3 receptor. ATF3-KO mice escape from PE-dependent maladaptive cardiac remodeling by suppressing the IFNγ-CXCL10-CXCR3 axis at multiple levels.

Overexpressed extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDAC) limits drug penetration into the tumor and is associated with poor prognosis. Here, we demonstrate that a pretreatment based on a proteolytic-enzyme nanoparticle system disassembles the dense PDAC collagen stroma and increases drug penetration into the pancreatic tumor. More specifically, the collagozome, a 100 nm liposome encapsulating collagenase, was rationally designed to protect the collagenase from premature deactivation and prolonged its release rate at the target site. Collagen is the main component of the PDAC stroma, reaching 12.8 ± 2.3% vol in diseased mice pancreases, compared to 1.4 ± 0.4% in healthy mice. Upon intravenous injection of the collagozome, ∼1% of the injected dose reached the pancreas over 8 h, reducing the level of fibrotic tissue to 5.6 ± 0.8%. The collagozome pretreatment allowed increased drug penetration into the pancreas and improved PDAC treatment. PDAC tumors, pretreated with the collagozome followed by paclitaxel micelles, were 87% smaller than tumors pretreated with empty liposomes followed by paclitaxel micelles. Interestingly, degrading the ECM did not increase the number of circulating tumor cells or metastasis. This strategy holds promise for degrading the extracellular stroma in other diseases as well, such as liver fibrosis, enhancing tissue permeability before drug administration.

Aims Obesity and type 2 diabetes (T2D) trigger a harmful stress-induced cardiac remodeling process known as cardiomyopathy. These diseases represent a serious and widespread health problem in the Western world; however the underlying molecular basis is not clear. ATF3 is an ‘immediate early’ gene whose expression is highly and transiently induced in response to multiple stressors such as metabolic, oxidative, endoplasmic reticulum and inflammation, stressors that are involved in T2D cardiomyopathy. The role of ATF3 in diabetic cardiomyopathy is currently unknown. Our research has aimed to study the effect of ATF3 expression on cardiomyocytes, heart function and glucose homeostasis in an obesity-induced T2D mouse model. Methods and results We used wild type mice (WT) as well as mutant mice with a cardiac-specific ATF3 deficiency (ATF3-cKO). Mice were fed a high-fat diet (HFD) for 15 weeks. HFD induced high ATF3 expression in cardiomyocytes. Mice were examined for cardiac remodeling processes and the diabetic state was assessed. HFD-fed ATF3-cKO mice exhibited severe cardiac fibrosis, higher levels of heart hypertrophic markers, increased inflammation and worse cardiac function, as compared to WT mice. Interestingly, HFD-fed ATF3-cKO mice display increased hyperglycemia and reduced glucose tolerance, despite higher blood insulin levels, as compared to HFD-fed WT mice. Elevated levels of the cardiac inflammatory cytokines IL-6 and TNFα leading to impaired insulin signalling may partially explain the peripheral glucose intolerance. Conclusions Cardiac ATF3 has a protective role in dampening the HFD-induced cardiac remodeling processes. ATF3 exerts both local and systemic effects related to T2D-induced cardiomyopathy. This study provides a strong relationship between heart remodeling processes and blood glucose homeostasis.

The ability of glioma cells to invade adjacent brain tissue remains a major obstacle to therapeutic disease management. Therefore, the development of novel treatment modalities that disrupt glioma cell motility could facilitate greater disease control. 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 and malignant pleural mesothelioma. TTFields are currently being investigated in other solid tumors in ongoing trials, including the phase 3 METIS trial (brain metastases from NSCLC; NCT02831959). Although established as an anti-mitotic treatment, the anti-metastatic potential of TTFields and its effects on cytoskeleton rapid dynamics during cellular motility warrant further investigation. Previous studies have demonstrated that TTFields inhibits metastatic properties of cancer cells. Identification of a unifying mechanism connecting the versatile TTFields-induced molecular responses is required to optimize the therapeutic potential of TTFields. In this study, confocal microscopy, computational tools, and biochemical analyses were utilized to show that TTFields disrupt glioma cellular polarity by interfering with microtubule assembly and directionality. Under TTFields application, changes in microtubule organization resulted in activation of GEF-H1, which led to an increase in active RhoA levels and consequent focal adhesion formation with actin cytoskeleton architectural changes. Furthermore, the optimal TTFields frequency for inhibition of invasion in glioma cells was 300 kHz, which differed from the optimal anti-mitotic frequency leading to glioma cell death of 200 kHz. The inhibitory effect of TTFields on migration was observed at fields intensities of 0.6 V/cm RMS (below the threshold of 1 V/cm RMS previously reported for cytotoxic effects). Together, these data identify discrete TTFields effects that disrupt processes crucial for glioma cell motility.

Rationale Pressure overload induces adaptive remodeling processes in the heart. However, when pressure overload persists, adaptive changes turn into maladaptive alterations leading to cardiac hypertrophy and heart failure. ATF3 is a stress inducible transcription factor that is transiently expressed following neuroendocrine stimulation. However, its role in chronic pressure overload dependent cardiac hypertrophy is currently unknown. Objective The objective of the study was to study the role of ATF3 in chronic pressure overload dependent cardiac remodeling processes. Methods and results Pressure overload was induced by phenylephrine (PE) mini-osmotic pumps in various mice models of whole body, cardiac specific, bone marrow (BM) specific and macrophage specific ATF3 ablations. We show that ATF3-KO mice exhibit a significantly reduced expression of cardiac remodeling markers following chronic pressure overload. Consistently, the lack of ATF3 specifically in either cardiomyocytes or BM derived cells blunts the hypertrophic response to PE infusion. A unique cross-talk between cardiomyocytes and macrophages was identified. Cardiomyocytes induce an ATF3 dependent induction of an inflammatory response leading to macrophage recruitment to the heart. Adoptive transfer of wild type macrophages, but not ATF3-KO derived macrophages, into wild type mice potentiates maladaptive response to PE infusion. Conclusions Collectively, this study places ATF3 as a key regulator in promoting pressure overload induced cardiac hypertrophy through a cross-talk between cardiomyocytes and macrophages. Inhibiting this cross-talk may serve as a useful approach to blunt maladaptive remodeling processes in the heart.

c-Jun dimerization protein (JDP2) and Activating Transcription Factor 3 (ATF3) are closely related basic leucine zipper proteins. Transgenic mice with cardiac expression of either JDP2 or ATF3 showed maladaptive remodeling and cardiac dysfunction. Surprisingly, JDP2 knockout (KO) did not protect the heart following transverse aortic constriction (TAC). Instead, the JDP2 KO mice performed worse than their wild type (WT) counterparts. To test whether the maladaptive cardiac remodeling observed in the JDP2 KO mice is due to ATF3, ATF3 was removed in the context of JDP2 deficiency, referred as double KO mice (dKO). Mice were challenged by TAC, and followed by detailed physiological, pathological and molecular analyses. dKO mice displayed no apparent differences from WT mice under unstressed condition, except a moderate better performance in dKO male mice. Importantly, following TAC the dKO hearts showed low fibrosis levels, reduced inflammatory and hypertrophic gene expression and a significantly preserved cardiac function as compared with their WT counterparts in both genders. Consistent with these data, removing ATF3 resumed p38 activation in the JDP2 KO mice which correlates with the beneficial cardiac function. Collectively, mice with JDP2 and ATF3 double deficiency had reduced maladaptive cardiac remodeling and lower hypertrophy following TAC. As such, the worsening of the cardiac outcome found in the JDP2 KO mice is due to the elevated ATF3 expression. Simultaneous suppression of both ATF3 and JDP2 activity is highly beneficial for cardiac function in health and disease.

Summary The NF-κB transcription factor controls diverse biological processes. According to the classical model, NF-κB is retained in the cytoplasm of resting cells via binding to inhibitory, IκB proteins and translocates into the nucleus upon their ligand-induced degradation. Here we reveal that Sef, a known tumor suppressor and inhibitor of growth factor signaling, is a spatial regulator of NF-κB. Sef expression is regulated by the proinflammatory cytokines tumor necrosis factor and interleukin-1, and Sef specifically inhibits "classical" NF-κB (p50:p65) activation by these ligands. Like IκBs, Sef sequesters NF-κB in the cytoplasm of resting cells. However, contrary to IκBs, Sef continues to constrain NF-κB nuclear entry upon ligand stimulation. Accordingly, endogenous Sef knockdown markedly enhances stimulus-induced NF-κB nuclear translocation and consequent activity. This study establishes Sef as a feedback antagonist of proinflammatory cytokines and highlights its potential to regulate the crosstalk between proinflammatory cytokine receptors and receptor tyrosine kinases.

Cardiac hypertrophy is an adaptive response to various mechanophysical and pathophysiological stresses. However, when chronic stress is sustained, the beneficial response turns into a maladaptive process that eventually leads to heart failure. Although major advances in the treatment of patients have reduced mortality, there is a dire need for novel treatments for cardiac hypertrophy. Accordingly, considerable efforts are being directed towards developing mice models and understanding the processes that lead to cardiac hypertrophy. A case in point is ATF3, an immediate early transcription factor whose expression is induced in various cardiac stress models but has been reported to have conflicting functional significance in hypertrophy. To address this issue, we generated a transgenic mouse line with tetracycline-regulated ATF3 cardiac expression. These mice allowed us to study the consequence of ATF3 expression in the embryo or during the adult period, thus distinguishing the effect of ATF3 on development versus pathogenesis of cardiac dysfunction. Importantly, ATF3 expression in adult mice resulted in rapid ventricles hypertrophy, heart dysfunction, and fibrosis. When combined with a phenylephrine-infusion pressure overload model, the ATF3 expressing mice displayed a severe outcome and heart dysfunction. In a complementary approach, ATF3 KO mice displayed a lower level of heart hypertrophy in the same pressure overload model. In summary, ectopic expression of ATF3 is sufficient to promote cardiac hypertrophy and exacerbates the deleterious effect of chronic pressure overload; conversely, ATF3 deletion protects the heart. Therefore, ATF3 may serve as an important drug target to reduce the detrimental consequences of heart hypertrophy.

JDP2 (c-Jun dimerization protein 2) is a member of the basic leucine zipper family of transcription factors that is ubiquitously expressed in all examined cell types. JDP2 is phosphorylated on Thr148 by JNK (c-Jun N-terminal kinase) and p38 kinase, although the functional role of its phosphorylation is unknown. In the present paper we show that the JDP2 protein level is dramatically reduced in response to serum stimulation, anisomycin treatment, ultraviolet light irradiation and cycloheximide treatment, all of which activate the JNK pathway. In addition, endogenous and overexpressed JDP2 are phosphorylated in response to these stimuli. Replacement of Thr148 with an alanine residue stabilizes ectopically expressed JDP2 in the presence of the stimuli; conversely, substitution with glutamic acid destabilizes it. Serum-induced phosphorylation and degradation of JDP2 are specific to JNK activation since a JNK inhibitor (SP600125) abolishes these effects, whereas p38 and MEK inhibitors (SB203580 and UO126) have no effect. In the presence of cycloheximide, JDP2 is rapidly phosphorylated and degraded due to the combined effects of protein synthesis inhibition and activation of JNK. Pre-treatment of cells with SP600125 prior to cycloheximide treatment significantly prolongs the half-life of JDP2 that is found mainly in the unphosphorylated form. Lastly, the proteasome inhibitor (MG132) rescues JDP2 degradation following cycloheximide treatment and increases the expression of the JDP2 phospho-mimetic T148E mutant. Collectively, these results suggest that phosphorylation of JDP2 on thr148 by JNK targets it to the proteasome for degradation.

We thankYang et al. for their important comments regarding our recent manuscript “ATF3-dependent cross-talk between cardiomyocytes and macrophages promotes cardiac maladaptive remodeling” by Koren et al. [1]. We completely agree with the authors that the discrepancy can be explained by the different models used in the studies. Our analysis is based on the phenylephrine (PE) infusion pressure overload model and ATF3 transgenic mice. The results from these studies are consistent with ATF3's role in promoting cardiac hypertrophy [1,2]. In contrast, studies using the transverse aortic constriction (TAC) model suggest a cardio-protective role for ATF3 [3–6]. The opposing functions of ATF3 observed in the two models may be explained by the timing and duration of ATF3 expression. In the PE model, ATF3 expression is induced transiently and involves the recruitment of macrophages to the heart in an ATF3 dependent manner [1]. In the TAC model, whereas the involvement of immune cells is yet to be determined, persistent ATF3 expression appears as cardioprotective [3]. However, prolonged adult cardiac ATF3 expression displays maladaptive cardiac remodeling [2]. So how can constitutive ATF3 expression lead to opposite outcome? Previously, we showed that ATF3 may switch between either an inhibition or an activation mode, depending on the cellular context and protein partner, i.e. ATF3 inhibits transcription as a homodimer from TPA response elements, but activates transcription as a heterodimer with CHOP10 [7]. Revealing whether ATF3 exhibits differential transcriptional activity in the TAC, ATF3-transgenic mice and PE models may provide an explanation for the resulting diverse outcomes. Collectively, in mice models, ATF3 can play either a promoting or an inhibiting role in cardiacmaladaptive remodeling processes. Significantly

Acidic pH in the tumor microenvironment is associated with cancer metabolism and creates a physiological barrier that prevents from drugs to penetrate cells. Specifically, ionizable weak-base drugs, such as doxorubicin, freely permeate membranes in their uncharged form, however, in the acidic tumor microenvironment these drugs become charged and their cellular permeability is retarded. In this study, 100-nm liposomes loaded with sodium bicarbonate were used as adjuvants to elevate the tumor pH. Combined treatment of triple-negative breast cancer cells (4T1) with doxorubicin and sodium-bicarbonate enhanced drug uptake and increased its anti-cancer activity. In vivo, mice bearing orthotropic 4T1 breast cancer tumors were administered either liposomal or free bicarbonate intravenously. 3.7 ± 0.3% of the injected liposomal dose was detected in the tumor after twenty-four hours, compared to 0.17% ± 0.04% in the group injected free non-liposomal bicarbonate, a 21-fold increase. Analyzing nanoparticle biodistribution within the tumor tissue revealed that 93% of the PEGylated liposomes accumulated in the extracellular matrix, while 7% were detected intracellularly. Mice administered bicarbonate-loaded liposomes reached an intra-tumor pH value of 7.38 ± 0.04. Treating tumors with liposomal bicarbonate combined with a sub-therapeutic dose of doxorubicin achieved an improved therapeutic outcome, compared to mice treated with doxorubicin or bicarbonate alone. Interestingly, analysis of the tumor microenvironment demonstrated an increase in immune cell’ population (T-cell, B-cell and macrophages) in tumors treated with liposomal bicarbonate. This study demonstrates that targeting metabolic adjuvants with nanoparticles to the tumor microenvironment can enhance anticancer drug activity and improve treatment.