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Extracellular vesicles (EVs) are effective mediators of intercellular communications between enterocytes and immune cells. The current study showed that EVs isolated from mouse and human intestinal organoids modulated inflammatory responses of various immune cells including mouse bone-marrow derived-macrophages, dendritic cells, microglia cells, and human monocytes. EVs suppressed LPS-elicited cytokine production in these cells while morphine abolished EVs' immune modulatory effects. Microarray analysis showed that various microRNAs, especially Let-7, contributed to EV-mediated immune modulation. Using murine models, we showed that injection of EVs derived from intestinal organoids reduced endotoxin-induced systemic inflammation and alleviated the symptoms of DSS-induced colitis. EVs derived from morphine-treated organoids failed to suppress the immune response in both these models. Our study suggests that EVs derived from intestinal crypt cells play crucial roles in maintaining host homeostasis and opioid use is a risk factor for exacerbating inflammation in patients with inflammatory diseases such as sepsis and colitis.

Heat shock protein 70 (Hsp70), a protein chaperone, is known to promote cell survival and tumor progression. However, its role in the tumor microenvironment (TME) is largely unknown. We specifically evaluated Hsp70 in the TME by implanting tumors in wild-type (WT) controls or Hsp70−/- animals, thus creating a TME with or without Hsp70. Loss of Hsp70 led to significantly smaller tumors; there were no differences in stromal markers, but interestingly, depletion of CD8 + T-cells abrogated this tumor suppressive effect, indicating that loss of Hsp70 in the TME affects tumor growth through the immune cells. Compared to WT, adoptive transfer of Hsp70−/- splenocytes exhibited greater antitumor activity in immunodeficient NSG and Rag 1−/- mice. Hsp70−/- dendritic cells showed increased expression of MHCII and TNF-α both in vitro and in vivo. These results suggest that the absence of Hsp70 in the TME inhibits tumors through increased dendritic cell activation. Hsp70 inhibition in DCs may emerge as a novel therapeutic strategy against pancreatic cancer.

BACKGROUND RHD is the leading cause of morbidity and mortality in the underdeveloped world. The burden of RHD has been estimated variably depending on the method and tool of examination. Recent echocardiography-based studies, generally accepted as the best tools to detect RHD, had shown 10-fold increased prevalence of RHD compared to clinical examination albeit fear for overestimation of the disease. While studies using more stringent criteria of WHF 2012 are limited and no such study has been conducted in this part of the country, we felt it necessary to conduct such a study in this hilly state of India. MATERIALS AND METHODS This is a community based cross sectional study in which each and every selected child aged 5-15 years from randomly selected schools of Manipur were examined physically and by 2D colour Doppler Echocardiography. The anthropometric parameters, clinical details and echocardiography findings were all recorded. Echocardiography loops were recorded for review by another cardiologist later. Analysis was done by using T test, descriptive statistics and with 95% confidence interval. All the analysis was done using STATA 13.0 (Stata Corp, USA). RESULTS 3600 children were screened in two years. The mean age was 11.77 years ± 2.50 SD, 1865 (51.8%) were male, 1442 (40%) were from government school and majority belonged to rural population (67%). Only 1 case of Clinical RHD with a combination of MR and AS was found giving a prevalence of 0.28/1000 (CI: 0.04-1.97). Echocardiography detected 3 cases of definite subclinical (prevalence rate of 0.83/1000 (CI: 0.27-2.58) and 14 cases of borderline subclinical RHD (prevalence rate of 3.9/1000 (CI: 2.30-6.56) befitting WHF 2012 criteria. CONCLUSION Prevalence of clinical RHD (0.28/1000) is very low compared to that of other Indian states. Using echocardiography, the prevalence becomes several folds higher compared to clinical examination alone, 0.83/1000 for Definite Subclinical and 3.9/1000 for Borderline Subclinical RHD. Further follow up studies using less stringent criteria (modified WHO criteria), may still have a role in detection of true burden of RHD in our community from the public health point of view.

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. Metastasis is the major cause of TNBC mortality. Angiogenesis facilitates TNBC metastases. Many TNBCs also form vascular channels lined by tumor cells rather than endothelial cells, known as ‘vasculogenic mimicry’ (VM). VM has been linked to metastatic TNBC behavior and resistance to anti-angiogenic agents. Epidermal growth factor receptor (EGFR) is frequently expressed on TNBC, but anti-EGFR antibodies have limited efficacy. We synthesized an anti-EGFR antibody–endostatin fusion protein, αEGFR IgG1-huEndo-P125A (αEGFR-E-P125A), designed to deliver a mutant endostatin, huEndo-P125A (E-P125A), to EGFR expressing tumors, and tested its effects on angiogenesis, TNBC VM, and motility in vitro, and on the growth and metastasis of two independent human TNBC xenograft models in vivo. αEGFR-E-P125A completely inhibited the ability of human umbilical vein endothelial cells to form capillary-like structures (CLS) and of TNBC cells to engage in VM and form tubes in vitro. αEGFR-E-P125A treatment reduced endothelial and TNBC motility in vitro more effectively than E-P125A or cetuximab, delivered alone or in combination. Treatment of TNBC with αEGFR-E-P125A was associated with a reduction in cytoplasmic and nuclear β-catenin and reduced phosphorylation of vimentin. αEGFR-E-P125A treatment of TNBC xenografts in vivo inhibited angiogenesis and VM, reduced primary tumor growth and lung metastasis of orthotopically implanted MDA-MB-468 TNBC cells, and markedly decreased lung metastases following intravenous injection of MDA-MB-231-4175 lung-tropic TNBC cells. Combined inhibition of angiogenesis, VM, and TNBC motility mediated by αEGFR-E-P125A is a promising strategy for the prevention of TNBC metastases.

SUMMARY Despite wide use of anti-vascular endothelial growth factor (VEGF) therapy for many solid cancers, most individuals become resistant to this therapy, leading to disease progression. Therefore, new biomarkers and strategies for blocking adaptive resistance of cancer to anti-VEGF therapy are needed. As described here, we demonstrate that cancer-derived small extracellular vesicles package increasing quantities of VEGF and other factors in response to anti-VEGF therapy. The packaging process of VEGF into small extracellular vesicles (EVs) is mediated by the tetraspanin CD63. Furthermore, small EV-VEGF (eVEGF) is not accessible to anti-VEGF antibodies and can trigger intracrine VEGF signaling in endothelial cells. eVEGF promotes angiogenesis and enhances tumor growth despite bevacizumab treatment. These data demonstrate a mechanism where VEGF is partitioned into small EVs and promotes tumor angiogenesis and progression. These findings have clinical implications for biomarkers and therapeutic strategies for ovarian cancer., Graphical abstract, In brief Ma et.al report that cancer-cell-derived small EVs contain increasing amounts of VEGF (eVEGF) and contribute to resistance to anti-VEGF therapy (AVT). CD63 is a potential mediator that regulates packaging of VEGF into small EVs. eVEGF can trigger intracrine VEGF signaling in endothelial cells and promote angiogenesis despite AVT.

Background and Aims Opioids are the most prescribed analgesics for pain in inflammatory bowel diseases [IBD]; however, the consequences of opioid use on IBD severity are not well defined. This is the first study investigating consequences of hydromorphone in both dextran sodium sulphate [DSS]-induced colitis and spontaneous colitis (IL-10 knockout [IL-10-/-]) mouse models of IBD. Methods To determine the consequences of opioids on IBD pathogenesis, wild-type [WT] mice were treated with clinically relevant doses of hydromorphone and colitis was induced via 3% DSS in drinking water for 5 days. In parallel we also determined the consequences of opioids in a spontaneous colitis model. Results Hydromorphone and DSS independently induced barrier dysfunction, bacterial translocation, disruption of tight junction organisation and increased intestinal and systemic inflammation, which were exacerbated in mice receiving hydromorphone in combination with DSS. Hydromorphone + DSS-treated mice exhibited significant microbial dysbiosis. Predictive metagenomic analysis of the gut microbiota revealed high abundance in the bacterial communities associated with virulence, antibiotic resistance, toxin production, and inflammatory properties. Hydromorphone modulates tight junction organisation in a myosin light chain kinase [MLCK]-dependent manner. Treatment with MLCK inhibitor ML-7 ameliorates the detrimental effects of hydromorphone on DSS-induced colitis and thus decreases severity of IBD. Similarly, we demonstrated that hydromorphone treatment in IL-10-/- mice resulted in accelerated clinical manifestations of colitis compared with control mice. Conclusions Opioids used for pain management in IBD accelerate IBD progression by dysregulation of the gut microbiota, leading to expansion of pathogenic bacteria, translocation of bacteria, immune deregulation and sustained inflammation.

BACKGROUND: Even after surgical resection, most patients with localized pancreatic ductal adenocarcinoma (PDAC) succumb to disease recurrence. Current animal models do not recapitulate this pattern of disease recurrence. Our goal was to develop a clinically relevant, immunocompetent model of PDAC resection to study recurrence and evaluate therapy. METHODS: Pancreatic cancer cells derived from tumors arising in KPC (LSL-Kras(G12D/+;) LSL-Trp53(R172H/+;) Pdx-1-Cre) mice were co-injected with stromal cells (pancreatic stellate cells) into the pancreas of immunocompetent mice to simulate the stroma rich tumors seen in human PDAC. After allowing tumors to form, we resected these localized tumors and followed the mice for tumor recurrence. Circulating tumor cells (CTCs) were isolated, and systemic chemotherapy or immunotherapy was administered following tumor resection. RESULTS: Tumors formed by co-injection of KPC cells and stromal cells demonstrated a dense desmoplastic reaction similar to that seen in human disease. Resection at days 15 and 21 after implantation revealed uniform tumor volumes of 92 ± 19 mm(3) on day 15 and 444 ± 54 mm(3) on Day 21. Histology of resected tumors showed negative margins. Resembling human PDAC, mice that underwent resection showed improved median survival (58 vs 47 days) but most animals developed intra-abdominal recurrence on follow up. Adjuvant chemotherapy (Median survival 69 vs 58 days), but not immunotherapy (Median Survival: 69 vs 65 days) tended towards improved survival as seen in human disease. Circulating-Tumor-Cells were reliably identified from mice with and without resection, suggesting utility of this model in studying tumor metastases and recurrence. CONCLUSION: We describe an immunocompetent animal model that recapitulates human disease in morphology and recurrence patterns. We show that it can be used to evaluate therapy in clinical scenarios associated with surgical resection and may help characterize factors responsible for disease recurrence.

Purpose: Pancreatic cancer stromal microenvironment is considered to be the major reason for failure of conventional and targeted therapy for this disease. The desmoplastic stroma, comprising mainly collagen and glycosaminoglycans like hyaluronan (HA), is responsible for compression of vasculature in the tumor resulting in impaired drug delivery and poor prognosis. Minnelide, a water-soluble prodrug of triptolide currently in phase I clinical trial, has been very effective in multiple animal models of pancreatic cancer. However, whether Minnelide will have efficacious delivery into the tumor despite the desmoplastic stroma has not been evaluated before. Experiment Design: Patient tumor-derived xenografts (PDX) and spontaneous pancreatic cancer mice were treated with 0.42 and 0.21 mg/kg body weight for 30 days. Stromal components were determined by IHC and ELISA-based assays. Vascular functionality and drug delivery to the tumor were assessed following treatment with Minnelide. Result: Our current study shows that treatment with Minnelide resulted in reduction of ECM components like HA and collagen in the pancreatic cancer stroma of both the spontaneous KPC mice as well as in patient tumor xenografts. Furthermore, treatment with Minnelide improved functional vasculature in the tumors resulting in four times more functional vessels in the treated animals compared with untreated animals. Consistent with this observation, Minnelide also resulted in increased drug delivery into the tumor compared with untreated animals. Along with this, Minnelide also decreased viability of the stromal cells along with the tumor cells in pancreatic adenocarcinoma. Conclusions: In conclusion, these results are extremely promising as they indicate that Minnelide, along with having anticancer effects is also able to deplete stroma in pancreatic tumors, which makes it an effective therapy for pancreatic cancer. Clin Cancer Res; 22(2); 415–25. ©2015 AACR.

Background & Aims Immunotherapies are ineffective against pancreatic cancer. We investigated whether the activity of nuclear factor (NF)κB in pancreatic stromal cells contributes to an environment that suppresses antitumor immune response. Methods Pancreata of C57BL/6 or Rag1–/– mice were given pancreatic injections of a combination of KrasG12D/+; Trp53 R172H/+; Pdx-1cre (KPC) pancreatic cancer cells and pancreatic stellate cells (PSCs) extracted from C57BL/6 (control) or mice with disruption of the gene encoding the NFκB p50 subunit (Nfkb1 or p50–/– mice). Tumor growth was measured as an endpoint. Other mice were given injections of Lewis lung carcinoma (LLC) lung cancer cells or B16-F10 melanoma cells with control or p50–/– fibroblasts. Cytotoxic T cells were depleted from C57BL/6 mice by administration of antibodies against CD8 (anti-CD8), and growth of tumors from KPC cells, with or without control or p50–/– PSCs, was measured. Some mice were given an inhibitor of CXCL12 (AMD3100) and tumor growth was measured. T-cell migration toward cancer cells was measured using the Boyden chamber assay. Results C57BL/6 mice coinjected with KPC cells (or LLC or B16-F10 cells) and p50–/– PSCs developed smaller tumors than mice given injections of the cancer cells along with control PSCs. Tumors that formed when KPC cells were injected along with p50–/– PSCs had increased infiltration by activated cytotoxic T cells along with decreased levels of CXCL12, compared with tumors grown from KPC cells injected along with control PSCs. KPC cells, when coinjected with control or p50–/– PSCs, developed the same-size tumors when CD8+ T cells were depleted from C57BL/6 mice or in Rag1–/– mice. The CXCL12 inhibitor slowed tumor growth and increased tumor infiltration by cytotoxic T cells. In vitro expression of p50 by PSCs reduced T-cell migration toward and killing of cancer cells. When cultured with cancer cells, control PSCs expressed 10-fold higher levels of CXCL12 than p50–/– PSCs. The CXCL12 inhibitor increased migration of T cells toward KPC cells in culture. Conclusions In studies of mice and cell lines, we found that NFκB activity in PSCs promotes tumor growth by increasing expression of CXCL12, which prevents cytotoxic T cells from infiltrating the tumor and killing cancer cells. Strategies to block CXCL12 in pancreatic tumor cells might increase antitumor immunity.

Minnelide is a water-soluble pro-drug of triptolide, a natural product. The goal of this study was to evaluate the effectiveness of Minnelide on ovarian cancer growth in vitro and in vivo.The effect of Minnelide on ovarian cancer cell proliferation was determined by real time electrical impedance measurements. Multiple mouse models with C200 and A2780 epithelial ovarian cancer cell lines were used to assess the efficacy of Minnelide in inhibiting ovarian cancer growth.Minnelide decreased cell viability of both platinum sensitive and resistant epithelial ovarian cancer cells in vitro. Minnelide with carboplatin showed additive effects in vitro. Minnelide monotherapy increased the survival of mice bearing established ovarian tumors. Minnelide, in combination with carboplatin and paclitaxel, improved overall survival of mice.Minnelide is a promising pro-drug for the treatment of ovarian cancer, especially when combined with standard chemotherapy.

Background: Recent reports have elaborated on how the gut microbiota shape vertebrate immune system during ontogeny. Additionally, tumor-promoting inflammation and gut dysbiosis are often associated with multiple cancers including pancreatic ductal adenocarcinoma (PDAC). We sought to investigate the gut microbiota-immune relationship in mouse models of cancer. Methods: To investigate if cancer-associated dysbiosis modulates cancer per se, neonatal mice with unstable, dynamic gut microbiota (guests) were cohoused with either adult cancer-naive mice or adult pancreatic cancer-bearing KPC (KrasG12D/+;Trp53R172H/+;Pdx-1-Cre) mice. After 4 weeks of co-housing, guest mice were subcutaneously implanted with KPC PDAC and monitored for cancer progression. In a parallel series of experiments, tumor progression was compared between control mice and mice given oral antibiotics and also, between germ free mice and conventionalized mice. Later, oral antibiotics were combined with checkpoint inhibitors in preclinical trials of efficacy. Monoclonal antibodies and knockout strains of mice were used to elucidate mechanism. Pancreatic tumors were probed for bacteria. Results: Co-housing with KPC mice significantly increased cancer progression in guest mice compared to that in guest mice co-housed with non-cancer-bearing cagemates. Bowel sterilization dramatically reduced cancer burden in subcutaneous, metastatic, and orthotopic models of pancreatic cancer as well as in subcutaneous and metastatic models of melanoma. Similarly, germ free mice had decreased subcutaneous PDAC burden compared to littermate conventionalized mice. Surprisingly and in findings contradictory to previously published reports, oral antibiotics also potentiated the efficacy of checkpoint inhibitors in shrinking tumors. The tumor decreasing effect of antibiotics disappeared in immunodeficient Rag1-/- mice, wild type mice depleted of IL-17a but not in Tlr4-/- mice. Immunophenotyping further confirmed microbiota-mediated infiltration of immunosuppressive cells inside tumor and antibiotics-mediated activation of intratumoral Th1/Tc1 immunity. Interestingly, pancreatic cancer liver metastases revealed a significant presence of 16S rDNA resembling the gut flora and subcutaneous pancreatic cancer tissue grew viable bacteria on culture medium and this was prevented by oral antibiotics, thereby suggesting a gut-to-tumor translocation of bacteria. Conclusion: Our data suggest that gut microbiota-cancer cross talk is mediated by the immune system and that oral antibiotics may be repurposed as potent anti-cancer immunotherapeutic agents. Citation Format: Vrishketan Sethi, Saba Kurtom, Irina Fernandez, Maria Abreu, Sabita Roy, Sundaram Ramakrishnan, Ashok Saluja, Vikas Dudeja. Oral antibiotics act as potent immunotherapeutic agents against pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4956.

Previous studies have shown that a single point mutation in endostatin at position 125 (P125A) can improve the biological activity of endostatin. Addition of an integrin-targeting moiety, R-G-D, resulted in better localization to tumor vasculature and improved the anti-angiogenic activity of endostatin. Since endostatin has relatively shorter serum half-life, frequent dosing was required for inhibiting tumor growth. In the present study, we have genetically fused RGD-P125A-endostatin to Fc of IgG4 isotype and evaluated its anti-angiogenic and anti-tumor effects in athymic mice. Two genetic constructs were made, RGD-P125A-endostatin-Fc (RE-Fc) and P125A-endostatin-RGD-Fc (ER-Fc). Both constructs were cloned and expressed in mammalian cells. Purified fusion proteins inhibited endothelial cell migration and proliferation better than yeast derived P125A-endostatin. Both RE-Fc and ER-Fc inhibited ovarian cancer growth and were found to be as effective as Bevacizumab treatment. Fusion protein showed marked increased half-life. Combination treatment with Bevacizumab and ER-Fc showed additive inhibition of ovarian cancer growth. These studies demonstrate that genetic fusion with human IgG4-Fc increases the half-life of P125A-endostatin and can be used along with Bevacizumab to improve anti-angiogenic and anti-tumor activity.

Human Papilloma Virus (HPV) infection is associated with the development of Cervical cancer. Therapeutic resistance and recurrence remain the major obstacle to successful treatment of cervical cancer patients. Recently, a novel pro-drug Minnelide, derived from the diterpenoid triepoxide, triptolide, was found to be very effective against several human cancers in experimental animal models. The goal of the present study is to determine the effect of triptolide (in vitro) or Minnelide (in vivo) on HPV positive cervical cancer cells. Triptolide inhibited the proliferation of human cervical cancer cell lines, Ca Ski, SiHa, Me-180, and C-33a with an IC50 of 50 - 100 nM. Furthermore, triptolide inhibited the expression of viral oncoproteins E6 and E7 transcripts significantly. Consequently, p53, tumor suppressor protein levels were increased in the treated cells which led to increased apoptosis and reduced proliferation. Subsequently, we determined the effect of Minnelide on cervical cancer growth in athymic, nude mice. Tumor (SiHa and CaSki) bearing mice were treated with either 0.2 or 0.4 ug/kg doses of Minnelide. These studies showed that Minnelide inhibited tumor growth by about 70 % when compared to control group of mice and improved survival significantly. In a parallel study, combination treatment with carboplatin and Minnelide showed more than additive inhibition of tumor growth. In summary, Minnelide treatment reduced the transcript levels of HPV oncoproteins and inhibited cellular proliferation by upregulating p53. Our studies further demonstrate that Minnelide can be used either as a monotherapy or in combination with platinum drugs to inhibit the growth of cervical cancers. Citation Format: Vivek Ramakrishnan, Bhuwan Giri, Urvashi Hooda, Peter Wilkinson, Christopher deHaydu, Janneth Oleas, Sabita Roy, Sundaram Ramakrishnan, Ashok Saluja. Effect of Minnelide, a prodrug, on cervical cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5879.

Introduction: Bacteria residing in the human body outnumber ‘human' cells and most of these bacteria exist in the gut. This gut microbiome forms a unique and dynamic metagenome which changes with nutrition and even disease state. A growing body of evidence has implicated the role of the gut microbiome in modulating our immune system. We aimed to investigate this association in murine models of cancer. Methods: C57BL/6 mice were either orally given saline or a gut sterilizing cocktail of poorly absorbable broad-spectrum antibiotics. These mice were used to model multiple cancers, viz. subcutaneous injection of melanoma cells derived from Tyr::CreER; Braf V600E/+;Ptenlox5/lox5 mice, subcutaneous injection of pancreatic cancer cells derived from KPC (Kras LSL.G12D/+; p53 R172H/+ ;Pdx::Cre) mice, intrasplenic injection of KPC pancreatic cancer cells and intrasplenic injection of B16-F10 melanoma cells. The tumors were immunophenotyped through flow cytometry and immunostained for multiple antigens. Mice plasma was used for chemokine analysis. To confirm the role of immunity, subcutaneous experiments were repeated in mice carrying a Rag1tm1Mom mutation (RAG1 knockout), thereby having an immature adaptive immune system. Results: Gut microbiome depletion reduced tumor burden across all four subcutaneous and hepatic metastases models. This effect disappeared when experiments were repeated in immunosuppressed RAG1 knockout mice. Antibiotic-treated tumors had increased TNF-secreting macrophages and NKT cells as well a higher number of iNOS staining cells in the microenvironment. Plasma from antibiotic-treated tumor-bearing mice had decreased levels of CXCL12, a chemokine known to cause tumor immunosuppression. Conclusion: Eradication of the gut microbiota decreases tumor burden by activating the anti-cancer immune system Citation Format: Vrishketan Sethi, Saba Kurtom, Mohd Tarique, Bhuwan Giri, Bharti Garg, Shweta Lavania, Anthony Ferrantella, Zoe Malchiodi, Leonor Hellmund, Harrys Charles Jacob, Rajinder Dawra, Sulagna Banerjee, Sabita Roy, Sundaram Ramakrishnan, Ashok Saluja, Vikas Dudeja. Eradication of the gut microbiota reduces cancer burden in multiple models by modulating the immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5127.

Objectives:Tumor infiltrating macrophages play an important role in tumor progression. Macrophage chemoattractant protein-1 (MCP-1) is one of the major chemokines responsible for inducing macrophage migration. Our objective was to investigate chemotherapy-induced modulation of MCP-1 in ovarian cancer by investigating macrophage infiltration, tumor vascularity, and MCP-1 expression after chemotherapy exposure.Methods:MA-148 ovarian cancer cells were treated with paclitaxel (43 pg/mL) and carboplatin (5μg/mL) alone or in combination. Reverse transcription-polymerase chain reaction determined MCP-1 transcript levels and enzyme-linked immunosorbent assay evaluated MCP-1 protein production at multiple time points. The effect of kinase inhibitors on MCP-1 expression was investigated. In vivo MCP-1 production was examined in tumor-bearing mice and immunohistochemistry with fluorescein isothiocyanate conjugated anti-mouse F4/80 antibody, phycoerythrin-anti-CD31, and terminal deoxynucleotide transferase dUTP nick-end labeling assay were performed.Results:Macrophage chemoattractant protein-1 transcript levels were up-regulated in MA-148 after treatment with paclitaxel and carboplatin individually and in combination. The greatest elevation was seen with combination therapy: 2.5-fold increase in the MCP-1 protein levels from baseline (P = 0.011) with the mitogen-activated protein kinase and janus kinases/signal transducers and activators of transcription pathways appearing to be involved in the regulation of MCP-1 production. In vivo mouse studies confirmed increased MCP-1 production after chemotherapy; however, there was no significant difference in macrophage, apoptosis, or vessel density.Conclusions:Macrophage chemoattractant protein-1 is up-regulated in ovarian cancer after chemotherapy in vitro and in vivo. Whether MCP-1 production is increased because of a stress-induced response or a scavenger response promoting macrophage infiltration remains unknown. Chemotherapy induction of MCP-1 in ovarian cancer suggests this chemokine plays an important role in the immune response occurring after chemotherapy exposure.

Objective Intimal hyperplasia (IH) is a clinical concern leading to failure of up to 50% of vein grafts and 10% of arterial grafts after 10 years with no known current treatment. Recent studies have shown that hypoxia differentially regulates proliferation of vein derived smooth muscle cells (V-SMC) compared to artery derived smooth muscle cells (A-SMC). The objective of this study is to evaluate the effect of hypoxia on cellular migration and the mechanisms underlying the differential effects of hypoxia on A-SMC and V-SMC migration. Methods and results Hypoxic treatment (3-5% O2) of Smooth Muscle Cells (SMC) resulted in differential migration in scratch wound and electric cell substrate impedance sensing (ECIS) assays. Hypoxia led to greater migration compared to normoxia with venous derived wound closure (V-SMC 30.8% Normoxia to 67% Hypoxia) greater than arterial wound closure (A-SMC 6.2% Normoxia to 24.7% Hypoxia). Paracrine factors secreted by hypoxic endothelial cells induced more migration in SMC compared to factors secreted by normoxic endothelial cells. Migration of V-SMC was greater than A-SMC in the presence of paracrine factors. Neutralizing antibody to Vascular Endothelial Growth Factor Receptor -1 (VEGFR-1) completely inhibited V-SMC migration while there was only partial inhibition of A-SMC migration. A-SMC migration was completely inhibited by Platelet Derived Growth Factor BB (PDGF-BB) neutralizing antibody. p38 Mitogen Activated Protein kinase (p38 MAPK) inhibitor pre-incubation completely inhibited migration induced by paracrine factors in both A-SMC and V-SMC. Conclusion Our study determines that SMC migration under hypoxia occurs via both an autocrine and paracrine mechanism and is dependent on Vascular Endothelial Growth Factor-A (VEGF-A) in V-SMC and PDGF-BB in A-SMC. Migration of both A-SMC and V-SMC is inhibited by p38 MAPK inhibitor. These studies suggest that pharmacotherapeutic strategies directed at modulating p38 MAPK activity can be exploited to prevent IH in vascular grafts.

A valid preclinical tumor model should recapitulate the tumor microenvironment. Immune and stromal components are absent in immunodeficient models of pancreatic cancer. While these components are present in genetically engineered models such as Kras(G12D); Trp53(R172H); Pdx-1Cre (KPC), immense variability in development of invasive disease makes them unsuitable for evaluation of novel therapies. We have generated a novel mouse model of pancreatic cancer by implanting tumor fragments from KPC mice into the pancreas of wild type mice. Three-millimeter tumor pieces from KPC mice were implanted into the pancreas of C57BL/6J mice. Four to eight weeks later, tumors were harvested, and stromal and immune components were evaluated. The efficacy of Minnelide, a novel compound which has been shown to be effective against pancreatic cancer in a number of preclinical murine models, was evaluated. In our model, consistent tumor growth and metastases were observed. Tumors demonstrated intense desmoplasia and leukocytic infiltration which was comparable to that in the genetically engineered KPC model and significantly more than that observed in KPC tumor-derived cell line implantation model. Minnelide treatment resulted in a significant decrease in the tumor weight and volume. This novel model demonstrates a consistent growth rate and tumor-associated mortality and recapitulates the tumor microenvironment. This convenient model is a valuable tool to evaluate novel therapies.

A human ovarian cancer cell line, which migrates to mouse ovaries and establishes peritoneal carcinomatosis, was used to evaluate the cooperative effect of an antiangiogenic gene therapy combined with chemotherapy. The ovarian carcinoma cell line MA148 was genetically modified by “Sleeping Beauty” transposon-mediated delivery of DsRed2 fluorescent protein. Stable, high-level expression of DsRed protein enabled in vivo imaging of peritoneal dissemination of ovarian cancer. Both external and internal imaging, along with histopathology, showed migration of i.p. injected human ovarian cancer cell line to mouse ovaries. Using this model, we evaluated the effect of adeno-associated virus (AAV)–mediated expression of a mutant endostatin either alone or in combination with carboplatin treatment. A single i.m. injection of recombinant AAV (rAAV)-mutant human endostatin with P125A substitution (P125A-endostatin) showed sustained expression of mutant endostatin. Antiangiogenic gene therapy inhibited orthotopic growth of ovarian cancer and resulted in 33% long-term tumor-free survival. A single cycle of carboplatin treatment combined with mutant endostatin gene therapy resulted in 60% of the animals remaining tumor free for >200 days, which was significantly better than rAAV-LacZ and/or carboplatin. Combination treatment delayed tumor appearance in 40% of the animals, wherein the residual tumors were smaller in size with limited or no peritoneal metastasis. These studies suggest that AAV-mediated gene therapy of P125A-endostatin in combination with carboplatin is a useful method to inhibit peritoneal dissemination of ovarian carcinoma. (Cancer Res 2006; 66(8): 4319-28)

Ovarian physiology is intricately connected to hormonally regulated angiogenic response. Recent advances in the post genomic revolution have significantly impacted our understanding of ovarian function. In an angiogenesis perspective, the ovary offers a unique opportunity to unravel the molecular orchestration of blood vessel development and regression under normal conditions. A majority of ovarian cancers develop from the single layer of epithelium surrounding the ovaries. Angiogenesis is critical for the development of ovarian cancer and its peritoneal dissemination. The present review summarizes recent findings on the angiogenic response in neoplastic ovaries and discusses the prospects of using anti-angiogenic approaches to treat ovarian cancer.

Vascular Endothelial Growth Factor (VEGF) functions as a key regulator in tumor angiogenesis. In addition, VEGF is an important survival factor for endothelial cells under chemical or physical stress. In our report, we show that treatment of endothelial cells with the chemotherapeutic agent carboplatin significantly increased the expression of VEGF. Furthermore, neutralization of secreted VEGF with specific polyclonal anti-VEGF antibodies or monoclonal antibody sensitized endothelial cells to carboplatin treatment and increased apoptosis several-fold. Interestingly, carboplatin treatment did not alter VEGF expression in tumor cells. Similarly, antibody to VEGF did not change the chemosensitivity of tumor cells to this drug. Most importantly, tumor-bearing animals treated with carboplatin showed an increase in VEGF immunoreactivity in the tumor vasculature, confirming the in vitro studies. Based on these observations, we determined whether neutralization of VEGF could enhance the anti-tumor activity of carboplatin in an in vivo ovarian cancer model system. A combination therapy consisting of a suboptimal dose of carboplatin (32.5 mg/kg/inj., q3d x 5; i.p.) and polyclonal anti-VEGF antibody (2 mg/inj., q3d x 10; i.p.) significantly enhanced solid tumor growth inhibition over individual monotherapies and included multiple complete responses. These findings suggest that VEGF is a critical endothelial cell specific survival factor that is induced by carboplatin and contributes to the protection of tumor vasculature during chemotherapy treatment. In addition, these results provide evidence for a potential mechanism that underlies enhanced anti-tumor activity achieved with chemotherapy and anti-VEGF antibody combination treatment regimens as recently reported in a number of clinical trials. We conclude that a similar type of combination therapy may be applicable to many types of malignancies since VEGF expression was differentially induced in the tumor host environment (i.e., tumor vasculature) and not in the tumor cells themselves; hence, this phenomenon may be independent of the type and origin of the primary cancer.

Hypoxia-driven changes in the tumor microenvironment facilitate cancer metastasis. In the present study, we investigated the regulatory cross talk between endocytic pathway, hypoxia, and tumor metastasis. Dynamin 2 (DNM2), a GTPase, is a critical mediator of endocytosis. Hypoxia decreased the levels of DNM2. DNM2 promoter has multiple hypoxia-inducible factor (HIF)-binding sites and genetic deletion of them relieved hypoxia-induced transcriptional suppression. Interestingly, DNM2 reciprocally regulated HIF. Inhibition of DNM2 GTPase activity and dominant-negative mutant of DNM2 showed a functional role for DNM2 in regulating HIF. Furthermore, the opposite strand of DNM2 gene encodes miR-199a, which is similarly reduced in cancer cells under hypoxia. miR-199a targets the 3'-UTR of HIF-1α and HIF-2α. Decreased miR-199a expression in hypoxia increased HIF levels. Exogenous expression of miR-199a decreased HIF, cell migration, and metastasis of ovarian cancer cells. miR-199a-mediated changes in HIF levels affected expression of the matrix-remodeling enzyme, lysyloxidase (LOX). LOX levels negatively correlated with progression-free survival in ovarian cancer patients. These results demonstrate a regulatory relationship between DNM2, miR-199a, and HIF, with implications in cancer metastasis.

The biologic processes of apoptosis and angiogenesis are linked in endothelial biology because some endothelial cell growth factors also exert anti-apoptotic effects. We studied whether apoptosis is occurring in circulating endothelial cells (CEC) that have lost the survival signals derived from anchorage to extracellular matrix. Consistent with this expectation, 64% ± 16% of CEC from normal donors showed evidence of apoptosis (by morphology and TdT-mediated dUTP nick end labeling [TUNEL] assay). However, only 30% ± 15% (P < .001 v normal) of CEC from donors with sickle cell anemia were apoptotic. Vascular endothelial growth factor (VEGF) levels were significantly (P = .001) higher in plasma of sickle donors (120.1 ± 81.4 pg/mL) than that of normal donors (37.6 ± 34.6 pg/mL), and there was an inverse correlation between VEGF and CEC apoptosis (r = .612,P = .001). Consistent with stimulation by VEGF, CEC from sickle donors exhibited increased expression of vβ3. In vitro experiments showed that VEGF inhibits apoptosis for cultured endothelial cells that are kept unanchored and not allowed to re-establish attachment to extracellular matrix, thus demonstrating that VEGF provides survival signals independent of its ability to promote matrix reattachment. These data suggest the hypothesis that sickle cell anemia is a state of enhanced anti-apoptotic tone for endothelial cells. If true, this has implications for disease pathobiology, particularly the development of neovascularizing retinopathy.

Vascular endothelial growth factor (VEGF) expression and microvessel density were studied in cases of advanced epithelial ovarian carcinoma to evaluate their usefulness as prognostic variables. Tumor samples from 18 patients with advanced stage serous epithelial ovarian cancer were evaluated for VEGF expression by reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. Immunohistochemical study of corresponding archival tissues with an antibody to von Willebrand factor (vWF; FVIII-RA) was used for tumor microvessel count determinations. The correlation of VEGF expression and mean microvessel counts was determined by an unpaired t-test. Survival analysis for known prognostic factors and VEGF expression was performed. Survival distributions were calculated by the product limit of Kaplan and Meier and significant differences between distributions were analyzed with a log rank test. From the RT-PCR analysis of tumor VEGF expression, 12 samples were found to be strongly positive, whereas six samples had low/negative VEGF expression. The median survival was 60 months for the VEGF-low/negative group and 28 months for the VEGF-positive group (P = 0.058). Other prognostic variables had minimal impact on survival, i.e. age < 65 years (P = 0.873), FIGO stage (P = 0.06), grade (P = 0.236) and debulking status (P = 0.842). Fourteen of 18 tumor specimens were suitable for microvessel counting. The mean microvessel counts of the VEGF-positive group and the VEGF-negative group were 27/hpf and 35/hpf, respectively (P = 0.16). In this preliminary analysis, high VEGF expression in epithelial ovarian carcinomas was associated with poor overall survival. Further study will be necessary to elucidate the lack of association of VEGF expression and tumor microvessel counts.

BACKGROUND Tumor angiogenesis is essential for solid tumor growth. Yet, the importance of any particular factor in neoplastic proliferation is poorly defined. This study examines the clinical significance of increased expression of one of the angiogenic factors, vascular endothelial growth factor (VEGF), in early stage ovarian carcinoma. METHODS Tumor specimens from 68 patients with International Federation of Gynecology and Obstetrics Stage I and II ovarian carcinoma were evaluated for VEGF expression. Antisense and corresponding sense (control) RNA probes were transcribed from the pCR™II construct (Invitrogen, San Diego, CA), which contained human VEGF cDNA. The antisense probe was designed to include a highly conserved region of the VEGF coding sequence and thus detect all known variants. After in situ hybridization, sections were assessed for overexpression of VEGF. RESULTS Twenty-nine of the tumor samples overexpressed VEGF, whereas 39 specimens did not. In patients whose tumors demonstrated elevated VEGF expression, 25% were without evidence of disease recurrence at last follow-up. In contrast, 75% of the patients whose tumors did not overexpress VEGF were without evidence of disease at last follow-up (P 108 months for the VEGF negative group (P 120 months for the VEGF negative group (P 0.05). Assignment to a high risk group, as defined by the Gynecologic Oncology Group of the National Cancer Institute, was somewhat predictive of a shorter relapse free interval (P = 0.056). In a multivariate analysis, however, only elevated VEGF expression was associated with poorer survival. CONCLUSIONS In this analysis, patients with early stage ovarian carcinoma with increased VEGF expression had a poorer prognosis. Further study of VEGF may ultimately lead to identification of patients with high risk lesions whose tumor biology portends a worse prognosis and who therefore may benefit from aggressive adjuvant therapy. Cancer 1997; 80:98-106. © 1997 American Cancer Society.

Development of tolerance to endotoxin prevents sustained hyper inflammation during systemic infections. Here we report for the first time that chronic morphine treatment tempers endotoxin tolerance resulting in persistent inflammation, septicemia and septic shock. Morphine was found to down-regulate endotoxin/LPS induced miR-146a and 155 in macrophages. However, only miR-146a over expression, but not miR-155 abrogates morphine mediated hyper-inflammation. Conversely, antagonizing miR-146a (but not miR-155) heightened the severity of morphine-mediated hyper-inflammation. These results suggest that miR-146a acts as a molecular switch controlling hyper-inflammation in clinical and/or recreational use of morphine.

OBJECTIVE: Our purpose was to test the ability of an antiserum to vascular permeability factor/vascular endothelial growth factor to inhibit postoperative adhesion formation in a murine model. STUDY DESIGN: After a standardized peritoneal injury, 28 Balb/c mice were randomized and treated intraperitoneally with either vascular permeability factor antiserum (n = 14) or preimmune serum (n = 14) at the time of abdominal closure. Mice were killed on postoperative day 14, and the development of intraabdominal adhesions was determined. Adhesion scoring was based on an overall assessment of the extent, location, and type of adhesions present. Statistical analyses were performed with the Mann-Whitney and Fisher's exact tests. RESULTS: The mice treated with the vascular permeability factor antiserum had significantly lower adhesion scores than did the control group (mean ± SD 1.5 ± 0.9, median 1.0, vs control 2.5 ± 0.7, median 3.0). When the groups were analyzed for the presence of grade 2 or 3 adhesions, the group treated with vascular permeability factor antiserum had a significantly lower incidence of advanced adhesions (38%, vs control 92%). CONCLUSION: This study demonstrates that the intraperitoneal administration of a neutralizing antiserum to vascular permeability factor/vascular endothelial growth factor limits postoperative adhesion formation. These observations, to the best of our knowledge, are the first to suggest a role for vascular permeability factor in the pathogenesis of adhesion formation. (AM J OBSTET GYNECOL 1996;174:1502-6.)

It has been more than 40 years since the initial prospect emerged that tumor angiogenesis could be targeted for cancer therapy. This proposal was based on the very simple observation that tumors are capable of stimulating new vascularization. Later the concept of an angiogenic switch was proposed; where cancer cells evolve from a dormant state into angiogenic phenotype attracting blood vessels growth and invading neighboring and distant sites. Since Folkman's landmark observations [1] a more complex picture of angiogenesis has emerged replete with a complex tumor microenvironment and interactions with multiple cell types within the tumor. Numerous anti angiogenic agents were developed successfully for the treatment of renal cell carcinoma, lung cancer, breast cancer, brain tumors, and colorectal carcinoma, but resistance to these therapies soon emerged. This stimulated additional research that led to the better understanding of the very complex interaction of tumor angiogenesis and the tumor microenvironment responsible for the lack of lasting response to angiogenesis targeted therapy. In this issue of Tumor Angiogenesis, several review papers discuss the current status of our understanding of and therapy for ovarian carcinoma, head, and neck cancer, melanoma, glioma, and leukemia (papers by A. Amini et al., C. Carla et al., U. Adamcic et al., K. Shirai et al., S. R. Choudhury et al., and A. Trujillo et al.) The role of pericytes by E. Fakhrejahani and M. Toi T. Duong et al.'s contribution on the role of lymphangiogenesis enhance the spectrum of different cellular and structural components contributing to cancer progression and metastases. The most novel contribution emerges from the role of external stimuli in the promotion of angiogenesis. Diet as a modifier of tumor angiogenesis by W. W. Li et al. and I. Urits et al., substantiates the role of external control of microenvironment by diet, which is complemented by the observations of K. Luk et al. on pericyte-endothelial interaction influenced by morphine generally used to treat severe pain in patients with cancer. Insights into newer signaling pathways such as, PTEN (S. Rodriguez, and U. Huynh-Do), c-Kit (K. Rupertus et al.) and transcription factor FoxC2 (T. Kume) provide an understanding of yet to be conquered targets to efficiently treat cancer. Additionally, imaging techniques for tumor angiogenesis discussed by K. Tanaka et al. also offer novel approaches to understand tumor angiogenesis. The interaction between tumor vasculature and the microenvironment is reviewed by E. Fakhrejahani, M. Toi, K. Rupertus et al., and S. Niland et al., and inflammation and cancer is examined by J. I. Arias et al. These publications provide an up to date comprehensive state of research on tumor angiogenesis. We therefore hope that this issue will help the readers understand the underlying complex biology and challenges in developing successful antiangiogenic therapy. Arkadiusz Z. Dudek Kalpna Gupta Sundaram Ramakrishnan Debabrata Mukhopadhyay

Advanced ovarian cancers contain 2 distinct phenotypic populations: (a) free-floating tumor cells in the ascitic fluid and (b) solid tumors. Ascites cells are derived from the solid tumors and spread throughout the peritoneum. Changes in cell-cell and cell-extracellular matrix interactions are thought to be responsible for the origin of ascites cells. Since E-cadherin molecules play a crucial role in the cell-cell interactions in epithelial cells, we investigated the expression of E-cadherin in these 2 phenotypic populations. Paired samples of ascites and solid tumors were obtained from patients. Both primary tumors and tumor cells isolated from an experimental model showed a marked decrease in E-cadherin expression in the ascites cells compared to the respective solid tumors. Semi-quantitative, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the steady-state levels of E-cadherin-specific mRNA. Results indicate that the primary tumors had significantly lower levels of E-cadherin transcript in ascites cells when compared to their solid tumor counterparts. Changes in E-cadherin expression were also reflected in the invasion capacity of tumor cells in vitro. Ascites cells were 4-fold more invasive then solid tumor cells, suggesting that ascites cells are a highly malignant phenotype.

GnRH agonists have been found to be clinically useful in several hormone-sensitive conditions, including cancer. However, there is controversy regarding a direct action of these agents on the pathologic tissue of a given disease process, in particular, tumors. In this study, we examined the effects of D-Trp(6) -LHRH, a potent GnRH agonist, on the expression of an increasingly important angiogenesis factor, VPF (vascular permeability factor)/VEGF (vascular endothelial growth factor). Ovarian carcinoma cell lines exposed to D-Trp(6) -LHRH in culture demonstrated a reversible inhibition of VPF mRNA expression and a parallel decrease in the endothelium-specific mitogenic activity of the conditioned media from these treated cultures. This study reports a novel activity of a GnRH agonist and provides a starting point to investigate the in vivo anti-angiogenic properties of GnRH peptide analogs.

Vascular endothelial growth factor (VEGF)/ vascular permeability factor (VPF) is emerging as an important growth factor in a variety of tumor types. As a potent endothelial cell mitogen and vascular permeabilizing agent, VEGF/VPF has the unique functional capacity to mediate the component events of solid tumor neovascularization and ascites tumor growth. In the present series of investigations, our experimental hypothesis was that VEGF/VPF is a critical mediator of ovarian carcinoma-associated ascites formation and solid tumor growth. Athymic nude mice xenotransplanted with human ovarian carcinoma cell lines received either a preimmune rabbit serum or VEGF/VPF antiserum. Compared with the control group receiving the preimmune serum, the antiserum-treated animals displayed a 10- and 12-fold reduction in ascites accumulation and solid tumor growth, respectively. The administration of a neutralizing antiserum to VEGF/VPF conferred a modest survival advantage to animals harboring intraperitoneal tumors. These data demonstrate the significance of VEGF/VPF in the pathogenesis of ovarian carcinoma and suggest that interventions targeting this growth factor and/or its receptor may be of therapeutic value in the management of ovarian cancer.

BACKGROUND Recent studies have shown that multiple cytokines are secreted by ovarian epithelial cancer cells. Previous studies have shown that the cancer cell lines secrete macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 (IL-1), interleukin-6 (IL-6), and transforming growth factor-alpha (TGF-alpha). Concomitantly, the serum levels of one of the growth factors (M-CSF) was found to be significantly elevated in patients with primary ovarian cancer and in second-look patients. The authors evaluated the serum levels of IL-1 alpha, IL-1 beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha) in patients with primary ovarian epithelial cancer. These levels were then compared with cytokine concentration found in normal peritoneal fluid. METHODS Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of cytokines in normal peritoneal fluid, ascites, and serum. RESULTS In serum, TNF-alpha and IL-6 were significantly increased in primary ovarian cancer patients when compared with control subjects (P < 0.0001 for both cytokines). TNF-alpha and IL-6 were also significantly higher than the levels found in second-look patients (P < 0.007 for TNF-alpha, and P = 0.0002 for IL-6). The levels of IL-1 alpha and beta were not elevated in ovarian cancer. TNF-alpha in the ascites was higher when compared with normal peritoneal fluid and was statistically significantly different when a cut-off point between 71-110 pg was selected (P < 0.005). The levels of IL-6 in ascites from patients with primary ovarian cancer also showed a marked increase (P < 0.0001) when compared with peritoneal fluid from control subjects. CONCLUSIONS Levels of IL-1, IL-6, and TNF-alpha were determined in normal peritoneal fluid, ovarian malignant ascites, normal serum, and serum from patients with ovarian cancer. This study showed that the patients with ovarian cancer have elevated levels of IL-6 and TNF-alpha in serum and ascitic fluid. A larger study would help in evaluating the potential use of cytokines as tumor markers in ovarian cancer.

Background. Haptoglobin is an acute-phase reactant protein involved in immune regulation. It has been isolated from malignant ovarian ascites and has been shown to be capable of mediating nonspecific immune suppression in vitro in humans and in vivo in experimental animals. The range of concentrations under which such a nonspecific suppression is observed lies well within concentrations of haptoglobin observed in vivo. This immune suppression could adversely affect patient outcome. Methods. Based on this information, ascites haptoglobin levels of 21 consecutive patients with ovarian cancer who underwent initial surgical debulking were determined. After the patients received chemotherapy, they were divided into those having a positive and those have a negative second-look operation. Results. Seventeen patients with positive pathology at second look had an initial mean ascites haptoglobin level of 99 ± 49 mg/dl. The mean haptoglobin level in the four patients with negative findings was 67 ± 42 mg/dl. The difference in these mean haptoglobin levels is not statistically significant (P > 0.05). Conclusion. Although the number of patients in this study is small, the wide range of values documented in both groups make it doubtful that an initial absolute ascites haptoglobin level will prove clinically prognostic of eventual disease status at the time of second look laparotomy.

Endostatin potentiates the antimitotic effects of paclitaxel (taxol) on endothelial cells (ECs). P125A-endostatin and taxol-treated ECs showed multipolar spindles and nuclear lobulation, leading to mitotic catastrophe and cell death. Induction of nuclear abnormalities was found to be dependent on β-catenin levels as wnt-mediated overexpression of β-catenin reversed the changes in nuclear morphology. These results prompted us to investigate whether antiangiogenic gene therapy and paclitaxel chemotherapy can synergistically inhibit angiogenesis and tumor growth. We first determined the effect of combination treatment in a transgenic mouse model of breast cancer. Intramuscular injection of recombinant adeno-associated virus type-2 virus induced sustained expression of P125A-endostatin. In vivo studies showed that combination therapy inhibited mammary cancer growth, delayed the onset of multifocal mammary adenocarcinomas, decreased tumor angiogenesis and increased survival in treated mice. In a second model, female athymic mice were orthotopically transplanted with a metastatic human breast cancer cell line. Antiangiogenic gene therapy in combination with paclitaxel inhibited tumor angiogenesis and lung/lymph-node metastasis in this model. These studies demonstrate cooperation between endostatin gene therapy and chemotherapy to inhibit tumor initiation, growth and metastasis.

Objective: The purpose of this study was to evaluate the prognostic significance of macrophage colony-stimulating factor serum levels in patients with ovarian cancer undergoing second-look laparotomy. Study design: The presurgical serum levels of macrophage colony-stimulating factor from 33 consecutive patients with ovarian cancer undergoing second-look laparotomy were determined and compared with those of 50 healthy control subjects. Mean differences in groups were evaluated with the Student f test. Results: Sixteen of 33 patients had a positive result at second look and a mean serum macrophage colony-stimulating factor level of 2.31 ± 1.45 ng/ml. Seventeen of 33 patients had a negative result at second look and a mean macrophage colony-stimulating factor level of 1.90 ± 0.86 ng/ml ( p > 0.05). The mean macrophage colony-stimulating factor level in the control group was 1.20 ± 0.51 ng/ml. This was statistically lower than the mean level found in patients with a negative second-look result ( p Conclusion: Regardless of tumor status, serum macrophage colony-stimulating factor levels tend to be elevated at the time of second-look laparotomy.

Macrophage colony-stimulating factor is a cytokine that stimulates proliferation and differentiation of phagocytic cells. Macrophage colony-stimulating factor is produced by ovarian epithelial cancer cell lines and might provide a useful serum marker for the disease. Among sera from 69 patients with clinically apparent epithelial ovarian cancer, 47 (68%) had at least 2.5 ng/ml macrophage colony-stimulating factor, whereas only two of 80 apparently healthy donors (2.5%) had a comparable elevation of macrophage colony-stimulating factor. Circulating levels of macrophage colony-stimulating factor did not correlate with serum levels of CA 125. Moreover, 14 of 25 ovarian cancer patients (56%) with clinically evident disease and normal levels of CA 125 (less than 35 U/ml) had elevated levels of macrophage colony-stimulating factor. Among 29 patients with serum CA 125 levels less than 35 U/ml before positive surgical surveillance procedures, 9 (31%) had at least 2.5 ng/ml macrophage colony-stimulating factor. Elevated levels of macrophage colony-stimulating factor were also found in patients with carcinomas from other primary sites and in 31% of 134 patients with benign diseases. If intercurrent benign disease can be taken into account, macrophage colony-stimulating factor deserves further evaluation in combination with CA 125 in monitoring ovarian cancer.

The peritoneal fluid (PF) of women with infertility (especially in the presence of endometriosis) contains increased numbers of leukocytes, 90% to 95% of which are macrophages. The high numbers of peritoneal macrophages presumably result from an influx of blood monocytes into the peritoneum, and/or from local proliferation of peritoneal macrophages. Once in the peritoneal cavity, monocytes differentiate into tissue macrophages. Mononuclear phagocyte proliferation and differentiation are influenced by different cytokines, including macrophage colony-stimulating factor (M-CSF). The purpose of this study was to determine the relationship of M-CSF levels in human PF and plasma to the macrophage content, and to the patient diagnoses. Mean concentrations of PF M-CSF were higher than plasma levels (2.44 +/- 0.13 v 0.95 +/- 0.06 ng/mL, respectively). The mean concentrations of plasma M-CSF did not differ in samples from women of different diagnostic groups (normal, peritoneal adhesions, endometriosis, inactive pelvic inflammatory disease, uterine fibroids, and idiopathic infertility), but the PF concentration was slightly higher in normal women. The absolute (total) amount of PF M-CSF in normal women was lower than in those of the other diagnostic groups. The total amount of PF M-CSF in all women correlated closely with the total number of peritoneal macrophages. The tubal patency status (open versus closed) did not influence the plasma and PF concentrations of M-CSF, nor the PF absolute amount of M-CSF. The PF M-CSF may have come from peritoneal macrophages, fibroblasts, mesothelial cells, or endothelial cells. PF M- CSF may play important roles in the proliferation and/or the differentiation of peritoneal mononuclear phagocytes.

Ovarian cancer cells use integrins to attach to the peritoneal wall. Integrin α5β1 is also the target for the angiogenesis inhibitor, endostatin. Therefore, the ability of endostatin to competitively inhibit tumor cell seeding of the peritoneum was investigated. An imaging method was developed to determine early phases of peritoneal dissemination of ovarian cancer cells. Using this method, endostatin was found to bind ovarian cancer cells through integrin α5β1 and inhibit vessel cooption efficiently. Although both angiostatin and endostatin are potent inhibitors of tumor angiogenesis, peritoneal attachment and vessel cooption was blocked only by the endostatin. Knocking down the expression of integrins α5 and β1 in ovarian cancer cells interfered with endostatin-mediated inhibition of peritoneal seeding. Furthermore, adenovirus-mediated in situ expression of endostatin either inside the peritoneum or by the ovarian tumor cells inhibited peritoneal seeding and dissemination in vivo. Endostatin treatment also prevented primary ovarian cancer cells from attaching to mouse peritoneal wall. These studies show a paraendothelial mechanism by which endostatin can inhibit peritoneal dissemination of ovarian cancer cells and raises the possibility of intraperitoneal expression of endostatin to reduce recurrence. [Cancer Res 2007;67(22):10813–22]

Endostatin, a C-terminal fragment of collagen type XVIII, is one of the well-characterized endogenous inhibitors of angiogenesis. Endostatin is known to bind integrin alpha(5)beta(1), which is upregulated on tumor endothelium. Most of the ovarian cancer cells express significant amounts of alpha(5)beta(1) integrin, which is important for ovarian cancer cells to attach to the peritoneal wall. Therefore we investigated whether endostatin could directly bind ovarian cancer cells and inhibit tumor cell attachment to extracellular matrix. Binding of endostatin to ovarian cancer cells was characterized by preincubation with function blocking antibodies to integrin subunits. These studies showed that ovarian cancer cell attachment to fibronectin-coated wells can be inhibited by alpha(5)beta(1) integrin specific antibodies as well as endostatin. Downregulation of integrin alpha(5) and beta(1) by siRNA abrogated the binding of OVCAR5 and human umbilical vein endothelial cell to endostatin. Although endostatin treatment did not affect ovarian cancer cell migration, treated cells failed to attach mouse peritoneal wall preparations. These studies suggest an extra-antiangiogenic role for endostatin, which can be used prevent peritoneal attachment and dissemination of ovarian cancer cells.