Correction: Davidi et al. Tumor Treating Fields (TTFields) Concomitant with Sorafenib Inhibit Hepatocellular Carcinoma In Vitro and In Vivo. Cancers 2022, 14, 2959.

The authors wish to make minor corrections to Figure 1 and Figure 2 of the following paper [...].

Tumor Treating Fields (TTFields) Concomitant with Immune Checkpoint Inhibitors Are Therapeutically Effective in Non-Small Cell Lung Cancer (NSCLC) In Vivo Model.

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.

Tumor Treating Fields (TTFields) Reversibly Permeabilize the Blood-Brain Barrier In Vitro and In Vivo.

Despite the availability of numerous therapeutic substances that could potentially target CNS disorders, an inability of these agents to cross the restrictive blood-brain barrier (BBB) limits their clinical utility. Novel strategies to overcome the BBB are therefore needed to improve drug delivery. We report, for the first time, how Tumor Treating Fields (TTFields), approved for glioblastoma (GBM), affect the BBB's integrity and permeability. Here, we treated murine microvascular cerebellar endothelial cells (cerebEND) with 100-300 kHz TTFields for up to 72 h and analyzed the expression of barrier proteins by immunofluorescence staining and Western blot. In vivo, compounds normally unable to cross the BBB were traced in healthy rat brain following TTFields administration at 100 kHz. The effects were analyzed via MRI and immunohistochemical staining of tight-junction proteins. Furthermore, GBM tumor-bearing rats were treated with paclitaxel (PTX), a chemotherapeutic normally restricted by the BBB combined with TTFields at 100 kHz. The tumor volume was reduced with TTFields plus PTX, relative to either treatment alone. In vitro, we demonstrate that TTFields transiently disrupted BBB function at 100 kHz through a Rho kinase-mediated tight junction claudin-5 phosphorylation pathway. Altogether, if translated into clinical use, TTFields could represent a novel CNS drug delivery strategy.

Tumor Treating Fields (TTFields) Concomitant with Sorafenib Inhibit Hepatocellular Carcinoma In Vitro and In Vivo.

Hepatocellular carcinoma (HCC), a highly aggressive liver cancer, is a leading cause of cancer-related death. Tumor Treating Fields (TTFields) are electric fields that exert antimitotic effects on cancerous cells. The aims of the current research were to test the efficacy of TTFields in HCC, explore the underlying mechanisms, and investigate the possible combination of TTFields with sorafenib, one of the few front-line treatments for patients with advanced HCC. HepG2 and Huh-7D12 human HCC cell lines were treated with TTFields at various frequencies to determine the optimal frequency eliciting maximal cell count reduction. Clonogenic, apoptotic effects, and autophagy induction were measured. The efficacy of TTFields alone and with concomitant sorafenib was tested in cell cultures and in an orthotopic N1S1 rat model. Tumor volume was examined at the beginning and following 5 days of treatment. At study cessation, tumors were weighed and examined by immunohistochemistry to assess autophagy and apoptosis. TTFields were found in vitro to exert maximal effect at 150 kHz, reducing cell count and colony formation, increasing apoptosis and autophagy, and augmenting the effects of sorafenib. In animals, TTFields concomitant with sorafenib reduced tumor weight and volume fold change, and increased cases of stable disease following treatment versus TTFields or sorafenib alone. While each treatment alone elevated levels of autophagy relative to control, TTFields concomitant with sorafenib induced a significant increase versus control in tumor ER stress and apoptosis levels, demonstrating increased stress under the multimodal treatment. Overall, TTFields treatment demonstrated efficacy and enhanced the effects of sorafenib for the treatment of HCC in vitro and in vivo, via a mechanism involving induction of autophagy.

Tumor Treating Fields (TTFields) downregulate the Fanconi Anemia-BRCA pathway and increase the efficacy of chemotherapy in malignant pleural mesothelioma preclinical models.

OBJECTIVES: Tumor Treating Fields (TTFields) are low intensity, intermediate frequency, alternating electric fields with antimitotic effects on cancerous cells. TTFields concomitant with pemetrexed and a platinum agent are approved in the US and EU as first line therapy for unresectable, locally advanced or metastatic malignant pleural mesothelioma (MPM). The goal of the current study was to characterize the mechanism of action of TTFields in MPM cell lines and animal models. METHODS: Human MPM cell lines MSTO-211H and NCI-H2052 were treated with TTFields to determine the frequency that elicits maximal cytotoxicity. The effect of TTFields on DNA damage and repair, and the cytotoxic effect of TTFields in combination with cisplatin and/or pemetrexed were examined. Efficacy of TTFields concomitant with cisplatin and pemetrexed was evaluated in orthotopic IL-45 and subcutaneous RN5 murine models. RESULTS: TTFields at a frequency of 150 kHz demonstrated the highest cytotoxicity to MPM cells. Application of 150 kHz TTFields resulted in increased formation of DNA double strand breaks, elevated expression of DNA damage induced cell cycle arrest proteins, and reduced expression of Fanconi Anemia (FA)-BRCA DNA repair pathway proteins. Co-treatment of TTFields with cisplatin or pemetrexed significantly increased treatment efficacy versus each modality alone, with additivity and synergy exhibited by the TTFields-pemetrexed and TTFields-cisplatin combinations, respectively. In animal models, tumor volume was significantly lower for the TTFields-cisplatin-pemetrexed combination compared to control, accompanied by increased DNA damage within the tumor. CONCLUSION: This research demonstrated that the efficacy of TTFields for the treatment of MPM is associated with reduced expression of FA-BRCA pathway proteins and increased DNA damage. This mechanism of action is consistent with the observed synergism for TTFields-cisplatin vs additivity for TTFields-pemetrexed, as cisplatin-induced DNA damage is repaired via the FA-BRCA pathway.

Tumor Treating Fields (TTFields) Hinder Cancer Cell Motility through Regulation of Microtubule and Acting Dynamics.

Tumor Treating Fields (TTFields) are noninvasive, alternating electric fields within the intermediate frequency range (100-300 kHz) that are utilized as an antimitotic cancer treatment. TTFields are loco-regionally delivered to the tumor region through 2 pairs of transducer arrays placed on the skin. This novel treatment modality has been FDA-approved for use in patients with glioblastoma and malignant pleural mesothelioma based on clinical trial data demonstrating efficacy and safety; and is currently under investigation in other types of solid tumors. TTFields were shown to induce an anti-mitotic effect by exerting bi-directional forces on highly polar intracellular elements, such as tubulin and septin molecules, eliciting abnormal microtubule polymerization during spindle formation as well as aberrant cleavage furrow formation. Previous studies have demonstrated that TTFields inhibit metastatic properties in cancer cells. However, the consequences of TTFields application on cytoskeleton dynamics remain undetermined. In this study, methods utilized in combination to study the effects of TTFields on cancer cell motility through regulation of microtubule and actin dynamics included confocal microscopy, computational tools, and biochemical analyses. Mechanisms by which TTFields treatment disrupted cellular polarity were (1) interference with microtubule assembly and directionality; (2) altered regulation of Guanine nucleotide exchange factor-H1 (GEF-H1), Ras homolog family member A (RhoA), and Rho-associated coiled-coil kinase (ROCK) activity; and (3) induced formation of radial protrusions of peripheral actin filaments and focal adhesions. Overall, these data identified discrete effects of TTFields that disrupt processes crucial for cancer cell motility.

Tumor-treating fields (TTFields) induce immunogenic cell death resulting in enhanced antitumor efficacy when combined with anti-PD-1 therapy.

Tumor-treating fields (TTFields) are alternating electric fields in a specific frequency range (100-300 kHz) delivered to the human body through transducer arrays. In this study, we evaluated whether TTFields-mediated cell death can elicit antitumoral immunity and hence would be effectively combined with anti-PD-1 therapy. We demonstrate that in TTFields-treated cancer cells, damage-associated molecular patterns including high-mobility group B1 and adenosine triphosphate are released and calreticulin is exposed on the cell surface. Moreover, we show that TTFields treatment promotes the engulfment of cancer cells by dendritic cells (DCs) and DCs maturation in vitro, as well as recruitment of immune cells in vivo. Additionally, our study demonstrates that the combination of TTFields with anti-PD-1 therapy results in a significant decline of tumor volume and increase in the percentage of tumor-infiltrating leukocytes in two tumor models. In orthotopic lung tumors, these infiltrating leukocytes, specifically macrophages and DCs, showed elevated expression of PD-L1. Compatibly, cytotoxic T-cells isolated from these tumors demonstrated increased production of IFN-γ. In colon cancer tumors, T-cells infiltration was significantly increased following long treatment duration with TTFields plus anti-PD-1. Collectively, our results suggest that TTFields therapy can induce anticancer immune response. Furthermore, we demonstrate robust efficacy of concomitant application of TTFields and anti-PD-1 therapy. These data suggest that integrating TTFields with anti-PD-1 therapy may further enhance antitumor immunity, hence achieve better tumor control.

AMPK-dependent autophagy upregulation serves as a survival mechanism in response to Tumor Treating Fields (TTFields).

Tumor Treating Fields (TTFields), an approved treatment modality for glioblastoma, are delivered via non-invasive application of low-intensity, intermediate-frequency, alternating electric fields. TTFields application leads to abnormal mitosis, aneuploidy, and increased cell granularity, which are often associated with enhancement of autophagy. In this work, we evaluated whether TTFields effected the regulation of autophagy in glioma cells. We found that autophagy is upregulated in glioma cells treated with TTFields as demonstrated by immunoblot analysis of the lipidated microtubule-associated protein light chain 3 (LC3-II). Fluorescence and transmission electron microscopy demonstrated the presence of LC3 puncta and typical autophagosome-like structures in TTFields-treated cells. Utilizing time-lapse microscopy, we found that the significant increase in the formation of LC3 puncta was specific to cells that divided during TTFields application. Evaluation of selected cell stress parameters revealed an increase in the expression of the endoplasmic reticulum (ER) stress marker GRP78 and decreased intracellular ATP levels, both of which are indicative of increased proteotoxic stress. Pathway analysis demonstrated that TTFields-induced upregulation of autophagy is dependent on AMP-activated protein kinase (AMPK) activation. Depletion of AMPK or autophagy-related protein 7 (ATG7) inhibited the upregulation of autophagy in response to TTFields, as well as sensitized cells to the treatment, suggesting that cancer cells utilize autophagy as a resistance mechanism to TTFields. Combining TTFields with the autophagy inhibitor chloroquine (CQ) resulted in a significant dose-dependent reduction in cell growth compared with either TTFields or CQ alone. These results suggest that dividing cells upregulate autophagy in response to aneuploidy and ER stress induced by TTFields, and that AMPK serves as a key regulator of this process.

Tumor treating fields (TTFields) delay DNA damage repair following radiation treatment of glioma cells.

BACKGROUND: Tumor Treating Fields (TTFields) are an anti-neoplastic treatment modality delivered via application of alternating electric fields using insulated transducer arrays placed directly on the skin in the region surrounding the tumor. A Phase 3 clinical trial has demonstrated the effectiveness of continuous TTFields application in patients with glioblastoma during maintenance treatment with Temozolomide. The goal of this study was to evaluate the efficacy of combining TTFields with radiation treatment (RT) in glioma cells. We also examined the effect of TTFields transducer arrays on RT distribution in a phantom model and the impact on rat skin toxicity. METHODS: The efficacy of TTFields application after induction of DNA damage by RT or bleomycin was tested in U-118 MG and LN-18 glioma cells. The alkaline comet assay was used to measure repair of DNA lesions. Repair of DNA double strand breaks (DSBs) were assessed by analyzing γH2AX or Rad51 foci. DNA damage and repair signaled by the activation pattern of phospho-ATM (pS1981) and phospho-DNA-PKcs (pS2056) was evaluated by immunoblotting. The absorption of the RT energy by transducer arrays was measured by applying RT through arrays placed on a solid-state phantom. Skin toxicities were tested in rats irradiated daily through the arrays with 2Gy (total dose of 20Gy). RESULTS: TTFields synergistically enhanced the efficacy of RT in glioma cells. Application of TTFields to irradiated cells impaired repair of irradiation- or chemically-induced DNA damage, possibly by blocking homologous recombination repair. Transducer arrays presence caused a minor reduction in RT intensity at 20 mm and 60 mm below the arrays, but led to a significant increase in RT dosage at the phantom surface jeopardizing the "skin sparing effect". Nevertheless, transducer arrays placed on the rat skin during RT did not lead to additional skin reactions. CONCLUSIONS: Administration of TTFields after RT increases glioma cells treatment efficacy possibly by inhibition of DNA damage repair. These preclinical results support the application of TTFields therapy immediately after RT as a viable regimen to enhance RT outcome. Phantom measurements and animal models imply that it may be possible to leave the transducer arrays in place during RT without increasing skin toxicities.

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.

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.

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.

Recruitment of Oligoclonal Viral-Specific T cells to Kill Human Tumor Cells Using Single-Chain Antibody-Peptide-HLA Fusion Molecules.

Tumor progression is often associated with the development of diverse immune escape mechanisms. One of the main tumor escape mechanism is HLA loss, in which human solid tumors exhibit alterations in HLA expression. Moreover, tumors that present immunogenic peptides via class I MHC molecules are not susceptible to CTL-mediated lysis, because of the relatively low potency of the tumor-specific CLTs. Here, we present a novel cancer immunotherapy approach that overcomes these problems by using the high affinity and specificity of antitumor antibodies to recruit potent antiviral memory CTLs to attack tumor cells. We constructed a recombinant molecule by genetic fusion of a cytomegalovirus (CMV)-derived peptide pp65 (NLVPMVATV) to scHLA-A2 molecules that were genetically fused to a single-chain Fv Ab fragment specific for the tumor cell surface antigen mesothelin. This fully covalent fusion molecule was expressed in E. coli as inclusion bodies and refolded in vitro. The fusion molecules could specifically bind mesothelin-expressing cells and mediate their lysis by NLVPMVATV-specific HLA-A2-restricted human CTLs. More importantly, these molecules exhibited very potent antitumor activity in vivo in a nude mouse model bearing preestablished human tumor xenografts that were adoptively transferred along with human memory CTLs. These results represent a novel and powerful approach to immunotherapy for solid tumors, as demonstrated by the ability of the CMV-scHLA-A2-SS1(scFv) fusion molecule to mediate specific and efficient recruitment of CMV-specific CTLs to kill tumor cells.

Blocking IL1ß 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 IL1ß is overexpressed in response to paclitaxel chemotherapy in macrophages, subsequently promoting the invasive properties of malignant cells. In accordance, blocking IL1ß, 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 IL1ß-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.

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.

Anti-Bv8 antibody and metronomic gemcitabine improve pancreatic adenocarcinoma treatment outcome following weekly gemcitabine therapy.

Weekly gemcitabine therapy is the major treatment offered for patients with pancreatic adenocarcinoma cancer; however, relative resistance of tumor cells to chemotherapy, rapid regrowth, and metastasis are the main causes of death within a year. Recently, the daily continuous administration of chemotherapy in low doses--called metronomic chemotherapy (MC)--has been shown to inhibit primary tumor growth and delay metastases in several tumor types; however, its use as a single therapy is still in question due to its moderate therapeutic benefit. Here, we show that the combination of weekly gemcitabine with MC of the same drug delays tumor regrowth and inhibits metastasis in mice implanted orthotopically with pancreatic tumors. We further demonstrate that weekly gemcitabine, but not continuous MC gemcitabine or the combination of the two drug regimens, promotes rebound myeloid-derived suppressor cell (MDSC) mobilization and increases angiogenesis in this tumor model. Furthermore, Bv8 is highly expressed in MDSCs colonizing pancreatic tumors in mice treated with weekly gemcitabine compared to MC gemcitabine or the combination of the two regimens. Blocking Bv8 with antibodies in weekly gemcitabine-treated mice results in a significant reduction in tumor regrowth, angiogenesis, and metastasis. Overall, our results suggest that pro-tumorigenic effects induced by weekly gemcitabine are mediated in part by MDSCs expressing Bv8. Therefore, both Bv8 inhibition and MC can be used as legitimate 'add-on' treatments for preventing post-chemotherapy pancreatic cancer recurrence, progression, and metastasis following weekly gemcitabine therapy.

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.

Differential therapeutic effects of anti-VEGF-A antibody in different tumor models: implications for choosing appropriate tumor models for drug testing.

We previously reported that the host response to certain chemotherapies can induce primary tumor regrowth, angiogenesis, and even metastases in mice, but the possible impact of anti-VEGF-A therapy in this context has not been fully explored. We, therefore, used combinations of anti-VEGF-A with chemotherapy on various tumor models in mice, including primary tumors, experimental lung metastases, and spontaneous lung metastases of 4T1-breast and CT26-colon murine cancer cell lines. Our results show that a combined treatment with anti-VEGF-A and folinic acid/5-fluorouracil/oxaliplatin (FOLFOX) but not with anti-VEGF-A and gemcitabine/cisplatinum (Gem/CDDP) enhances the treatment outcome partly due to reduced angiogenesis, in both primary tumors and experimental lung metastases models. However, neither treatment group exhibited an improved treatment outcome in the spontaneous lung metastases model, nor were changes in endothelial cell numbers found at metastatic sites. As chemotherapy has recently been shown to induce tumor cell invasion, we tested the invasion properties of tumor cells when exposed to plasma from FOLFOX-treated mice or patients with cancer. While plasma from FOLFOX-treated mice or patients induced invasion properties of tumor cells, the combination of anti-VEGF-A and FOLFOX abrogated these effects, despite the reduced plasma VEGF-A levels detected in FOLFOX-treated mice. These results suggest that the therapeutic impact of antiangiogenic drugs varies in different tumor models, and that anti-VEGF-A therapy can block the invasion properties of tumor cells in response to chemotherapy. These results may implicate an additional therapeutic role for anti-VEGF-A when combined with chemotherapy.

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.

Lysyl oxidase-like-2 promotes tumour angiogenesis and is a potential therapeutic target in angiogenic tumours.

Lysyl oxidase-like 2 (LOXL2), a secreted enzyme that catalyzes the cross-linking of collagen, plays an essential role in developmental angiogenesis. We found that administration of the LOXL2-neutralizing antibody AB0023 inhibited bFGF-induced angiogenesis in Matrigel plug assays and suppressed recruitment of angiogenesis promoting bone marrow cells. Small hairpin RNA-mediated inhibition of LOXL2 expression or inhibition of LOXL2 using AB0023 reduced the migration and network-forming ability of endothelial cells, suggesting that the inhibition of angiogenesis results from a direct effect on endothelial cells. To examine the effects of AB0023 on tumour angiogenesis, AB0023 was administered to mice bearing tumours derived from SKOV-3 ovarian carcinoma or Lewis lung carcinoma (LLC) cells. AB0023 treatment significantly reduced the microvascular density in these tumours but did not inhibit tumour growth. However, treatment of mice bearing SKOV-3-derived tumours with AB0023 also promoted increased coverage of tumour vessels with pericytes and reduced tumour hypoxia, providing evidence that anti-LOXL2 therapy results in the normalization of tumour blood vessels. In agreement with these data, treatment of mice bearing LLC-derived tumours with AB0023 improved the perfusion of the tumour-associated vessels as determined by ultrasonography. Improved perfusion and normalization of tumour vessels after treatment with anti-angiogenic agents were previously found to improve the delivery of chemotherapeutic agents into tumours and to result in an enhancement of chemotherapeutic efficiency. Indeed, treatment with AB0023 significantly enhanced the anti-tumourigenic effects of taxol. Our results suggest that inhibition of LOXL2 may prove beneficial for the treatment of angiogenic tumours.