Antiangiogenic activity of trabectedin in myxoid liposarcoma: Involvement of host TIMP‐1 and TIMP‐2 and tumor thrombospondin‐1

Trabectedin is a marine natural product, approved in Europe for the treatment of soft tissue sarcoma and relapsed ovarian cancer. Clinical and experimental evidence indicates that trabectedin is particularly effective against myxoid liposarcomas where response is associated to regression of capillary networks. Here, we investigated the mechanism of the antiangiogenic activity of trabectedin in myxoid liposarcomas. Trabectedin directly targeted endothelial cells, impairing functions relying on extracellular matrix remodeling (invasion and branching morphogenesis) through the upregulation of the inhibitors of matrix metalloproteinases TIMP‐1 and TIMP‐2. Increased TIMPs synthesis by the tumor microenvironment following trabectedin treatment was confirmed in xenograft models of myxoid liposarcoma. In addition, trabectedin upregulated tumor cell expression of the endogenous inhibitor thrombospondin‐1 (TSP‐1, a key regulator of angiogenesis‐dependent dormancy in sarcoma), in in vivo models of myxoid liposarcomas, in vitro cell lines and primary cell cultures from patients' myxoid liposarcomas. Chromatin Immunoprecipitation analysis showed that trabectedin displaced the master regulator of adipogenesis C/EBPβ from the TSP‐1 promoter, indicating an association between the up‐regulation of TSP‐1 and induction of adipocytic differentiation program by trabectedin. We conclude that trabectedin inhibits angiogenesis through multiple mechanisms, including directly affecting endothelial cells in the tumor microenvironment—with a potentially widespread activity—and targeting tumor cells' angiogenic activity, linked to a tumor‐specific molecular alteration.     

Clinical significance of programmed death‐1 and programmed death‐ligand 1 expression in the tumor microenvironment of clear cell renal cell carcinoma

Recently, immunotherapy based on blocking immune checkpoints with programmed death‐1 (PD‐1) or PD‐ligand 1 (PD‐L1) Abs has been introduced for the treatment of advanced clear cell renal cell carcinoma (ccRCC), especially tumors resistant to vascular endothelial growth factor‐tyrosine kinase inhibitors (VEGF‐TKIs), but the significance of their expression in the tumor microenvironment is unclear. We investigated these immune checkpoint markers in tumor cells and tumor‐infiltrating immune cells (TIIC) in the tumor microenvironment of 100 untreated and 25 VEGF‐TKI‐treated primary ccRCC tissues. Upregulated expression of PD‐1 and PD‐L1 by TIIC, and PD‐L1 by tumor cells was associated with the histological grade and unfavorable prognosis of RCC patients. High PD‐1 and PD‐L1 expression by TIIC was associated with a poorer response to VEGF‐TKI, whereas PD‐L1 expression by tumor cells did not affect the efficacy of the treatment. Furthermore, increased PD‐1‐positive TIIC and PD‐L1‐positive TIIC were observed in tumors treated with VEGF‐TKIs compared with those in untreated tumors. Our data suggest that PD‐1 and PD‐L1 expression by TIIC in the tumor microenvironment is involved in treatment resistance, and that sequential therapy with immune checkpoint inhibitors could be a promising therapeutic strategy for ccRCC resistant to VEGF‐TKI treatment.     

Activating NK cell receptor ligands are differentially expressed during progression to cervical cancer

Human papillomavirus-induced cervical carcinomas often show impaired expression of MHC class I molecules resulting in the inability of tumor cells to directly present viral peptides to cytotoxic T lymphocytes. Loss of MHC class I expression combined with the expression of activating NK cell receptor ligands renders tumor cells potentially susceptible to NK cell attack. Thus, in this study, we analyzed the expression of activating NK cell receptor ligands, NK cell accumulation and activation status in situ in normal ectocervical tissue (NCT), cervical intraepithelial neoplasia (CIN) and squamous cervical carcinoma (CxCa). We observed that expression of the DNAM-1 ligand CD155 was frequently upregulated in CxCa, but not in CIN. The NKG2D ligand MICA was upregulated in fewer CxCa biopsies. In contrast, another NKG2D ligand ULBP2 was preferentially expressed in differentiated epithelial cells of NCT. Increased numbers of NK cells were detected in CIN as compared to NCT and CxCa. Expression of activating NK cell receptor ligands combined with loss of MHC class I was not correlated with enhanced NK cell accumulation or activation status. Furthermore, we demonstrate that cervical cancer cell lines are killed by the NK cell line, NKL, in a NKG2D- and DNAM-1-dependent manner in vitro. Since a significant number of CxCa biopsies showed low MHC class I expression combined with high expression of one or more of the tested activating NK cell receptor ligands, we conclude that CxCa might be a promising target for NK cell-based adoptive immunotherapy.     

Intratumoural expression of TNF-R1 and EMAP-II in relation to response of patients treated with TNF-based isolated limb perfusion

Tumour necrosis factor-alpha (TNF) has been used in the clinic for more than 10 years in an isolated limb perfusion (ILP). However, intra-tumoural expression of TNF receptor-1 (TNF-R1) and TNF-R1 upregulating factors are unknown. We determined the expression of TNF-R1, proEMAP and endothelial monocyte-activating polypeptide-II (EMAP-II) before and after ILP and evaluated this against clinical response. Tumour biopsies were taken before and after ILP of patients (n = 27) with advanced sarcoma or metastatic melanoma. Biopsies were randomly analysed by western blotting for proEMAP/EMAP-II and TNF-R1 expression. Appropriate melanoma biopsies were stained for EMAP-II, TNF-R1, CD31 and CD68. For melanomas we found that an up-regulation of EMAP-II, in contrast to proEMAP or TNF-R1, directly after ILP significantly correlated with a complete tumour response. No correlation was found for sarcoma patients. In a comparative analysis we found that the overall proEMAP and EMAP-II expression was higher in melanoma as compared to sarcoma cases and measurements in cell lines revealed high proEMAP expression by melanoma cells. We report high EMAP-II expression by endothelial cells and association with macrophages. In addition, macrophages are recruited to vessel-remnants after ILP. An upregulation of EMAP-II directly after ILP of melanoma patients correlates with and might predict a complete response to TNF-based ILP. The association of macrophages with EMAP-II expression and vascular damage suggests a role for EMAP-II in regulating the TNF-based anti-tumour effects observed with an ILP. Analysis of EMAP-II expression in melanoma biopsies should be implemented in the ILP procedure.     

Intra-tumoral cytolytic cells: Pattern of distribution in B-cell non Hodgkin’s lymphoma

Non-Hodgkin s lymphomas (NHLs) constitute a heterogeneous group of lymphoid neoplasms and a majority of them in India are of B-cell phenotype. Varying numbers of T lymphocytes and natural killer (NK) cells are consistently present within the lymph nodes (LNs). The role of these reactive cells is becoming understood. TIA-1 is a cytotoxic granule associated RNA binding protein, the expression of which is restricted to cytotoxic T lymphocytes (CTLs) and NK cells. Snap frozen lymph node biopsies obtained from 41 B-cell NHLs were localized for intra-tumoral TIA-1 + cytolytic cells by immunohistochemistry. Distribution of T cell subsets and NK cells were also quantified. Cells expressing TIA-1 antigen was observed in all the cases, seen as a strong granular cytoplasmic signal. Results indicate significantly higher number of TIA-1 cytolytic cells outside (periphery of the follicle and interfollicular areas) than within the neoplastic follicle in follicular lymphomas (p<0.001). In small lymphocytic lymphomas, cytolytic cells were mainly seen as uniformly scattered single cells, distributed throughout the tumor environment. In mantle cell and diffuse large B-cell lymphomas these were most often seen as small clusters and less frequently as singly scattered cells. Higher numbers of CD4 + than the CD8 + T cells were observed in most cases. Contrary to the follicles in follicular hyperplasia, CD57 + NK cells were predominantly observed outside the neoplastic follicle in follicular lymphomas (FLs). These results outline specific interactions between the potential anti-tumoral cytolytic and the malignant cells of B-cell NHLs.     

The inflammatory cytokine tumor necrosis factor-alpha regulates chemokine receptor expression on ovarian cancer cells

Epithelial ovarian cancer cells express the chemokine receptor, CXCR4, which may be associated with increased survival and metastatic potential, but the regulation of this receptor is not understood. The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is found in ovarian cancer biopsies and is associated with increased tumor grade. In this report, we show that CXCR4 expression on human epithelial ovarian cancer cells is associated with, and can be modulated by, TNF-alpha. Ovarian cancer cells with high endogenous expression of TNF-alpha expressed higher levels of CXCR4 mRNA and protein than cells with low TNF-alpha expression. Stimulation of ovarian cancer cell lines and primary epithelial cancer cells with TNF-alpha resulted in increased CXCR4 mRNA and protein. The TNF-alpha-stimulated increase in CXCR4 mRNA was due partly to de novo synthesis, and up-regulation of CXCR4 cell surface protein increased migration to the CXCR4 ligand CXCL12. CXCR4 mRNA and protein was down-regulated by anti-TNF-alpha antibody or by targeting TNF-alpha mRNA using RNAi. TNF-alpha stimulation activated components of the nuclear factor kappaB pathway, and overexpression of the inhibitor of kappaB also reduced CXCR4 expression. Coculture of macrophages with ovarian cancer cells also resulted in cancer cell up-regulation of CXCR4 mRNA in a TNF-alpha-dependent manner. Finally, there was a correlation between the levels of TNF-alpha and CXCR4 mRNA in clinical biopsies of ovarian cancer, and TNF-alpha protein was expressed in CXCR4-positive tumor cells. TNF-alpha is a critical mediator of tumor promotion in a number of experimental cancers. Our data suggest that one mechanism may be through nuclear factor kappaB-dependent induction of CXCR4.     

Copper binding by tetrathiomolybdate attenuates angiogenesis and tumor cell proliferation through the inhibition of superoxide dismutase 1.

PURPOSE: A second-generation tetrathiomolybdate analogue (ATN-224; choline tetrathiomolybdate), which selectively binds copper with high affinity, is currently completing two phase I clinical trials in patients with advanced solid and advanced hematologic malignancies. However, there is very little information about the mechanism of action of ATN-224 at the molecular level. EXPERIMENTAL DESIGN: The effects of ATN-224 on endothelial and tumor cell growth were evaluated in cell culture experiments in vitro. The antiangiogenic activity of ATN-224 was investigated using the Matrigel plug model of angiogenesis. RESULTS: ATN-224 inhibits superoxide dismutase 1 (SOD1) in tumor and endothelial cells. The inhibition of SOD1 leads to inhibition of endothelial cell proliferation in vitro and attenuation of angiogenesis in vivo. The inhibition of SOD1 activity in endothelial cells is dose and time dependent and leads to an increase in the steady-state levels of superoxide anions, resulting in the inhibition of extracellular signal-regulated kinase phosphorylation without apparent induction of apoptosis. In contrast, the inhibition of SOD1 in tumor cells leads to the induction of apoptosis. The effects of ATN-224 on endothelial and tumor cells could be substantially reversed using Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, a catalytic small-molecule SOD mimetic. CONCLUSIONS: These data provide a distinct molecular target for the activity of ATN-224 and provide validation for SOD1 as a target for the inhibition of angiogenesis and tumor growth.     

Mechanism of antitumor activity of tumor necrosis factor alpha with hyperthermia in a tumor necrosis factor alpha-resistant tumor

Cells from a radiation-induced fibrosarcoma (RIF-1) are exceedingly resistant to tumor necrosis factor alpha (TNF-alpha) in vitro. We tested whether the addition of mild hyperthermia (42.5 degrees C, 30 minutes) could enhance TNF-alpha activity against RIF-1 tumors growing in syngeneic hosts (C3H mice). TNF-alpha was administered intratumorally. Tumor cell killing essentially was not measurable following TNF-alpha, hyperthermia, or a combination of the two. Single-modality treatments also had no effect on tumor growth delay or on the x-ray dose (given 24 hours after the primary treatment) required to sterilize 50% of the tumors. The combination of TNF-alpha and hyperthermia, however, resulted in a marked increase in tumor doubling time and a highly significant reduction in the x-ray dose required to sterilize the tumors. Syngeneic lymph nodal lymphocytes and blood leukocytes did not appear to mediate the action of TNF-alpha on RIF-1 cells in vitro. Necrosis and hemorrhage were the most prominent histopathological alterations in the treated tumors. Electron microscopic studies 6 hours after therapy showed increased damage to capillary endothelial cells and accumulation of neutrophils in the capillaries of tumors treated with TNF-alpha with or without heat, suggesting that neutrophils may mediate the endothelial cell injury. These observations indicate a greater than additive tumoricidal effect of TNF-alpha with hyperthermia. Furthermore, they support the concept that the interaction between the two agents damages the vasculature, compromising the microcirculation and ultimately causing ischemic tumor necrosis.     

Inhibition of tumor growth by systemic treatment with thrombospondin-1 peptide mimetics

Many normal human cells produce thrombospondin-1 (TSP-1), a potent antiangiogenic protein that promotes vascular quiescence. In various organ systems, including the brain, breast and bladder and in fibroblasts, TSP-1 secretion is reduced during tumorigenesis, thereby allowing induction of the vigorous neovascularization required for tumor growth and metastasis. Full-length and short TSP-1-derived peptides inhibit angiogenesis by inducing endothelial cell apoptosis and thus disrupting the vasculature of the growing tumor. CD36 expressed on the surface of endothelial cells functions as the primary antiangiogenic receptor for TSP-1. A D-isoleucyl enantiomer of a TSP-1 heptapeptide specifically inhibits the proliferation and migration of capillary endothelial cells. DI-TSP, an approximately 1 kDa capped version of this peptide, is also antiangiogenic in vitro, with a specific activity approaching that of the 450 kDa parental molecule. Here, we show that DI-TSP delivered systemically dose-dependently inhibits the growth of murine melanoma metastases in syngeneic animals and that its more soluble isomer, DI-TSPa, similarly blocks the progression of primary human bladder tumors in an orthotopic model in immune-deficient mice. Like intact TSP-1, these peptide mimetics had no effect on cancer cells growing in vitro but markedly suppressed the growth of endothelial cells by inducing receptor-dependent apoptosis. Antibodies raised against CD36 blocked the ability of peptides to induce apoptosis in endothelial cells but had no effect on tumor necrosis factor-alpha-induced apoptosis. In vivo, the peptide mimetics were associated with a significantly reduced microvessel density and increased apoptotic indices in both the endothelial and tumor cell compartments. Such short peptides targeted to a specific antiangiogenic receptor, potent and easy to synthesize, show great promise as lead compounds in clinical antiangiogenic strategies.     

Phase II study of the antiangiogenic agent SU5416 in patients with advanced soft tissue sarcomas.

PURPOSE: SU5416 (semaxanib) is a small molecule inhibitor of the vascular endothelial growth factor (VEGF) receptor-2 and KIT receptor tyrosine kinases. This Phase II study was conducted to investigate the safety and efficacy of SU5416 for patients with soft tissue sarcomas. EXPERIMENTAL DESIGN: Thirteen patients with locally advanced or metastatic soft tissue sarcomas were treated with SU5416 via intravenous infusion at a dose of 145 mg/m(2) twice weekly. In selected cases tumor biopsies were taken before and after 2 months of treatment. RESULTS: The median progression-free survival was 1.8 months. Median overall survival was 22.8 months. No objective tumor responses were observed. There was evidence of shorter survival among patients with high baseline urine VEGF levels (P = 0.04). No grade 4 toxicities were observed. The most common grade 3 toxicities were headache and thrombosis. Other less serious toxicities included fatigue, nausea, and abdominal pain. The median systolic blood pressure increased from 118 mmHg at baseline to 133 after 1 month of treatment (P = 0.01). Post-treatment tumor biopsies showed no significant decreases in VEGF receptor phosphorylation compared with baseline in 3 evaluable patients. One patient with gastrointestinal stromal tumor who had rapid progression during SU5416 treatment was subsequently treated with another KIT inhibitor, imatinib mesylate, and had a partial response lasting >36 months. CONCLUSIONS: SU5416 was relatively well tolerated but did not demonstrate significant antitumor activity against advanced soft tissue sarcoma. Correlative studies suggest that VEGF receptor or KIT inhibition was incomplete in at least some cases, providing a possible explanation for the observed lack of activity.     

Production of tumor necrosis factor-alpha and interferon-gamma from human peripheral blood lymphocytes by MGN-3, a modified arabinoxylan from rice bran, and its synergy with interleukin-2 in vitro

Recently, we presented evidence for the role of MGN-3, an enzymatically modified arabinoxylan extracted from rice bran, in potent activation of human natural killer (NK) cell function in vivo and in vitro. In the current study, we examined the mechanism by which MGN-3 elevated NK cytotoxic activity. We did this by testing the action of MGN-3 on the levels of both tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) secretions and MGN-3 function on the expression of key cell surface receptors. Peripheral blood lymphocytes were treated with MGN-3 at concentrations of 0.1 mg/ml and 1 mg/ml, and supernatants were subjected to enzyme-linked immunosorbent assay. Results showed that MGN-3 is a potent TNF-alpha inducer. The effect was dose-dependent. MGN-3 concentration at 0.1 and 1 mg/ml increased TNF-alpha production by 22.8- and 47. 1-fold, respectively. MGN-3 also increased production of IFN-gamma but at lower levels as compared to TNF-alpha With respect to key cell surface receptors, MGN-3 increases the expression of CD69, an early activation antigen at 16 hours after treatment. Furthermore, the interleukin-2 receptor CD25 and the adhesion molecule ICAM-1 (CD54) were upregulated after treatment with MGN-3. Treating highly purified NK cells with MGN-3 also resulted in increased levels of TNF-alpha and IFN-gamma secretion in conjunction with augmentation of NK cell cytotoxic function. Furthermore, addition of MGN-3 to interleukin-2-activated NK cells resulted in a synergistic induction of TNF-alpha and IFN-gamma secretion. Overall, our data suggest that MGN-3, a novel biological response modifier, can be used as a safe alternative or as an adjuvant to the existing immunotherapeutic modalities.     

Modulatory effects of adiponectin on the polarization of tumor‐associated macrophages

The plasticity of macrophages with selective functional phenotypes partially arises in respective to their microenvironment. Tumor‐associated macrophages (TAMs) may promote disease progression with tumor specific manner. Here we report that in pediatric malignant soft‐tissue tumors, the presence of TAMs and expression of adiponectin (APN) are heterogeneous. Both APN and TAMs had high expression in rhabdomyosarcoma, especially in the malignant subtype, alveolar rhabdomyosarcoma. To investigate the mode of action of APN on TAM activation, a murine MN/MCA1 sarcoma model was used. The Results revealed that exogenous APN had no effect on MN/MCA1 proliferation but tumor size was markedly reduced in apn^?/? mice versus WT controls. The accumulation of TAMs in apn^?/? mice was also reduced which correlated to downregulated serum levels of MCP‐1. Likewise, TAMs in apn^?/? mice exhibited a M1‐like phenotype, characterized by increase in MHC II^high population and M1 phenotypic markers, such as iNOS gene and serum TNF‐α accompanied by a decrease in M2 markers, namely YM1 gene and serum IL‐10. In addition, APN deficiency increased the number of CD4⁺ T cells, CD8⁺ T cells and NK cells in tumors and reduced tumor metastasis. The altered phenotype of TAMs in apn^?/? mice was associated with a marked decrease in phospho‐p38 and treatment with a p38 MAPK inhibitor significantly reduced tumor size and increased MHC II expression on TAMs in WT mice, implying p38 MAPK signaling pathway may contribute to APN‐mediated TAM polarization. Collectively, our findings suggest that APN may have a potential role in regulating soft tissue sarcoma growth.     

Lymphokine-activated killer activity of tumor-associated and peripheral blood lymphocytes isolated from patients with ascites ovarian tumors

Peripheral blood lymphocytes (PBLs) and tumor-associated lymphocytes (TALs) were isolated from 36 patients with advanced ovarian adenocarcinoma and peritoneal effusions for study of lymphokine-activated killer activity. PBLs and TALs cultured in vitro for 3-5 days in the presence of interleukin-2 (IL-2, supernatant of the MLA 144 gibbon cell line, or human recombinant IL-2) expressed higher levels of cytotoxicity as compared to cells cultured in medium alone, against natural killer (NK)-susceptible (K562) or NK-resistant targets (Daudi and the human ovarian carcinoma cell line SW626). When ovarian tumor cells, freshly isolated from carcinomatous ascites or surgical specimens, were used as target cells in the cytotoxicity assay, 8 of 14 PBLs and 5 of 7 TAL preparations lysed the autologous tumor after treatment with IL-2, while no spontaneous reactivity was observed in any of the 14 patients tested. Although levels of lysis were usually relatively low, these data demonstrate that PBLs and TALs from ovarian cancer patients (TALs usually exhibiting low NK activity) when stimulated in vitro by IL-2 acquire some cytotoxic potential against the autologous tumor.