Search

Suppl. Figure legends Suppl. Fig. 1. The structure of H1001, a small molecule heparanase inhibitor. H1001 inhibit heparanase activity inside and outside the cell, and attenuates cellular invasion Suppl. Fig. 2. Medium conditioned by PCT-treated macrophages promote cell migration; Matrigel containing PCT-treated macrophages recruits far more host macrophages into the plug. Suppl. Fig. 3. Injecting LLC cells with PCT-treated macrophages modestly increase tumor growth in WT C57Bl/6 mice Suppl. Fig. 4. Differential cytokine induction in WT vs KO macrophages is not mediated by TLR2 and TLR4 or MLL1-4, SET1A or RBBP5 Suppl. Fig. 5. Cytokine induction by PCT is attenuated by OICR-9429, a WDR5 inhibitor Suppl. Fig. 6. WDR5 silencing attenuates while WDR5 over expression promote cytokine induction. Cytokine induction by PCT is attenuated by CCG-203971, a MKL1 inhibitor Suppl. Fig. 7. PCT enhances the phosphorylation of JNK in WT but not KO macrophages, and cytokine induction by PCT is attenuated by inhibitor of JNK Suppl. Table 1. Mouse primers sets utilized in this study Suppl. Table 2. Antibodies utilized for FACS analyses

The emerging role of heparanase in tumor initiation, growth, metastasis, and chemoresistance is well recognized, encouraging the development of heparanase inhibitors as anticancer drugs. Unlike the function of heparanase in cancer cells, little attention has been given to heparanase contributed by cells composing the tumor microenvironment. Here, we focused on the cross-talk between macrophages, chemotherapy, and heparanase and the combined effect on tumor progression. Macrophages were markedly activated by chemotherapeutics paclitaxel and cisplatin, evidenced by increased expression of proinflammatory cytokines, supporting recent studies indicating that chemotherapy may promote rather than suppress tumor regrowth and spread. Strikingly, cytokine induction by chemotherapy was not observed in macrophages isolated from heparanase-knockout mice, suggesting macrophage activation by chemotherapy is heparanase dependent. paclitaxel-treated macrophages enhanced the growth of Lewis lung carcinoma tumors that was attenuated by a CXCR2 inhibitor. Mechanistically, paclitaxel and cisplatin activated methylation of histone H3 on lysine 4 (H3K4) in wild-type but not in heparanase-knockout macrophages. Furthermore, the H3K4 presenter WDR5 functioned as a molecular determinant that mediated cytokine induction by paclitaxel. This epigenetic, heparanase-dependent host-response mechanism adds a new perspective to the tumor-promoting functions of chemotherapy, and offers new treatment modalities to optimize chemotherapeutics.Significance:Chemotherapy-treated macrophages are activated to produce proinflammatory cytokines, which are blunted in the absence of heparanase.

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.

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.

Radiotherapy induces immune-related responses in cancer patients by various mechanisms. Here, we investigate the immunomodulatory role of tumor-derived microparticles (TMPs)—extracellular vesicles shed from tumor cells—following radiotherapy. We demonstrate that breast carcinoma cells exposed to radiation shed TMPs containing elevated levels of immune-modulating proteins, one of which is programmed death-ligand 1 (PD-L1). These TMPs inhibit cytotoxic T lymphocyte (CTL) activity both in vitro and in vivo, and thus promote tumor growth. Evidently, adoptive transfer of CTLs pre-cultured with TMPs from irradiated breast carcinoma cells increases tumor growth rates in mice recipients in comparison with control mice receiving CTLs pre-cultured with TMPs from untreated tumor cells. In addition, blocking the PD-1-PD-L1 axis, either genetically or pharmacologically, partially alleviates TMP-mediated inhibition of CTL activity, suggesting that the immunomodulatory effects of TMPs in response to radiotherapy is mediated, in part, by PD-L1. Overall, our findings provide mechanistic insights into the tumor immune surveillance state in response to radiotherapy and suggest a therapeutic synergy between radiotherapy and immune checkpoint inhibitors.

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.

Bone marrow derived cells (BMDCs) play a wide variety of pro- and anti-tumorigenic roles in the tumor microenvironment (TME) and in the metastatic process. In response to chemotherapy, the anti-tumorigenic function of BMDCs can be enhanced due to chemotherapy-induced immunogenic cell death. However, in recent years, a growing body of evidence suggests that chemotherapy or other anti-cancer drugs can also facilitate a pro-tumorigenic function in BMDCs. This includes elevated angiogenesis, tumor cell proliferation and pro-tumorigenic immune modulation, ultimately contributing to therapy resistance. Such effects do not only contribute to the re-growth of primary tumors but can also support metastasis. Thus, the delicate balance of BMDC activities in the TME is violated following tumor perturbation, further requiring a better understanding of the complex crosstalk between tumor cells and BMDCs. In this review, we discuss the different types of BMDCs that reside in the TME and their activities in tumors following chemotherapy, with a major focus on their pro-tumorigenic role. We also cover aspects of rationally designed combination treatments that target or manipulate specific BMDC types to improve therapy outcomes.

Author “Uzi Avner” asked to remove the name in the list of authors of the paper. The request was agreed by the co-authors. Given in this article is the corrected list.

BackgroundImmunomodulatory agents that induce antitumor immunity have great potential for treatment of cancer. We have previously shown that interleukin (IL)-31, a proinflammatory cytokine from the IL-6 family, acts as an antiangiogenic agent. Here, we characterize the immunomodulatory effect of IL-31 in breast cancer.MethodsIn vivo breast carcinoma models including EMT6 and PyMT cell lines were used to analyze the effect of IL-31 on the composition of various immune cells in the tumor microenvironment using high-throughput flow cytometry. In vitro studies using isolated cytotoxic T cells, CD4+ T cells, myeloid-derived suppressor cells (MDSCs) and macrophages were carried out to study IL-31 immunological activity. The generation of recombinant IL-31 bound to IgG backbone was used to test IL-31 therapeutic activity.ResultsThe growth rate of IL-31-expressing breast carcinomas is decreased in comparison with control tumors due, in part, to antitumor immunomodulation. Specifically, cytotoxic T cell activity is increased, whereas the levels of CD4+ T cells, MDSCs, and tumor-associated macrophages are decreased in IL-31-expressing tumors. These cellular changes are accompanied by a cytokine profile associated with antitumor immunity. In vitro, IL-31 directly inhibits CD4+ Th0 cell proliferation, and the expression of Th2 canonical factors GATA3 and IL-4. It also promotes CD8+ T cell activation through inhibition of MDSC activity and motility. Clinically, in agreement with the mouse data, alterations in immune cell composition in human breast cancer biopsies were found to correlate with high expression of IL-31 receptor A (IL-31Ra) . Furthermore, high coexpression of IL-31Ra, IL-2 and IL-4 in tumors correlates with increased survival. Lastly, to study the therapeutic potential of IL-31, a recombinant murine IL-31 molecule was fused to IgG via a linker region (IL-31-L-IgG). This IL-31-L-IgG therapy demonstrates antitumor therapeutic activity in a murine breast carcinoma model.ConclusionsOur findings demonstrate that IL-31 induces antitumor immunity, highlighting its potential utility as a therapeutic immunomodulatory agent.

As part of the archaeological study of Nahal Amram in the ‘Araba valley, copper slag samples from a mining camp dating from the 5th millennium BCE until the 1st millennium AD were chemically analyzed by X-ray fluorescence (XRF) in an attempt to determine the technological level of production in each of the site’s occupation. The method provides a tool to distinguish between slag of various technological phases and periods. Thus, the slags from different sites in Nahal Amram related roughly to four stages of metallurgy development. These metallurgical results indicate the reuse of slag from earlier periods in later periods in order to produce more copper from the slag as a result of more complex production methods that were available in the later periods. Thus, in Nahal Amram, we can observe a real technological development of metallurgical production from earlier to later periods based almost exclusively upon the results of slag analyses. The results were compared with other smelting sites in the region.

// Shiri Davidi 1, * , Ella Fremder 1, * , Tal Kan 1 , Ziv Raviv 1 , Michael Timaner 1 , Nathan Karin 2 , Dov Hershkovitz 3 , Ami Arohneim 1, * , Yuval Shaked 1, * 1 Cell Biology and Cancer Science, Rappaport Faculty of Medicine, Technion, Haifa, Israel 2 Department of Immunology, Rappaport Faculty of Medicine, Technion, Haifa, Israel 3 Department of Pathology, Rambam Medical Center, Haifa, Israel * These authors contributed equally to this work Correspondence to: Yuval Shaked, email: yshaked@tx.technion.ac.il Ami Aronheim, email: aronheim@tx.technion.ac.il Keywords: metastasis, angiogenesis, cancer therapy, host response Received: December 14, 2016 Accepted: January 16, 2017 Published: January 27, 2017 ABSTRACT Pro-inflammatory cytokines in the tumor microenvironment are known for their ability to either inhibit or promote cancer progression. Here we evaluated the role of Interleukin-31 (IL31), a protein belonging to the pro-inflammatory IL-6 cytokine family which has been characterized in autoimmune disease, in tumorigenesis. We show that IL31 and its receptor, IL31RA, are highly expressed in various human and mouse cancer cell lines, as well as in tumor specimens from cancer patients. MC38 murine colon carcinoma cells depleted of IL31 exhibit an increase in invasive and migratory properties in vitro , effects that are reversed by supplementing the cells with exogenous IL31. In vivo , IL31-depleted MC38 tumor cells implanted to mice grow faster than control tumors. In contrast, MC38 tumor-bearing mice infused with recombinant IL31, exhibit a significant reduction in tumor growth than control mice. Furthermore, IL31 infusion reduces the number of metastatic lesions in the lungs of mice bearing 4T1 murine metastatic breast carcinoma. Lastly, injecting tumor-bearing, chemotherapy-treated mice with a long-lived IL31-IgG fusion protein reduces tumor growth, angiogenesis and pulmonary metastasis to a greater extent than when chemotherapy is used alone. The IL31 anti-tumor activity is explained, in part, by the anti-angiogenic effects demonstrated both in vitro and in vivo highlighting the potential use of IL31 as an anti-cancer drug.

The emerging role of heparanase in tumor initiation, growth, metastasis, and chemoresistance is well recognized, encouraging the development of heparanase inhibitors as anticancer drugs. Unlike the function of heparanase in cancer cells, little attention has been given to heparanase contributed by cells composing the tumor microenvironment. Here, we focused on the cross-talk between macrophages, chemotherapy, and heparanase and the combined effect on tumor progression. Macrophages were markedly activated by chemotherapeutics paclitaxel and cisplatin, evidenced by increased expression of proinflammatory cytokines, supporting recent studies indicating that chemotherapy may promote rather than suppress tumor regrowth and spread. Strikingly, cytokine induction by chemotherapy was not observed in macrophages isolated from heparanase-knockout mice, suggesting macrophage activation by chemotherapy is heparanase dependent. paclitaxel-treated macrophages enhanced the growth of Lewis lung carcinoma tumors that was attenuated by a CXCR2 inhibitor. Mechanistically, paclitaxel and cisplatin activated methylation of histone H3 on lysine 4 (H3K4) in wild-type but not in heparanase-knockout macrophages. Furthermore, the H3K4 presenter WDR5 functioned as a molecular determinant that mediated cytokine induction by paclitaxel. This epigenetic, heparanase-dependent host-response mechanism adds a new perspective to the tumor-promoting functions of chemotherapy, and offers new treatment modalities to optimize chemotherapeutics. Significance: Chemotherapy-treated macrophages are activated to produce proinflammatory cytokines, which are blunted in the absence of heparanase.

Cancer stem cells, also termed tumor initiating cells (TICs), are a rare population of cells within the tumor mass which initiate tumor growth and metastasis. In pancreatic cancer, TICs significantly contribute to tumor re-growth after therapy, due to their intrinsic resistance. Here we demonstrate that copper oxide nanoparticles (CuO-NPs) are cytotoxic against TIC-enriched PANC1 human pancreatic cancer cell cultures. Specifically, treatment with CuO-NPs decreases cell viability and increases apoptosis in TIC-enriched PANC1 cultures to a greater extent than in standard PANC1 cultures. These effects are associated with increased reactive oxygen species (ROS) levels, and reduced mitochondrial membrane potential. Furthermore, we demonstrate that CuO-NPs inhibit tumor growth in a pancreatic tumor model in mice. Tumors from mice treated with CuO-NPs contain a significantly higher number of apoptotic TICs in comparison to tumors from untreated mice, confirming that CuO-NPs target TICs in vivo. Overall, our findings highlight the potential of using CuO-NPs as a new therapeutic modality for pancreatic cancer.