Tali Voloshin

Mitotic Spindle Disruption by Alternating Electric Fields Leads to Improper Chromosome Segregation and Mitotic Catastrophe in Cancer Cells

Tumor Treating Fields (TTFields) are low intensity, intermediate frequency, alternating electric fields. TTFields are a unique anti-mitotic treatment modality delivered in a continuous, noninvasive manner to the region of a tumor. It was previously postulated that by exerting directional forces on highly polar intracellular elements during mitosis, TTFields could disrupt the normal assembly of spindle microtubules. However there is limited evidence directly linking TTFields to an effect on microtubules. Here we report that TTFields decrease the ratio between polymerized and total tubulin, and prevent proper mitotic spindle assembly. The aberrant mitotic events induced by TTFields lead to abnormal chromosome segregation, cellular multinucleation, and caspase dependent apoptosis of daughter cells. The effect of TTFields on cell viability and clonogenic survival substantially depends upon the cell division rate. We show that by extending the duration of exposure to TTFields, slowly dividing cells can be affected to a similar extent as rapidly dividing cells.

Alternating Electric Fields (Tumor-Treating Fields Therapy) Can Improve Chemotherapy Treatment Efficacy in Non-Small Cell Lung Cancer Both In Vitro and In Vivo

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths worldwide. Common treatment modalities for NSCLC include surgery, radiotherapy, chemotherapy, and, in recent years, the clinical management paradigm has evolved with the advent of targeted therapies. Despite such advances, the impact of systemic therapies for advanced disease remains modest, and as such, the prognosis for patients with NSCLC remains poor. Standard modalities are not without their respective toxicities and there is a clear need to improve both efficacy and safety for current management approaches. Tumor-treating fields (TTFields) are low-intensity, intermediate-frequency alternating electric fields that disrupt proper spindle microtubule arrangement, thereby leading to mitotic arrest and ultimately to cell death. We evaluated the effects of combining TTFields with standard chemotherapeutic agents on several NSCLC cell lines, both in vitro and in vivo. Frequency titration curves demonstrated that the inhibitory effects of TTFields were maximal at 150 kHz for all NSCLC cell lines tested, and that the addition of TTFields to chemotherapy resulted in enhanced treatment efficacy across all cell lines. We investigated the response of Lewis lung carcinoma and KLN205 squamous cell carcinoma in mice treated with TTFields in combination with pemetrexed, cisplatin, or paclitaxel and compared these to the efficacy observed in mice exposed only to the single agents. Combining TTFields with these therapeutic agents enhanced treatment efficacy in comparison with the respective single agents and control groups in all animal models. Together, these findings suggest that combining TTFields therapy with chemotherapy may provide an additive efficacy benefit in the management of NSCLC.

Tumor‐derived microparticles induce bone marrow‐derived cell mobilization and tumor homing: A process regulated by osteopontin

Acute chemotherapy can induce rapid bone‐marrow derived pro‐angiogenic cell (BMDC) mobilization and tumor homing, contributing to tumor regrowth. To study the contribution of tumor cells to tumor regrowth following therapy, we focused on tumor‐derived microparticles (TMPs). EMT/6 murine‐mammary carcinoma cells exposed to paclitaxel chemotherapy exhibited an increased number of TMPs and significantly altered their angiogenic properties. Similarly, breast cancer patients had increased levels of plasma MUC‐1+TMPs following chemotherapy. In addition, TMPs from cells exposed to paclitaxel induced higher BMDC mobilization and colonization, but had no increased effect on angiogenesis in Matrigel plugs and tumors than TMPs from untreated cells. Since TMPs abundantly express osteopontin, a protein known to participate in BMDC trafficking, the impact of osteopontin‐depleted TMPs on BMDC mobilization, colonization, and tumor angiogenesis was examined. Although EMT/6 tumors grown in mice inoculated with osteopontin‐depleted TMPs had lower numbers of BMDC infiltration and microvessel density when compared with EMT/6 tumors grown in mice inoculated with wild‐type TMPs, no significant difference in tumor growth was seen between the two groups. However, when BMDCs from paclitaxel‐treated mice were injected into wild‐type EMT/6‐bearing mice, a substantial increase in tumor growth and BMDC infiltration was detected compared to osteopontin‐depleted EMT/6‐bearing mice injected with BMDCs from paclitaxel‐treated mice. Collectively, our results suggest that osteopontin expressed by TMPs play an important role in BMDC mobilization and colonization of tumors, but is not sufficient to enhance the angiogenic activity in tumors.

Blocking IL1β Pathway Following Paclitaxel Chemotherapy Slightly Inhibits Primary Tumor Growth but Promotes Spontaneous Metastasis

Acquired resistance to therapy is a major obstacle in clinical oncology, and little is known about the contributing mechanisms of the host response to therapy. Here, we show that the proinflammatory cytokine IL1β is overexpressed in response to paclitaxel chemotherapy in macrophages, subsequently promoting the invasive properties of malignant cells. In accordance, blocking IL1β, or its receptor, using either genetic or pharmacologic approach, results in slight retardation of primary tumor growth; however, it accelerates metastasis spread. Tumors from mice treated with combined therapy of paclitaxel and the IL1 receptor antagonist anakinra exhibit increased number of M2 macrophages and vessel leakiness when compared with paclitaxel monotherapy-treated mice, indicating a prometastatic role of M2 macrophages in the IL1β-deprived microenvironment. Taken together, these findings demonstrate the dual effects of blocking the IL1 pathway on tumor growth. Accordingly, treatments using “add-on” drugs to conventional therapy should be investigated in appropriate tumor models consisting of primary tumors and their metastases. Mol Cancer Ther; 14(6); 1385–94. ©2015 AACR.

Small But Mighty: Microparticles as Mediators of Tumor Progression

A wide spectrum of both normal and diseased cell types shed extracellular vesicles that facilitate intercellular communication without direct cell-to-cell contact. Microparticles (MPs) are a subtype of extracellular vesicles that participate in multiple biological processes. They carry abundant bioactive molecules including different forms of nucleic acids and proteins that can markedly modulate cellular behavior. MPs are involved in several hallmarks of cancer such as drug resistance, thrombosis, immune evasion, angiogenesis, tumor invasion and metastasis. Such MPs originate from either cancer or other host cells. As MPs are secreted and can be detected in various body fluids, they can be used as potential diagnostic and prognostic biomarkers as well as vehicles for delivery of cytotoxic drugs. This review summarizes accumulating evidence on the biological properties of MPs in cancer, with reference to their potential usage in clinical settings.

Tumor‐Initiating Cells of Various Tumor Types Exhibit Differential Angiogenic Properties and React Differently to Antiangiogenic Drugs

Tumor‐initiating cells (TICs) are a subtype of tumor cells believed to be critical for initiating tumorigenesis. We sought to determine the angiogenic properties of TICs in different tumor types including U‐87MG (glioblastoma), HT29 (colon), MCF7 (breast), A549 (non‐small‐cell lung), and PANC1 (pancreatic) cancers. Long‐term cultures grown either as monolayers (“TIC‐low”) or as nonadherent tumor spheres (“TIC‐high”) were generated. The TIC‐high fractions exhibited increased expression of stem cell surface markers, high aldehyde dehydrogenase activity, high expression of p21, and resistance to standard chemotherapy in comparison to TIC‐low fractions. Furthermore, TICs from U‐87MG and HT29 but not from MCF7, A549, and PANC1 tumor types possess increased angiogenic activity. Consequently, the efficacy of vascular endothelial growth factor‐A (VEGF‐A) neutralizing antibody is limited only to those tumors that are dependent on VEGF‐A activity. In addition, such therapy had little or reversed antiangiogenic effects on tumors that do not necessarily rely on VEGF‐dependent angiogenesis. Differential angiogenic activity and antiangiogenic therapy sensitivity were also observed in TICs of the same tumor type, suggesting redundant angiogenic pathways. Collectively, our results suggest that the efficacy of antiangiogenic drugs is dependent on the angiogenic properties of TICs and, therefore, can serve as a possible biomarker to predict antiangiogenic treatment efficacy. Stem Cells2012;30:1831–1841

Alternating electric fields (TTFields) in combination with paclitaxel are therapeutically effective against ovarian cancer cells in vitro and in vivo.

Long‐term survival rates for advanced ovarian cancer patients have not changed appreciably over the past four decades; therefore, development of new, effective treatment modalities remains a high priority. Tumor Treating Fields (TTFields), a clinically active anticancer modality utilize low‐intensity, intermediate frequency, alternating electric fields. The goal of this study was to evaluate the efficacy of combining TTFields with paclitaxel against ovarian cancer cells in vitro and in vivo. In vitro application of TTFields on human ovarian cancer cell lines led to a significant reduction in cell counts as compared to untreated cells. The effect was found to be frequency and intensity dependent. Further reduction in the number of viable cells was achieved when TTFields treatment was combined with paclitaxel. The in vivo effect of the combined treatment was tested in mice orthotopically implanted with MOSE‐LTICv cells. In this model, combined treatment led to a significant reduction in tumor luminescence and in tumor weight as compared to untreated mice. The feasibility of effective local delivery of TTFields to the human abdomen was examined using finite element mesh simulations performed using the Sim4life software. These simulations demonstrated that electric fields intensities inside and in the vicinity of the ovaries of a realistic human computational phantom are about 1 and 2 V/cm pk‐pk, respectively, which is within the range of intensities required for TTFields effect. These results suggest that prospective clinical investigation of the combination of TTFields and paclitaxel is warranted.

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields (TTFields)

Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), alternating electric fields in the frequency range of several hundred kHz. The study of TTFields in tissue culture is carried out using the TTFields in vitro application system, which allows for the application of electric fields of varying frequencies and intensities to ceramic Petri dishes with a high dielectric constant (Ɛ > 5,000). Cancerous cell lines plated on coverslips at the bottom of the ceramic Petri dishes are subjected to TTFields delivered in two orthogonal directions at various frequencies to facilitate treatment outcome tests, such as cell counts and clonogenic assays. The results presented in this report demonstrate that the optimal frequency of the TTFields with respect to both cell counts and clonogenic assays is 200 kHz for both ovarian and glioma cells.

Abstract B79: Translational study of tumor treating fields in combination with paclitaxel in ovarian cancer.

Tumor Treating Fields (TTFields), a clinically active anticancer modality, are based on low intensity intermediate frequency alternating electric fields that exert their cytotoxicity by disrupting mitosis. The present study examines whether concomitant paclitaxel and TTFields have a beneficial impact on ovarian cancer growth both in vitro and in vivo. Moreover, on the basis of the preclinical observations, an open-label pilot clinical study evaluating the effect of the combined modalities in 30 patients with recurrent ovarian cancer was initiated. Preclinical studies: To investigate the inhibitory effect of TTFields on ovarian cancer cell growth in vitro and determine optimal therapeutic frequency of TTFields in ovarian cancer, human ovarian cancer cell lines were treated with TTFields (100-400 kHz) for 72 hours using the inovitro system (Novocure, Haifa, Israel). To assess whether adding TTFields to paclitaxel increases the response of ovarian cancer cells to paclitaxel, we treated these cell lines with paclitaxel alone and in combination with TTFields. In vivo efficacy of the combined treatment was tested in female C57Bl/6 mice, orthotopically implanted with MOSE-L FFL luciferase positive cells. The feasibility of effective regional delivery of TTFields therapy to the ovaries, pelvis and abdomen of human subjects was examined using Finite Element Mesh (FEM) simulations performed using the Sim4life software. The FEM simulations demonstrated effective distribution of fields at intensities of 1-2 V/cm, which is above the minimal threshold required for TTFields response. The INNOVATE Trial (NCT02244502): Based on positive preclinical studies demonstrating the combined efficacy of TTFields and paclitaxel in different ovarian cancer models, a pilot clinical trial was initiated to evaluate this therapeutic combination in recurrent ovarian carcinoma patients. In this prospective, pilot, single arm study, 30 patients will receive bi-directional TTFields at 200 kHz applied to the ovaries and surrounding intra-abdominal tissues using 4 transducer arrays located on the surface of the lower abdominal region. In addition, patients will receive concomitant paclitaxel at a standard regimen and dose. The combined treatment will be administered until further radiological progression. Inclusion criteria include ECOG score of 0-1 and no serious co-morbidities. The trial9s primary endpoint is adverse events frequency and severity. The study will also collect preliminary efficacy data through the analysis of progression-free survival, 1-year survival rate and overall survival. Compliance data will be analyzed as an additional secondary endpoint. The INNOVATE study started to enroll patients in October 2014, and is currently accruing patients in Switzerland, Belgium and Spain. So far the trial has enrolled half of the planned 30 patients. In summary, we present the first preclinical evidence in ovarian cancer of the combined efficacy of paclitaxel and TTFields, a new anticancer treatment modality. Our results suggest that it may represent a novel, effective therapeutic strategy against ovarian cancer. Pilot clinical testing is ongoing. Citation Format: Mijal Munster, Roni Blat, Paul C. Roberts, Eva M. Schmelz, Moshe Giladi, Rosa S. Schneiderman, Yaara Porat, Zeev Bomzon, Noa Urman, Aviran Itzhaki, Tali Voloshin, Shay Cahal, Eilon D. Kirson, Uri Weinberg, Yoram Palti. Translational study of tumor treating fields in combination with paclitaxel in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B79.

Abstract 5361: In vitro results and electric fields simulations suggest Tumor Treating Fields (TTFields) to be an effective treatment against Mesothelioma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Mesothelioma is an aggressive cancer affecting the membrane lining of the lungs and abdomen, and is associated with poor prognosis. Current available treatment options for Mesothelioma, including surgery, radiation therapy and chemotherapy, offer only modest improvement in survival, therefore emphasizing the desperate need for new treatment options. Tumor Treating Fields (TTFields) is a treatment modality based on non-invasive application of low intensity intermediate frequency alternating electric fields that disrupt mitosis. The goal of the present work was to determine whether TTFields in combination with standard of care drugs, could serve as an effective treatment against Mesothelioma. TTFields were applied to Mesothelioma cell cultures using the inovitro system. Cytotoxicity and effect on clonogenicity were determined following 72 hours of TTFields treatment. Combination index (CI) was calculated for the treatment of TTFields with each of the following drugs: Paclitaxel, Cisplatin, Pemetrexed and Vinorelbine. Finite Element Mesh (FEM) simulations were performed using the Sim4life software package (ZMT, Zurich, Switzerland) for the calculations of the electric fields intensities in the mesothelium of a realistic human computational phantom. Frequency titration experiments revealed 150 kHz to be the optimal TTFields frequency for Mesothelioma cell cultures, leading to 69% reduction in the number of cells (p<0.001) and 78% reduction in clonogenicity (p<0.05) as compared to control. The combined treatment of TTFields with Paclitaxel, Cisplatin, Pemetrexed and Vinorelbine resulted in combination index of 0.99, 0.88, 1.29 and 1.13, respectively. FEM simulations demonstrated that electric field intensities within the mesothelium are approximately 2 V/cm RMS which is above the minimal threshold required for TTFields effect. Taken together, these results suggest that combining TTFields therapy with systemic chemotherapy may be an approach that offers enhanced treatment efficacy for Mesothelioma. A clinical study testing the combined efficacy of TTFields Cisplatin and Pemetrexed is under development. Citation Format: Moshe Giladi, Mijal Munster, Roni Blat, Rosa Schneiderman, Yaara Porat, Zeev Bomzon, Noa Urman, Tali Voloshin, Eilon David Kirson, Uri Weinberg, Yoram Palti. In vitro results and electric fields simulations suggest Tumor Treating Fields (TTFields) to be an effective treatment against Mesothelioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5361. doi:10.1158/1538-7445.AM2015-5361