Tumor Metastasis Formation Research Articles

Targeting Adenosine in BRAF-Mutant Melanoma Reduces Tumor Growth and Metastasis.

Increasing evidence exists for the role of immunosuppressive adenosine in promoting tumor growth and spread in a number of cancer types, resulting in poor clinical outcomes. In this study, we assessed whether the CD73-adenosinergic pathway is active in melanoma patients and whether adenosine restricts the efficacy of clinically approved targeted therapies for commonly mutated BRAFV600E melanoma. In AJCC stage III melanoma patients, CD73 expression (the enzyme that generates adenosine) correlated significantly with patients presenting nodal metastatic melanoma, suggesting that targeting this pathway may be effective in advanced stage disease. In addition, dabrafenib and trametinib treatment of CD73+ BRAFV600E-mutant melanomas caused profound CD73 downregulation in tumor cells. Inhibition of BRAF and MEK in combination with the A2A adenosine receptor provided significant protection against tumor initiation and metastasis formation in mice. Our results suggest that targeting adenosine may enhance therapeutic responses for melanoma patients receiving targeted or immune-based therapies. Cancer Res; 77(17); 4684-96. ©2017 AACR.

The Distinct Role of Extracellular Vesicles Derived from Normal and Cancer Stem Cells

Extracellular vesicles (EVs) are a powerful tool for intercellular messages. EVs display many characteristics of the cell of origin and may mimic the biological activities of cells. In this review, we summarize the updated knowledge regarding the biological role of EVs released by both types of healthy somatic stem cells and cancer stem cells (CSCs). Adult somatic stem cells influence the regeneration and healing of damaged tissues in various models of tissue injury; on the other hand, CSCs retain some characteristics of healthy stem cells, however, they sustain tumor progression and metastasis formation. Recent studies highlight the role of EVs in biological processes. EVs contain proteins (receptors, enzymes, transcription and growth factors), genetic materials (mRNAs, miRNAs, and DNAs), and a specific lipid pattern that together induce epigenetic changes in recipient cells. Most cell types release EVs influencing physiological or pathological conditions. EV activities depend on their cargo. EVs released by healthy stem cells appear to accelerate the recovery of an injured tissue in several experimental conditions, while EVs shed by CSCs promote tumor progression by stimulating angiogenesis, tumor growth, tumor immune escape, and metastasis.

HBx-mediated decrease of AIM2 contributes to hepatocellular carcinoma metastasis.

Tumor metastasis is responsible for the high mortality rates in patients with hepatocellular carcinoma (HCC). Absent in melanoma 2 (AIM2) has been implicated in inflammation and carcinogenesis, although its role in HCC metastasis remains unknown. In the present study, we show that AIM2 protein expression was noticeably reduced in HCC cell lines and clinical samples. A reduction in AIM2 was closely associated with higher serum AFP levels, vascular invasion, poor tumor differentiation, an incomplete tumor capsule and unfavorable postsurgical survival odds. In vitro studies demonstrated that AIM2 expression was modulated by hepatitis B virus X protein (HBx) at transcriptional and post‐translational levels. HBx overexpression markedly blocked the expression of AIM2 at mRNA and protein levels by enhancing the stability of Enhancer of zeste homolog 2 (EZH2). Furthermore, HBx interacted with AIM2, resulting in an increase of AIM2 degradation via ubiquitination induction. Functionally, knockdown of AIM2 enhanced cell migration, formation of cell pseudopodium, wound healing and tumor metastasis, whereas reintroduction of AIM2 attenuated these functions. The loss of AIM2 induced the activation of epithelial‐mesenchymal transition (EMT). Fibronectin 1 (FN1) was found to be a downstream effector of AIM2, with its expression reversely modulated by AIM2. Silencing of FN1 significantly halted cell migration induced by AIM2 depletion. These data demonstrate that HBx‐induced loss of AIM2 is associated with poor outcomes and facilitates HCC metastasis by triggering the EMT process. The results of the present study therefore suggest that AIM2 is a potential prognostic biomarker in hepatitis B virus‐related HCC, as well as a possible therapeutic target for tumor metastasis.

Abstract 5932: Targeting tumor acidity with the LDHA inhibitor (FX11) and CAIX inhibitor (DH348) overcomes resistance to PD-1 blockade and inhibits metastasis in a pancreatic cancer model

Abstract Prior work by us and others has demonstrated that the extracellular pH (pHe) of solid tumors is acidic, due to a combination of increased fermentative metabolism, resulting in lactic acid production and poor perfusion. This acidity promotes tumor progression and metastasis formation. Recently we have shown in melanoma and pancreatic cancer models that acidity inhibits antitumor immunity by preventing T-cell activation. Reversal of acidity with buffer therapy (200mM NaHCO3) synergized with checkpoint blockade (anti-CTLA4 and anti-PD1) and adoptive T-cell therapy has resulted in cures. While this is promising, concerns are high regarding the ingestion of such large amounts of sodium bicarbonate, which makes clinical translation a challenge. Hence, we hypothesize that alternative pharmacological interventions can neutralize the pHe of tumors and remove this immunosuppressive effect. To study this we first investigated a series of agents for their ability to inhibit metastasis in the PC3 prostate cancer model, which is exquisitely sensitive to inhibition with buffer therapy. In this study, male SCID mice were grouped in to 6 groups (n=5) and treated with tap water, 200 mM bicarbonate ad lib, 30 mg/kg daily (q.d.) intraperitoneal (i.p.) Acetazolamide (CA inhibitor), 1.2 mg/kg q.d.i.p. Furosemide (diuretic), 10 mg/kg q.d.i.p. DH348 (selective CAIX inhibitor) or 2.1 mg/kg q.d.i.p. FX-11 (LDHA inhibitor). Mice were intravenously injected with 5*10^6 PC3M-luc cells and ventral bioluminescence images were acquired at time 0 and weekly thereafter. Our results showed that FX-11, acetazolamide, DH348 and bicarbonate were able to effectively (p<0.004) suppress metastasis formation in this system, whereas furosemide was not. Based on these results, a subsequent study investigated the combination of bicarbonate, FX-11 or DH348 with immune checkpoint blockade in a Panc02 mouse model of pancreatic cancer. Animals were inoculated orthotopically with Panc02 cells and randomized into 8 groups (n=10). Once tumors reached 0.3 cc in size, mice were treated with either 15 mg/kg anti-PD-1 antibody or normal rat IgG controls twice weekly alone or in combination with bicarbonate; FX-11 or DH348 and tumor response was evaluated using US 3D-Mode imaging and volume analysis (FUJIFILM VisualSonics) and histopathology. Anti-PD-1 antibody was ineffective as a monotherapy in this system. Both bicarbonate and DH348 neutralized tumor acidity and reduced tumor growth as monotherapies. Notably, despite the fact that FX-11 was shown to inhibit LDH-A in pancreatic cancer models, it did not affect pH and had no effect on tumor growth as monotherapy. However, FX-11 significantly reduced tumor growth (p <0.01) and metastasis formation in combination with anti-PD-1 antibody, suggesting that inhibition of LDH-A might be effective in combination with checkpoint blockade. Citation Format: Arig A. Ibrahim Hashim, Dominique Abrahams, Liping Xu, Barbra Centeno, Enakshi Sunassee, Rasha Abddelgader, Ludwig Dubois, Philippe Lambin, Robert A. Gatenby, Robert J. Gillies. Targeting tumor acidity with the LDHA inhibitor (FX11) and CAIX inhibitor (DH348) overcomes resistance to PD-1 blockade and inhibits metastasis in a pancreatic cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5932. doi:10.1158/1538-7445.AM2017-5932

Abstract 1915: Deconstructing cancer stem cells: IL-6, G-CSF and Activin-A as mediators of stroma-orchestrated epithelial cells’ dedifferentiation

Abstract Intercellular communication in cancer is a deceiving and extremely efficient process through which the corporal machinery is hijacked by a panoply of cellular and molecular strategies turning the entire human body into an evolutionary arena. The extensive crosstalk mediated by cytokines and chemokines overcome the inefficiency of the invasion-metastasis cascade, thus allowing the development of the often-fatal metastatic disease. Cancer stem cells (CSCs) have recently been implicated in major steps of the tumorigenic process, namely in tumor initiation, metastasis formation and tumor relapse following therapy. Yet, despite their relevance, the exact mechanisms underlying their formation are still unclear. After observing that the malignant human bronchial epithelial RenG2 cells dedifferentiated following culture in the subcutaneous mouse lumbar region, co-cultures of surgically isolated mice lumbar stromal cells with RenG2 cells were established and the conditioned media studied by multiplex and ELISA. Consequently, Interleukin-6 (IL-6), Granulocyte colony-stimulating factor (G-CSF) and Activin-A were identified as the paracrine mediators of the intercellular communication process. Aiming to ascertain the individual role of each cytokine in the dedifferentiation process, as well as to access the involvement of exosomes as transport vehicle, the same co-cultures were reproduced in the presence of specific cytokine-communication blockers, either individually or in combinations of up to three blockers, and exosome-mediated communication inhibitors. Finally, exosomes were also collected from control co-cultures and their cargo screened for the target cytokines. ELISA showed that the three cytokines were present inside fibroblasts-secreted exosomes. Moreover, whenever exosomes’ release was blocked, dedifferentiation was abrogated, further proving the role of the aforementioned cytokines and of exosomes in the dedifferentiation process. Additionally, the cytokine-blocking experiments revealed that only IL-6 and Activin-A were endowed with the potential to orchestrate dedifferentiation, as when at least one of these cytokines was present a stem cell population developed inside RenG2 cells. Finally, G-CSF appeared to be decisive in maintaining the undifferentiated phenotype, as a larger pool of CSCs was attained whenever this cytokine and either IL-6 or Activin-A were present. Altogether the attained results implicated IL-6 and Activin-A in the formation of CSCs by dedifferentiation, and G-CSF as a potent keeper of the dedifferentiation status. Subsequent studies are now required to access the use of these cytokines as therapeutic targets so the tumorigenic process may be abrogated in its initial steps, improving patients’ prognosis and survival. Work sponsored by FEDER, POFC-COMPETE and the FCT grants PTDC/BBB-BQB/2450/2012 and SFRH/BD/33884/2009. Citation Format: Carlos F. Rodrigues, Eurico Serrano, Marco Cunha, Maria I. Patrício, Mariana Val, João Fonseca, Célia Gomes, Antero Abrunhosa, Artur Paiva, Filomena Botelho, Lina Carvalho, Isabel M. Carreira, Alpoim Carmen. Deconstructing cancer stem cells: IL-6, G-CSF and Activin-A as mediators of stroma-orchestrated epithelial cells’ dedifferentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1915. doi:10.1158/1538-7445.AM2017-1915

Abstract 956: Novel oral small molecule CXCR4 inhibitor improves activity of immune checkpoint blockade in ovarian cancer mouse model

Abstract Ovarian cancer continues to be the most lethal gynecologic malignancy with no real cure for patients presenting with advanced stage disease. Immune check point blockade showed modest clinical response in patient with recurrent ovarian cancer, thus additional therapeutic strategies for combination therapy are needed. As chemokines and their receptors drive both immune cell migration and tumor growth, angiogenesis and metastasis formation, they are an attractive target for combinatorial cancer therapy. CXCR4 is the most highly expressed chemokine receptor in advanced stage high grade serous ovarian cancer, thus the objective of this study was to evaluate the efficacy of a novel oral small molecule CXCR4 inhibitor (X4-136) alone and in combination with immune checkpoint inhibition and the anti-angiogenic agent bevacizumab, and characterize the changes in circulating immune cells during treatment in murine ovarian cancer model. The ID8 cell line was used in C57BL/6J mice to establish an immune competent murine model and to compare single agent and combination therapy with oral X4-136 CXCR4 inhibitor, bevacizumab, and anti-PD1. During treatment blood sampling was performed and immune cells were analyzed by flow cytometry. Our results demonstrated that single agent therapies alone with either drug had no significant effect on tumor progression or survival. Combination therapy with the CXCR4 inhibitor and anti-PD1 improved survival compared to control animals and the other combination therapy groups. The addition of bevacizumab to the dual combination did not further prolong survival. Analysis of circulating immune cells revealed elevations in CD11b+Ly6C+ and the ratio of CD11b+Ly6C+ to CD11b+Ly6G+ in groups treated with the CXCR4 inhibitor, indicating an increase in circulating myeloid cells. Bevacizumab had no activity in this mouse model as a single agent, and did not have synergistic effect in combination therapy. For the first time we demonstrated that novel CXCR4 inhibitor X4-136 in combination therapy with anti-PD1 showed improved survival in murine ovarian cancer model. CXCR4 blockade increased the proportion of circulating myeloid cells during active treatment, thus further investigation into this novel therapeutic approach is warranted. Citation Format: Kristen Starbuck, Robert McGray, Samar Masoumi-Moghaddam, Ariel Francois, Kunle Odunsi, Emese Zsiros. Novel oral small molecule CXCR4 inhibitor improves activity of immune checkpoint blockade in ovarian cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 956. doi:10.1158/1538-7445.AM2017-956

Pancreatic cancer-derived exosomes promote tumor metastasis and liver pre-metastatic niche formation.

Exosomes play important roles in cell-cell communication, and are likely mediators of the metastatic cascade in cancer. This study examined the role of exosomes in pancreatic cancer cell adhesion, migration, and invasion. We isolated and purified exosomes from two isogenic pancreatic cancer cell lines with different metastatic potentials. Uptake of exosomes from highly metastatic Panc02-H7 cells decreased adhesion and increased migration and invasion capacity in weakly metastatic Panc02 cells in vitro. Exosomes from highly metastatic pancreatic cancer cells induced liver pre-metastatic niche formation in naïve mice and promoted primary tumor growth and liver metastasis in vivo. We identified 4,517 proteins in exosomes from Panc02 and Panc02-H7 cells via iTRAQ quantitative proteomic analyses, 79 of which were differentially expressed between the two cell lines. Bioinformatics analyses showed that most of the differentially expressed proteins were involved in pancreatic cancer growth, invasion, and metastasis, and that metabolism-related signaling pathways were involved in exosome-mediated intracellular communication. Further studies will be needed to determine whether these proteins are potential pancreatic cancer diagnostic/prognostic markers or novel therapeutic targets.

IL-33 restricts tumor growth and inhibits pulmonary metastasis in melanoma-bearing mice through eosinophils

ABSTRACT The alarmin IL-33 is an IL-1 family member that stimulates pleiotropic immune reactions depending on the target tissue and microenvironmental factors. In this study, we have investigated the role of IL-33/ST2 axis in antitumor response to melanoma. Injection of IL-33 in mice-bearing subcutaneous B16.F10 melanoma resulted in significant tumor growth delay. This effect was associated with intratumoral accumulation of CD8+ T cells and eosinophils, decrease of immunosuppressive myeloid cells, and a mixed Th1/Th2 cytokine expression pattern with local and systemic activation of CD8+ T and NK cells. Moreover, intranasal administration of IL-33 determined ST2-dependent eosinophil recruitment in the lung that prevented the onset of pulmonary metastasis after intravenous injection of melanoma cells. Accordingly, ST2-deficient mice developed pulmonary metastasis at higher extent than wild-type counterparts, associated with lower eosinophil frequencies in the lung. Of note, depletion of eosinophils by in vivo treatment with anti-Siglec-F antibody abolished the ability of IL-33 to both restrict primary tumor growth and metastasis formation. Finally, we show that IL-33 is able to activate eosinophils resulting in efficient killing of target melanoma cells, suggesting a direct antitumor activity of eosinophils following IL-33 treatment. Our results advocate for an eosinophil-mediated antitumoral function of IL-33 against melanoma, thus opening perspectives for novel cancer immunotherapy strategies.

Spontaneous lung metastasis formation of human Merkel cell carcinoma cell lines transplanted into scid mice.

Merkel cell carcinoma (MCC) is an aggressive skin cancer entity that frequently leads to rapid death due to its high propensity to metastasize. The etiology of most MCC cases is linked to Merkel cell polyomavirus (MCPyV), a virus which is monoclonally integrated in up to 95% of tumors. While there are presently no animal models to study the role of authentic MCPyV infection on transformation, tumorigenesis or metastasis formation, xenograft mouse models employing engrafted MCC-derived cell lines (MCCL) represent a promising approach to study certain aspects of MCC pathogenesis. Here, the two MCPyV-positive MCC cell lines WaGa and MKL-1 were subcutaneously engrafted in scid mice. Engraftment of both MCC cell lines resulted in the appearance of circulating tumor cells and metastasis formation, with WaGa-engrafted mice showing a significantly shorter survival time as well as increased numbers of spontaneous lung metastases compared to MKL-1 mice. Interestingly, explanted tumors compared to parental cell lines exhibit an upregulation of MCPyV sT-Antigen expression in all tumors, with WaGa tumors showing significantly higher sT-Antigen expression than MKL-1 tumors. RNA-Seq analysis of explanted tumors and parental cell lines furthermore revealed that in the more aggressive WaGa tumors, genes involved in inflammatory response, growth factor activity and Wnt signalling pathway are significantly upregulated, suggesting that sT-Antigen is the driver of the observed differences in metastasis formation.

Tumor-associated myeloid cells as guiding forces of cancer cell stemness.

Due to their ability to differentiate into various cell types and to support tissue regeneration, stem cells simultaneously became the holy grail of regenerative medicine and the evil obstacle in cancer therapy. Several studies have investigated niche-related conditions that favor stemness properties and increasingly emphasized their association with an inflammatory environment. Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) are major orchestrators of cancer-related inflammation, able to dynamically express different polarized inflammatory programs that promote tumor outgrowth, including tumor angiogenesis, immunosuppression, tissue remodeling and metastasis formation. In addition, these myeloid populations support cancer cell stemness, favoring tumor maintenance and progression, as well as resistance to anticancer treatments. Here, we discuss inflammatory circuits and molecules expressed by TAMs and MDSCs as guiding forces of cancer cell stemness.

Sox2 is dispensable for primary melanoma and metastasis formation.

Tumor initiation and metastasis formation in many cancers have been associated with emergence of a gene expression program normally active in embryonic or organ-specific stem cells. In particular, the stem cell transcription factor Sox2 is not only expressed in a variety of tumors, but is also required for their formation. Melanoma, the most aggressive skin tumor, derives from melanocytes that during development originate from neural crest stem cells. While neural crest stem cells do not express Sox2, expression of this transcription factor has been reported in melanoma. However, the role of Sox2 in melanoma is controversial. To study the requirement of Sox2 for melanoma formation, we therefore performed CRISPR-Cas9-mediated gene inactivation in human melanoma cells. In addition, we conditionally inactivated Sox2 in a genetically engineered mouse model, in which melanoma spontaneously develops in the context of an intact stroma and immune system. Surprisingly, in both models, loss of Sox2 did neither affect melanoma initiation, nor growth, nor metastasis formation. The lack of a tumorigenic role of Sox2 in melanoma might reflect a distinct stem cell program active in neural crest stem cells and during melanoma formation.

MicroRNA-1258: An invasion and metastasis regulator that targets heparanase in gastric cancer.

Numerous microRNAs (miRNAs/miRs) are involved in suppressing or promoting the formation of cancer. However, to the best of our knowledge, the role of miRNA-1258 in gastric cancer (GC) has not previously been investigated. Our previous study demonstrated an increased expression of heparanase (HPSE) in GC tissues and HPSE-facilitated invasion and metastasis of GC cells. Consequently, in the present study, the function of miR-1258 in the invasion and metastasis of GC cells was investigated to determine whether miR-1258 is associated with GC through HPSE. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine miR-1258 expression in GC cell lines and tissues. A Transwell cell invasion assay and an MTT proliferation assay were used to investigate the effects of miR-1258 on the invasion and proliferation of one of the cell lines, SGC-7901 cells, in vitro. The effect of miR-1258 on SGC-7901 cell metastasis was investigated using an in vivo tumor metastasis formation assay. Western blot analysis and RT-qPCR were used to investigate whether HPSE was a target of miR-1258 in GC. The expression of miR-1258 was significantly downregulated in 3 GC cell lines and 116 GC tissues compared with controls (all P<0.001). An association was identified between decreased miR-1258 expression level and increased age (P=0.042), advanced pathological tumor stage (P=0.027) and positive lymphatic vessel invasion (P=0.044) in patients with GC. Furthermore, the upregulation of miR-1258 expression suppressed SGC-7901 cell invasion in vitro (P<0.001) and inhibited SGC-7901 cell metastasis in vivo (P=0.016). The western blot analysis and RT-qPCR results indicated that miR-1258 downregulated the expression of HPSE at the translational level. The results of the present study indicate that miR-1258 acts as a tumor suppressor to inhibit invasion and metastasis by targeting HPSE. Therefore, miR-1258 may serve as a novel biomarker and therapeutic target in the treatment of GC.

PDPN gene promotes the proliferation of immature Bovine Sertoli cells in vitro

Podoplanin (PDPN) is a transmembrane receptor which is involved in various physiological and pathological processes, such as cell motility, invasion, tumor metastasis and blood vessels formation. Although there are reports on the involvement of PDPN in Sertoli cells in human and mice, the role of PDPN on the development of bovine Sertoli cells has not been reported. In the present study, Sertoli cells were isolated from 1-day-old bovine testes by two steps enzyme digestion method. Feulgen staining of satellite karyosomes and inhibin immunofluorescence staining suggested that the isolated immature Sertoli cells were very pure. Transfection with overexpression plasmid pBI-CMV3-PDPN and interference shRNA plasmid indicated that PDPN could significantly promote Sertoli cells cycle progression, cells proliferation and androgen-binding protein (ABP) production. Our results indicated that PDPN gene plays a significant role in the proliferation and maturation of bovine Sertoli cells.

AB030. PS01.12. A phase II study of regorafenib in patients with thymoma and thymic carcinoma previously treated with chemotherapy

Background: Available evidence suggests that angiogenesis has an important role in thymic epithelial tumours; VEGF is overexpressed in these cancers, and VEGF expression and microvessel density are associated with invasiveness and stage. Higher serum concentrations of VEGF and b-FGF have been noted in patients with thymic carcinoma. Overexpression of KIT has been reported in about 80% of thymic carcinomas, and mutations in the gene encoding this receptor are noted in about 10% of these cancers. PDGF and PDGFRα are also overexpressed in thymic epithelial cells. Recently, in a phase II trial, Sunitinib showed to be active in patients with thymic carcinoma achieving PR and SD in 26% and 65% of patients, respectively. Regorafenib potently inhibits the angiogenic and stromal RTKs VEGFR1, 2, and 3, TIE2 and PDGFR-β that promote tumor neo-vascularization, vessel stabilization and lymphatic vessel formation and play an important role in the tumor microenvironment, which all contribute to tumor development and metastasis formation. Regorafenib demonstrated nanomolar inhibition (3–200 nM) of the RTKs VEGFR2, PDGFR-β and FGFR in biochemical and cellular assays. The aim of this study is to determine the activity of Regorafenib as monotherapy in patients with advanced or recurrent thymoma (type B2–B3) and thymic carcinoma previously treated with cisplatin-containing chemotherapy.

Matrix Metalloproteinases Polymorphisms as Prognostic Biomarkers in Malignant Pleural Mesothelioma.

Background Malignant pleural mesothelioma (MPM) is a rare disease with a relatively short overall survival (OS). Metalloproteinases (MMPs) have a vast biological effect on tumor progression, invasion, metastasis formation, and apoptosis. MMP expression was previously associated with survival in MPM. Our aim was to evaluate if genetic variability of MMP genes could also serve as a prognostic biomarker in MPM. Methods We genotyped 199 MPM patients for ten polymorphisms: rs243865, rs243849 and rs7201, in MMP2; rs17576, rs17577, rs20544, and rs2250889 in MMP9; and rs1042703, rs1042704, and rs743257 in MMP14. We determined the influence on survival using Cox regression. Results Carriers of polymorphic MMP9 rs2250889 allele had shorter time to progression (TTP) (6.07 versus 10.03 months, HR = 2.45, 95% CI = 1.45–4.14, p = 0.001) and OS (9.23 versus 19.2 months, HR = 2.39, 95% CI = 1.37–4.18, p = 0.002). In contrast, carriers of at least one polymorphic MMP9 rs20544 allele had longer TTP (10.93 versus 9.40 months, HR = 0.57, 95% CI = 0.38–0.86 p = 0.007) and OS (20.67 versus 13.50 months, HR = 0.56, 95% CI = 0.37–0.85, p = 0.007). MMP14 rs1042703 was associated with nominally shorter TTP (8.7 versus 9.27 months, HR = 2.09, 95% CI = 1.06–4.12, p = 0.032). Conclusions Selected MMP SNPs were associated with survival and could be used as potential genetic biomarkers in MPM.

In Surgical Colon Cancer Patients Extended-Duration Thromboprophylaxis (30 days) with the Highest Dose of Tinzaparin (4,500 IU s.c./q.d.) Normalizes the Postoperative VEGF Levels.

Background/Purpose: In colon cancer (CC) patients preoperative (pre-op) levels of VEGF-A165 (VEGF) is a strong predictor for disease recurrence. Elevated postoperative (post-op) VEGF levels could have undesirable effects by enhancing tumor growth and metastasis formation. It has been suggested that thromboprophylaxis with a Low Molecular Weight Heparin (LMWH) in surgical cancer patients, further to thromboembolic protection, may exert some anti-neoplastic properties, as well. The aim of our study was to assess the potential impact of the LMWH Tinzaparin (Innohep® - Leo Pharma, Copenhagen, Denmark), given at different doses and for different perioperative (peri-op) periods, upon the post-op variability of serum VEGF levels in surgical CC patients.Methods: A total of 54 consecutive CC patients who underwent a curative resection were randomized in four groups according to their peri-op thromboprophylaxis scheme, which was based on administrating Tinzaparin in different doses and at different periods, as follows: group I: 3,500 IU for 10 days, group II: 3,500 IU for 30 days, group III: 4,500 IU for 10 days and group IV: 4,500 IU for 30 days. Serum VEGF concentrations were evaluated on the pre-op day (Day 0) and on the 10th and 30th post-op days (Day 10 and Day 30, respectively). For statistical analyses the mixed design ANOVA was used. P < 0.05 was considered significant.Results: On Day 0, VEGF didn't differ between groups I, II, III and IV (p>0.05, for every comparison). On Day 10, VEGF was increased in all groups. Between Day 10 and Day 30, VEGF remained stable in groups I (p=0.031) and II (p=1.000) and increased significantly in group III (p=0.005). On the contrary, VEGF decreased significantly in group IV (p<0.001). The most remarkable finding was observed when we compared VEGF between Day 0 and Day 30: while in groups I, II and III, VEGF remained significantly higher compared to Day 0 (p<0.001, p=0.041 and p<0.001, respectively), on the contrary, in group IV (extended-duration with the highest dose of 4,500 IU of tinzaparin) it was comparable to Day 0 (p=1.000).Conclusions: In surgical CC patients only the recommended thromboprophylaxis scheme with the highest prophylactic dose of Tinzaparin (4,500 IU) for extended-duration (30 days) normalizes VEGF levels at the end of the first post-op month by reducing them to the pre-op levels.

Functionalization of Ruthenium(II) Terpyridine Complexes with Cyclic RGD Peptides To Target Integrin Receptors in Cancer Cells

The lack of selectivity for cancer cells and the resulting negative impact on healthy tissue is a severe drawback of actual cancer chemotherapy. Tethering of cytotoxic drugs to targeting vectors such as peptides, which recognize receptors overexpressed on the surface of tumor cells, is one possible strategy to overcome such a problem. The pentapeptide cyc(RGDfK) targets the integrin receptor αvβ3, important for tumor growth and metastasis formation. In this work, two terpyridine‐based RuII complexes were prepared and for the first time conjugated to cyc(RGDfK) through amide bond formation, which resulted in a monomeric and a dimeric bioconjugate. Both RuII complexes were found to bind strongly and selectively to integrin αvβ3, and the dimeric molecule displayed a 20‐fold higher affinity to the receptor than the monomeric one. However, the cytotoxicity of the complexes and related bioconjugates against human A549 and SKOV‐3 cell lines is still not sufficient for application as anticancer agents. Nevertheless, considering the high selectivity for integrin receptor αvβ3, the synthesis of Ru‐based bioconjugates with cyc(RGDfK) paves a promising way towards the design of effective targeted anticancer agents.

A circulating tumor cell cluster-based model for tumor metastasis (Hypothesis).

Metastasis is the major cause of mortality in patients with malignancies; however, the mechanisms of tumor cell dissemination and metastasis formation are obscure. Circulating tumor cells (CTCs) are believed to be a critical step for distant metastasis and are associated with a poor patient prognosis. The precise processes of metastasis formation from CTCs are vague. In the present study, we hypothesize that two CTC cluster-based mechanisms of tumor cell inoculation in ectopic organs may be viable: i) Formation of a micro-cancer embolus due to interception of CTC clusters by small vessels; and ii) formation of micrometastasis in an extravasation-dependent or -independent manner. Pathological evidence of micro-cancer emboli is critical for the verifications of this hypothesis. If proved true, this hypothesis will provide a novel perspective for cancer metastasis and has valuable clinical implications.

Characterizing ErbB-2-Mediated Tyrosine Phosphorylation and Activation of Plexins.

Plexins comprise a family of transmembrane receptors for semaphorins. Plexins of the B- and D-subfamily interact with the receptor tyrosine kinase ErbB-2, and this interaction has been shown to be functionally relevant for various biological processes including tumor metastasis and bone formation. Binding of semaphorins to B- and D-subfamily plexins results in the activation of ErbB-2, which in turn phosphorylates these plexins. This phosphorylation triggers the activation of the small GTPases RhoA and RhoC downstream of B-subfamily plexins. Here we describe a methodology that allows the analysis of ErbB-2-mediated plexin phosphorylation and signaling.

Inhibitory effect of migration-inducing gene-7-shRNA recombinant retrovirus combined with endostatin on growth and metastasis of hepatoma xenograft

Objective: To investigate the inhibitory effect of migration-inducing gene-7(Mig-7)interfered with retrovirus-mediated RNA(shRNA)combined with recombinant human endostatin(ES)on the growth and metastasis of subcutaneous xenograft of human hepatoma cells in nude mice. Methods: Two Mig-7-mRNA oligonucleotide sequences(Mig-7-shRNA-1 and Mig-7-shRNA-2)and one sequence as a negative control(Mig-7-shRNA-N)were designed. The specific Mig-7-shRNA recombinant retrovirus expression vector plasmid was constructed and used for the transfection of human hepatoma MHCC-97H cells with high expression of Mig-7. The subcutaneous xenograft tumor model of human hepatocellular carcinoma(HCC)in nude mice was established, and according to the condition of transfection and administration, the nude mice were divided into pSIREN-M1 group, pSIREN-MN group, ES group, and pSIREN-M1+ES group. The xenograft tumor volume, mass, and metastasis were compared between groups. Immunohistochemistry was used to observe the formation of vasculogenic mimicry(VM)in xenograft tumor and the difference in tumor microvascular density(MVD), and Western blot was used to measure the expression of Mig-7 and vascular endothelial growth factor(VEGF)in each group. A one-way analysis of variance was used for comparison between groups, and the Fisher's exact test was used for comparison of continuous data between groups. Results: Compared with the pSIREN-MN group, the pSIREN-M1 group had significantly lower xenograft tumor volume, mass, and metastasis rate, Mig-7 expression, and formation of VM(P < 0.05), as well as significantly higher VEGF expression and MVD(P < 0.05). Compared with the pSIREN-MN group, the ES group had significantly lower xenograft tumor volume, mass, and metastasis rate, VEGF expression, and MVD(P < 0.05), as well as significantly higher Mig-7 expression and formation of VM(P < 0.05). Compared with the pSIREN-M1 group and the ES group, the pSIREN-M1+ES group had significantly lower xenograft tumor volume, mass, and metastasis rate, Mig-7 expression, formation of VM, VEGF expression, and MVD(P < 0.05). Conclusion: Mig-7-shRNA recombinant retrovirus combined with ES has a better inhibitory effect on the growth and metastasis of HCC xenograft tumor than Mig-7-shRNA recombinant retrovirus or ES alone. The anti-tumor angiogenesis therapy alone, which targets vascular endothelial cells in vivo, has a limited effect, since it may promote the formation of VM.