Formation Of Liver Metastases Research Articles

Abstract A121: Complement activation is critical for tumor progression in an orthotopic immunocompetent model of lung cancer

Abstract Background: The complement system is an important arm of the innate immune system, and complement proteins are produced by both cancer cells and cells of the TME. A primary function of the complement system is to remove pathogens and injured cells, and it had been assumed that complement activation would result in elimination of cancer cells. However, recent work has revealed new biologic effects of the complement activation fragments C3a and C5a. For example, C3a and C5a directly stimulate the growth of some tissues. Furthermore, inflammatory cells as well as CD4 and CD8 T cells express receptors for both of these activation fragments (C3aR and C5aR), and a recent study reported that C5a reduces the anti-tumor function of CD8+ T cells. Nevertheless, the role of complement activation in the growth and spread of non-small cell lung cancer is incompletely understood. While complement activation can proceed by multiple pathways, a central event is the deposition of C3 on the cell surface. The goal of this study was to examine the role of complement in lung cancer progression employing an immunocompetent orthotopic model which recapitulates many of the features of human lung cancer. Methods: Two murine lung cancer cell lines derived from C57BL/6 mice were used in an orthotopic model of tumor progression: Lewis Lung Carcinoma (LLC) cells, which are a de-differentiated cell line in which driver mutations have not been identified; and CMT167 cells, which are an epithelial lung cancer cell line that express oncogenic K-Ras. Tumor cells were implanted in the left lungs of syngeneic mice WT C57BL/6 mice, or mice which were globally knocked out for C3 (C3-KO). Primary tumor growth and formation of liver metastases were quantitated in the two groups of mice. Complement activation in the plasma was determined by ELISA. Single cell suspensions of the left lung of tumor-bearing mice were prepared and populations of myeloid cells and immune cells quantitated by flow cytometry. Results: Implantation of cancer lead to systemic complement activation as assessed by ELISA in plasma. Primary tumors and liver metastases were decreased in both cell lines implanted into C3-KO mice compared to controls. However, the degree of inhibition was much greater in CMT167 tumors. LLC cells expressed C3 in vitro, whereas CMT167 cells did not, suggesting that autonomous C3 production by cancer cells may contribute to tumor progression. Recruitment of inflammatory myeloid cells was not altered in tumors growing in C3-KO mice. At early time points, we observed equal increases in T cells (CD4, CD8) in the two groups of mice. However, at later time points (3 weeks), the number of T cells had decreased in WT mice, but remained elevated in C3-KO mice. Conclusions: Complement activation occurs in the setting of tumors. C3 plays a critical role in tumor progression, with strong inhibition observed in a subset of cancer cells. The differential response to complement inhibition may depend on the ability of the cancer cells to produce C3. Effects of complement activation may be mediated by inhibiting cytotoxic T cells. These data suggest that complement inhibitors may represent novel immunotherapeutic targets for lung cancer, and suggest interactions between the complement system and checkpoint inhibitors. Citation Format: Jeff Kwak, Howard Li, Jennifer Laskowski, Mary Weiser-Evans, Joshua Thurman, Raphael A. Nemenoff. Complement activation is critical for tumor progression in an orthotopic immunocompetent model of lung cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A121.

Abstract 4119: Tyrosine phosphorylation of MACC1 is essential and druggable for colorectal cancer metastasis restriction

Abstract We identified the previously undescribed gene Metastasis Associated in Colon Cancer 1 (MACC1). MACC1 is a metastasis inducer and prognostic biomarker for colorectal cancer (CRC). MACC1 has meanwhile been confirmed as decisive driver for tumorigenesis and metastasis for a broad variety of solid cancers. Here we aim to identify the impact of defined tyrosine phosphorylations on the function of the metastasis inducer MACC1. In silico identified MACC1 tyrosine phosphorylation (pY) sites were ranked and most promising positions were site-directed mutated in wildtype (wt) MACC1 constructs. Human CRC cells were transduced with these constructs. Tyrosine phosphorylation was quantified by selected reaction monitoring (SRM)-mass spectrometry. Effects of pY-MACC1 mutants on cell migration, proliferation, dissemination and colony formation were assessed in cell culture as well as on liver metastasis formation in human CRC-xenografted mice. To identify MACC1-phosphorylating kinases the MACC1 interactome was identified using mass spectrometry. Most promising binding partners were validated by immunoprecipitation. Impact of inhibitors targeting kinases phosphorylating MACC1 was assessed on MACC1-induced tumor growth and liver metastasis in human CRC-xenografted mice by in vivo imaging. We report that tyrosine phosphorylation of MACC1 is hierarchical and essential for motility and proliferation in cell culture, as well as for tumor growth and metastasis in mice. We identified kinases for MACC1 phosphorylation. Targeting these kinases using inhibitors employed in clinical trials restricts MACC1-induced tumor growth and metastasis in mice. MACC1 tyrosine phosphorylation is crucial for metastasis and is druggable by small molecule inhibitors. This fundamental finding opens new therapeutic options for inhibition of MACC1-induced cancer metastasis. Citation Format: Dennis Kobelt, Gunnar Dittmar, Claudia Fleuter, Janice Smith, Mathias Dahlmann, Rebekka Migotti, Peter M. Schlag, Ulrike S. Stein. Tyrosine phosphorylation of MACC1 is essential and druggable for colorectal cancer metastasis restriction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4119. doi:10.1158/1538-7445.AM2015-4119

Imaging of tumor clones with differential liver colonization.

We present a model of hepatic colorectal metastases which represents monoclonal cell lines double-labeled by luciferase and tdTomato. These cells form liver metastasis in varying numbers and patterns similar to those observed in patients. Using in vivo and ex vivo luminescent and fluorescent imaging we determine the growth kinetics and clonogenic frequency of tumor cells colonizing liver. Molecular profiling detected stable expressional differences between clones consistent with their phenotypes. The data indicate that clinically relevant phenotypes of liver metastases can be modeled in vivo.

Mechanisms involved in breast cancer liver metastasis.

Liver metastasis is a frequent occurrence in patients with breast cancer; however, the available treatments are limited and ineffective. While liver-specific homing of breast cancer cells is an important feature of metastasis, the formation of liver metastases is not random. Indeed, breast cancer cell factors contribute to the liver microenvironment. Major breakthroughs have been achieved recently in understanding breast cancer liver metastasis (BCLM). The process of liver metastasis consists of multiple steps and involves various factors from breast cancer cells and the liver microenvironment. A further understanding of the roles of breast cancer cells and the liver microenvironment is crucial to guide future work in clinical treatments. In this review we discuss the contribution of breast cancer cells and the liver microenvironment to liver metastasis, with the aim to improve therapeutic efficacy for patients with BCLM.

MicroRNA-146a inhibits cancer metastasis by downregulating VEGF through dual pathways in hepatocellular carcinoma.

Growing evidence indicates that miR-146a is involved in carcinogenesis and tumor progression in several human malignancies. However, the molecular details underlying miR-146a mediated regulation of its target genes and its precise biological function in cancer, especially in hepatocellular carcinoma (HCC) remains unclear.MethodsThe expression levels of genes including miR-146a, APC, VEGF and HAb18G were examined in HCC cell lines and patient specimens were compared with control levels using quantitative reverse transcription-PCR. The functions of miR-146a and HAb18G in migration/invasion and liver metastasis formation were determined by transwell and spleen injection assays, respectively. miR-146a related genes were determined by PCR array. The potential regulatory targets of miR-146a were determined by bioinformatics and prediction tools, correlation with target protein expression, and luciferase reporter assay. DNA methylation status of miR-146a promoter were performed by PCR analysis of bisulfite-modified genomic DNA.ResultsWe demonstrated that miR-146a expression was markedly downregulated in hepatoma cells and hepatoma tissues compared to immortalized normal liver epithelial cells and normal hepatic tissues. DNA methylation of miR-146a promoter correlated with its downexpression and with liver cancer metastasis. The restoration of miR-146a dramatically suppressed HCC cell invasion and metastasis by repressing VEGF expression through upregulating APC, which inhibits β-catenin accumulation in nucleus, and downregulating NF-κB p65 by targeting HAb18G. In human HCC, miR-146a expression was negative correlated with increased HAb18G, VEGF, NF-κB p65 and beneficial prognosis.ConclusionThis study identified a novel target of miR-146a and defined miR-146a as a crucial tumor suppressor in human HCC that acts through multiple pathways and mechanisms to suppress HCC invasion or metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-14-5) contains supplementary material, which is available to authorized users.

CCR1-Mediated Formation of Liver Microenvironment for Cancer Metastasis

Accumulating evidence suggests that bone marrow-derived myeloid cells constitute the major components of cancer microenvironment and play significant roles in the progression of cancer. We have previously shown that myeloid cells expressing CCR1, a chemokine receptor, are recruited to the microenvironment of disseminated colon cancer cells in the liver (PNAS 2010). These CCR1+ cells promote metastatic colonization of cancer cells in the liver through producing metalloproteinases MMP9 and MMP2. However, the cell lineage and precise characteristics of these myeloid cells were largely unknown. The aim of this study is to characterize the cell types among these myeloid cells expressing CCR1 and/or MMPs.In order to characterize the CCR1-expressing myeloid cells accumulating at liver metastatic foci, we have established bacterial artificial chromosome (BAC)-based transgenic mouse lines in which membrane-targeted Venus fluorescent protein (mVenus) was expressed under the control of Ccr1cis-regulatory elements. A flow cytometric analysis of adult transgenic mouse bone marrow cells showed that expression of the reporter mVenus fluorescence was restricted to the CD11b+ Gr-1+ cells and the level of endogenous Ccr1 mRNA correlated well with that of mVenus fluorescence, suggesting that expression of the reporter precisely reflected that of the endogenous CCR1.Inoculation of CCL9-expressing mouse colon cancer cell line CMT93 into the spleen of syngeneic hosts causes efficient metastatic colonization of the cancer cells in the liver. Employing this model, we monitored the behavior of the Ccr1-mVenus+ myeloid cells in the cancer metastasis microenvironment, and found that CD45+ CD11b+Ccr1-mVenus+ cells purified from liver metastatic foci were mostly neutrophils. In contrast, within CD45+ CD11b+Ccr1-mVenus– fraction, we identified eosinophils, monocytes and fibrocytes. Notably, the major cell types in the metastatic foci were monocytes and fibrocytes, with the latter producing abundant collagen.Chronologically, accumulation of neutrophils and monocytes preceded that of eosinophils and fibrocytes. In Ccr1−/− mice, early accumulation of Ccr1-mVenus+ Gr-1+ cells was significantly compromised and later recruitment of fibrocytes to the metastatic foci was reduced. Antibody-mediated depletion of neutrophils caused a significant reduction in the fibrocyte number at the metastatic foci. These results suggest that CCR1 plays critical roles in early accumulation of neutrophils and subsequent recruitment of fibrocytes.After having identified four types of myeloid cells in the metastatic foci, we determined the expression levels of CCR1, MMP9 and MMP2 mRNAs in each purified population, and found that CCR1 and MMP9 are expressed preferentially by neutrophils, whereas MMP2 is expressed exclusively by monocytes/fibrocytes. Considering the chronological difference in the appearance of myeloid cells in liver metastatic foci, these results suggest that production of MMP9 and MMP2 in succession by different myeloid cells is necessary for disseminated colon cancer cells to colonize in the liver.In summary, these results suggests that CCR1 is required for recruitment of neutrophils in the early phase of colon cancer dissemination and the subsequent accumulation of fibrocytes, and that MMP9 and MMP2 are produced by neutrophils and fibrocytes, respectively, in a successive manner during the formation of liver metastasis. Further investigation of these cancer-driven host reactions in human colon cancer dissemination may lead to effective preventive measures against cancer metastasis. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3367: Understanding the role of metabolic reprogramming in breast cancer progression and metastasis

Abstract Background: The emergence of metastatic breast cancer is the most deadly aspect of the disease and once it has spread from the primary site, it is largely incurable. Important metabolic changes have been correlated with breast cancer progression and acquisition of the metastatic phenotype. One prominent example includes the well-established Warburg effect, which stipulates that cancer cells preferentially utilize rapid glycolytic metabolism over mitochondrial respiration to produce energy. Such a metabolic shift ensures that cancer cells can meet the bioenergetics and biosynthetic demands associated with increased proliferation. Objectives: The energetic requirements of individual cancer cells will greatly vary as the tumor grows and acquires malignant characteristics. The metabolic demands during tumor initiation will differ from those that occur during tumor growth at the primary site and dissemination to distant metastatic sites. Therefore, we hypothesize that distinct metabolic signatures are associated with each one of these steps and that a better characterization of these transitions, as well as the key regulators governing this process, will help identifying potential therapeutic targets. Results: Using a series of mouse mammary cancer cells derived from a spontaneous mammary tumor, which includes 67NR (tumorigenic/non-metastatic), 66cl4 (tumorigenic/lung metastatic) and 4T1 (tumorigenic/metastatic to multiple sites), we examined changes in several parameters of cellular metabolism that could be associated with different stages of tumor progression. Using mass spectrometry, we assessed the uptake and metabolic flux of labeled glucose through the cellular metabolic pathways. Our results suggest that there is a further shift towards a “Warburg-like” phenotype as tumor cells acquire aggressive characteristics. Using an in vivo selection approach on the 4T1 breast cancer cells, we have established subpopulations that aggressively form liver metastases. Glucose tracing experiments reveal an accumulation of lactate at the expense of reduced allocation of glucose towards citrate production suggesting that these cells have a further increased glycolytic phenotypes compared to the 4T1 parental cells. These results raise the possibility that unique metabolic processes and checkpoints are engaged in metastatic populations that contribute to their metastatic ability. PDK1 is an enzyme that prevents the uptake of pyruvate into the TCA cycle. We show that PDK1 is upregulated with the increase in metastatic potential. This has led us to hypothesize that the “Warburg-like” phenotype observed is mediated by a PDK1-induced switch favouring glycolysis. We demonstrate that PDK1 is required to form liver metastases in vivo following splenic injections. Ongoing experiments are being pursued to uncover the mechanism by which PDK1 is regulated and how it influences the metastatic process. Citation Format: Fanny Dupuy, Julianna Blagih, Sébastien Tabariès, Julie St-Pierre, Russell G. Jones, Peter M. Siegel. Understanding the role of metabolic reprogramming in breast cancer progression and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3367. doi:10.1158/1538-7445.AM2014-3367

Abstract 2094: Regulation of site-specific liver metastasis by collagen IV-conveyed signals

Abstract The liver is a primary site of metastasis for some of the most common human malignancies. At present, surgical resection is the most effective curative option for liver metastases, but most patients with liver metastases still succumb to their disease. A better understanding of the underlying biology is essential for design of more effective therapy. We previously identified basement membrane type IV collagen α1/α2 expression levels as determinants of the liver colonizing potential in a murine lung carcinoma model (1) and observed high collagen IV expression levels in surgical specimens of liver metastases from diverse tumor types. The aim of this study was to elucidate the functional relevance of increased collagen IV expression to liver metastases formation. We reported that collagen IV α1/α2 overexpression in non-metastatic lung carcinoma M-27 (M27colIV) cells increased specifically their liver (but not lung) metastasizing potential. A transcriptome analysis was therefore performed on these cells in order to identify changes to gene expression that could account for the newly acquired metastatic potential. This analysis revealed that type IV collagen levels regulated the expression of multiple genes. Prominent among them were genes encoding for chemokines such as CCL-5 and CCL-7 and for growth factors such as amphiregulin (AREG) that were all upregulated by ≥ 3 fold. These changes were validated at the RNA and protein levels using qPCR and Western blotting, respectively. When the expression of these genes in M27colIV cells was subsequently silenced using shRNA, a significant decrease (5-10 folds) was observed in their ability to generate experimental liver metastases, as compared to wild type or mock-transfected cells and they failed to develop metastases in 40-50 % of injected mice. A similar correlation between CCL-5 and CCL-7 expression levels and liver metastases formation was also noted in human colon carcinoma cell lines. The results identify a type IV collagen-regulated gene expression signature that promotes liver metastasis and suggest that collagen IV-induced changes in chemokine and growth factor production levels mediate this effect. They provide a rationale for further exploration of the clinical utility of CCL-5, CCL-7 and AREG as targets in the management of liver metastases. Supported by a grant from the Canadian Institute for Health Research (to PB) and a Henry R. Shibata Cedars Cancer Fellowship (to RR). REFERENCES 1. Burnier JV, Wang N, Michel RP, Hassanain M, Li S, Lu Y, et al. Type IV collagen-initiated signals provide survival and growth cues required for liver metastasis. Oncogene. 2011;30:3766-83. Citation Format: Roni F. Rayes, Ni Wang, Julia V. Burnier, France Bourdeau, Pnina Brodt. Regulation of site-specific liver metastasis by collagen IV-conveyed signals. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2094. doi:10.1158/1538-7445.AM2014-2094

Abstract 4807: Zoledronic acid overcomes the resistance to the anti-angiogenic therapy and normalizes tumor vessels by switching from a M2- to a M1-like macrophages phenotype in a mouse model of spontaneous cervical cancer

Abstract Recent studies described a resistance to the anti-angiogenic therapy both in pre-clinical and clinical settings. Several mechanisms that contribute to this resistance has been so far proposed and, among these, the recruitment of bone marrow-derived cells to the tumors, seems to be critical. We have previously showed that an amino-bisphosphonate, Zoledronic acid (ZA), in a mouse model of spontaneous cervical carcinogenesis (HPV16/E2) by targeting MMP-9+ tumor-associated macrophages (TAM), strongly inhibited cancer progression and angiogenesis. Stemming from these data we sought to investigate whether ZA by acting on TAM, was able to overcome the resistance to anti-angiogenic treatments. We treated tumor-bearing HPV16/E2 mice with ZA alone or combined with the small molecule tyrosine kinase inhibitor Sunitinib or with an anti-VEGFR-2 antibody, DC101, to assess the ability of ZA to block metastasis dissemination. Notably ZA synergized with Sunitinib and DC101 to inhibit primary tumor growth. Interestingly, while these compounds increased the incidence and the number of liver and lung metastasis, ZA alone inhibited basal tumor metastasis in parallel to angiogenesis inhibition. ZA combined with Sunitinib or DC101 showed a greater effect in impairing liver and lung metastasis formation. Confocal analysis of the vasculature revealed that while Sunitinib- or DC101-treated cancers displayed poorly functional vessels, both ZA alone or combined with Sunitinib or DC101 efficiently induced a normalized vasculature highly covered by pericytes and better perfused. Remarkably, gene and protein expression analysis of FACS-sorted macrophages derived from tumors of the different treatment groups, showed that ZA alone or combined with Sunitinib or DC101 inhibited the expression of M2-markers such as MRC-1, Il-10, CCL22 and MMP-9 and simultaneously enhanced the levels of M1-markers such as Il12 and CXCL9 in these cells. We conclude that ZA, by promoting a M1-like macrophages phenotype in HPV16/E2 cervical cancers and consequently inducing a normalized vasculature, efficiently overcomes the resistance to angiogenesis inhibition by blocking metastasis dissemination. Citation Format: Stefania Capano, Federica Maione, Oriol Casanovas, Federico Bussolino, Enrico Giraudo. Zoledronic acid overcomes the resistance to the anti-angiogenic therapy and normalizes tumor vessels by switching from a M2- to a M1-like macrophages phenotype in a mouse model of spontaneous cervical cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4807. doi:10.1158/1538-7445.AM2014-4807

Abstract 1996: Inhibitor of differentiation-1 (Id1) expression deficiency in the tumor microenvironment impairs experimental hepatic metastasis of lung cancer

Abstract Background: Liver metastasis (LM) occurs in 30% of non-small cell lung cancer patients but the contribution of the patient's genetic background to the hepatic metastasis development is unclear. Previously, we reported that inflammation contributes to the prometastatic microenvironment of the liver. Upregulation of transcriptional regulator Id1 gene has been associated with inflammation while ablation of Id1 in mice reduced inflammation. In this study Id1 deficient mice were used to analyze the role of host Id1 in the hepatic colonization of an experimental lung cancer. Methods: Lewis lung carcinoma cells were used for the experimental production of hepatic metastasis via intrasplenic injection of cancer cells in C57BL/6 wild-type and Id1-knockout (KO) mice. Animals were splenectomized to avoid flank tumor formation and weekly FDG-micro-positron emission tomographies (PET) were performed to monitor LM formation. Animals were sacrificed at the time of LM occurrence and total RNA was purified from LM. A microarray gene expression analysis (Affymetrix) with the support of Ingenuity Pathways Analysis (IPA) was performed to evaluate the potential impact of Id1 deficiency on the regulation of genes mediating cancer cell invasion and proliferation, angiogenesis and metastasis. Results: By week two after cancer cell injection, 70% wild-type and 10% Id1-KO mice had detectable hepatic metastasis by FDG-PET (p=0.02). Three weeks after injection, when 100% wild-type had LM, still just 20% Id1-KO mice had LM (p=0.015). Moreover, 50% Id1-KO mice remained LM-free >4 weeks after cancer cell injection. No other metastasis sites were detected at necropsy. A microarray gene expression analysis of LM from Id1-KO animals uncovered a remarkable downregulation (p<0.05) of specific genes involved in the activation of cancer cell proliferation (Myc, Cdc20, Smc2, Aurora kinase B, Cyclin B1, CDK1, TIMP1, Epiregulin), migration (Ccl7, Serpine1/PAI-1, VIM, Anxa2, S100A4, S100A6, Akt3, Adrenomedullin), angiogenesis (Hif1a, PGF, Nestin) and metastasis (FGFR1, Src, IL-18, MMP3, MMP12, MMP13, Amphiregulin, PDGFA, FoxM1, Hsp90AA1, MIF, Ccl2, RhoC). Conclusion: Our results demonstrate that Id1 expression deficiency impaired the metastatic process of lung cancer cells to the liver through the specific downregulation of key metastasis-associated genes and suggest that Id1-dependent mechanisms are new targets for hepatic metastasis therapeutic. This study has been partially funded by “UTE project CIMA” and an ISCIII-FIS grant 2011. Citation Format: Ignacio Gil-Bazo, Eduardo Castanon, Inés López, Victor Segura, Mariano Ponz-Sarvise, José M. López-Picazo, Maria Collantes, Margarita Ecay, Isabel Gil-Aldea, Alfonso Calvo, Fernando Vidal-Vanaclocha. Inhibitor of differentiation-1 (Id1) expression deficiency in the tumor microenvironment impairs experimental hepatic metastasis of lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1996. doi:10.1158/1538-7445.AM2014-1996

Kupffer cells decrease metastasis of colon cancer cells to the liver in the early stage.

Although Kupffer cells (KCs) play an important role in the liver's immune response, their role in colon cancer metastasis to the liver is unclear. We here analyzed the relationship between KCs and tumor cells (TCs) in colon cancer metastasis to the liver. Fischer 344 (F344) rats were divided into control group (KC+ group) and KC elimination group (KC‑ group), in which KC elimination was induced by Cl2MDP liposome injection. RCN‑H4 colon cancer cells were injected into the rats of both groups, and the relationship between the two types of cells was observed by intravital microscopy (IVM) for 6 h. Moreover, to investigate the effect of KCs on liver metastasis formation, KCs were eliminated at different time points before and after the TC injection. The number of metastatic nodules 2 weeks after the injection was evaluated. In the KC‑ group, IVM revealed that the number of adherent TCs had increased 1.5‑fold at 6 h after the TC injection as compared with in the KC+ group. Moreover, in the KC+ group, 74% of the TCs adhered to the KCs, and KC activation and KC phagocytosis of the TCs were observed. Two weeks after the injection, the number of metastatic nodules was significantly increased in rats in which the KCs had been eliminated before the injection, but not in rats in which the KCs had been eliminated after the injection. KC activation and KC phagocytosis of TCs decreased colon cancer cell metastasis to the liver.

Inhibitory effect of gene combination in a mouse model of colon cancer with liver metastasis.

The aim of the present study was to establish an animal liver metastasis model with human colon cancer and investigate the inhibitory effect of the wild type (WT) p53 gene combined with thymidine kinase/ganciclovir (TK/GCV) and cytosine deaminase/5-fluorocytosine (CD/5-FC) systems on liver metastasis of colon cancer. A nude mouse liver metastasis model with human colon cancer was established via a spleen cultivation method. A total of 32 nude mice were randomly divided into four groups, each group with eight mice. Group 1 mice received splenic injections of SW480 cells (control group), while group 2 mice were injected with SW480/p53 cells in the spleen. Group 3 mice were administered splenic injections of SW480/TK-CD cells, and GCV and 5-FC were injected into the abdominal cavity. Finally, group 4 mice received splenic injections of SW480/p53 cells mixed in equal proportion with SW480/TK-CD cells, as well as GCV and 5-FC injections in the abdominal cavity. These cells described were constructed in our laboratory and other laboratories. The number of liver metastatic tumors, the liver metastasis rate, conventional pathology, electron microscopy and other indicators in the nude mice of each group were compared and observed. The nude mouse liver metastasis model with human colon cancer was successfully established; the liver metastasis rate of the control group was 100%. The results demonstrated that the rate of liver metastasis in the nude mice in each treatment group decreased, as well as the average number of liver metastatic tumors. Furthermore, the effect of the treatment group with genetic combination (group 4) was the most effective, demonstrating that WTp53 had a synergistic effect with TK/GCV and CD/5-FC. Therefore, the present study successfully established a mouse model of liver metastasis with colon cancer by injecting human colon cancer cells in the spleen. Combined gene therapy was shown to have a synergistic effect, which effectively inhibited the formation of liver metastasis from colon cancer.

Repression of liver colorectal metastasis by the serpin Spn4A a naturally occurring inhibitor of the constitutive secretory proprotein convertases.

Liver is the most common site of metastasis from colorectal cancers, and liver of patients with liver colorectal metastasis have abnormal levels of the proprotein convertases (PCs). These proteases are involved in the activation and/or expression of various colon cancer-related mediators, making them promising targets in colorectal liver metastasis therapy. Here, we revealed that the serpin Spn4 from Drosophila melanogaster inhibits the activity of all the PCs found in the constitutive secretory pathway and represses the metastatic potential of the colon cancer cells HT-29 and CT-26. In these cells, Spn4A inhibited the processing of the PCs substrates IGF-1R and PDGF-A that associated their reduced anchorage-independent growth, invasiveness and survival in response to apoptotic agents. In vivo, Spn4A-expressing tumor cells showed repressed subcutaneous tumor development and liver metastases formation in response to their intrasplenic inoculation. In these cells Spn4A induced the expression of molecules with anti-metastatic functions and inhibited expression of pro-tumorigenic molecules. Taken together, our findings identify Spn4A as the only endogenous inhibitor of all the constitutive secretory pathway PCs, which is able to repress the metastatic potential of colon cancer cells. These results suggest the potential use of Spn4A and/or derivates as a useful adduct colorectal liver metastasis prevention.

Suppression of Colorectal Cancer Liver Metastasis by Apolipoprotein(a) Kringle V in a Nude Mouse Model through the Induction of Apoptosis in Tumor-Associated Endothelial Cells

The formation of liver metastases in colorectal cancer patients is the primary cause of patient death. Current therapies directed at liver metastasis from colorectal cancer have had minimal impact on patient outcomes. Therefore, the development of alternative treatment strategies for liver metastasis is needed. In the present study, we demonstrated that recombinant human apolipoprotein(a) kringle V, also known as rhLK8, induced the apoptotic turnover of endothelial cells in vitro through the mitochondrial apoptosis pathway. The interaction of rhLK8 with glucose-regulated protein 78 (GRP78) may be involved in the induction of apoptosis because the inhibition of GRP78 by GRP78-specific antibodies or siRNA knockdown inhibited the rhLK8-mediated apoptosis of human umbilical vein endothelial cells in vitro. Next, to evaluate the effects of rhLK8 on angiogenesis and metastasis, an experimental model of liver metastasis was established by injecting a human colorectal cancer cell line, LS174T, into the spleens of BALB/c nude mice. The systemic administration of rhLK8 significantly suppressed liver metastasis from human colorectal cancer cells and improved host survival compared with controls. The combination of rhLK8 and 5-fluorouracil substantially increased these survival benefits compared with either therapy alone. Histological observation showed significant induction of apoptosis among tumor-associated endothelial cells in liver metastases from rhLK8-treated mice compared with control mice. Collectively, these results suggest that the combination of rhLK8 with conventional chemotherapy may be a promising approach for the treatment of patients with life-threatening colorectal cancer liver metastases.

S100P antibody-mediated therapy as a new promising strategy for the treatment of pancreatic cancer

Despite progresses in diagnosis and treatment, pancreatic cancer continues to have the worst prognosis of all solid malignant tumors. Recent evidences suggest that the metastasis-promoting protein S100P stimulates pancreatic tumor proliferation, survival, invasion and metastasis progression through extracellular functions. Moreover, its expression is strongly correlated with poor prognosis in patients with several types of cancer although the entire molecular mechanism responsible for the diverse biological functions is not fully understood. We showed that extracellular S100P stimulates pancreatic carcinoma BxPC3 cell line by promoting cell proliferation. We also demonstrated that S100P induces, in this cell line, the phosphorylation of IκBα and the secretion of matrix metalloproteinase 9 (MMP-9). In addition, treatment with S100P protected cells from injuries induced by the cytotoxic agent Gemcitabine. On the basis of these results, we developed function-blocking anti-S100P monoclonal antibodies (mAbs) that abolished all of its in vitro activities. Furthermore, in vivo treatment with the candidate 2H8 antibody decreased tumor growth and liver metastasis formation in a subcutaneous and orthotopic BxPC3 tumor model. We conclude here that a therapeutic strategy blocking the extracellular activity of S100P by means of specific mAbs could be an attractive therapeutic approach as a single agent or in combination with target-directed or chemotherapeutic drugs to treat pancreatic cancer.

Expression of N-acetylglucosaminyltransferase V in the subserosal layer correlates with postsurgical survival of pathological tumor stage 2 carcinoma of the gallbladder

N-Acetylglucosaminyltransferase V (GnT-V), an enzyme that catalyzes the β1-6 branching of N-acetylglucosamine on asparagine-linked oligosaccharides of cellular proteins, enhances the malignant behaviors of carcinoma cells in experimental models. The aim of this study was to determine clinical significance of GnT-V expression in human pT2 gallbladder carcinoma with simple in vitro experiments. Ninety patients with pT2 gallbladder carcinoma were included for this study. The in vitro and in vivo biological effects of GnT-V were investigated using gallbladder carcinoma cells with variable GnT-V expression levels induced by a small interfering RNA. Of the 90 cases, 57 showed positive staining and the remaining 33 demonstrated negative staining, the subcellular localization in the 57 cases was classified into the granular-type in 31 cases and the diffuse-type in 26 cases. In 76 cases with curative resection, postsurgical survival was significantly poorer in those showing positive staining than in those showing negative staining (P=0.028). In all of the 76 cases, postsurgical recurrence was significantly more frequent in those showing diffuse-type localization than in those showing negative staining. Experimental analyses demonstrated that the down-regulation of GnT-V expression in gallbladder carcinoma cells induced suppression of cell growth in vitro. The expression levels of GnT-V in the cells were highly correlated with the rapid in vivo growth coupled with the enhanced angiogenesis, and the tendency to form liver metastasis. GnT-V expression in the subserosal layer of pT2 gallbladder carcinoma is correlated with the aggressiveness of the disease.

Transforming growth factor‐beta‐induced protein ig‐h3 is essential for the formation of human colorectal carcinoma liver metastasis in vivo

Transforming growth factor‐beta‐induced protein ig‐h3 (TGFBI) is extracellular matrix component important for cell‐collagen interaction. We and others have reported elevated expression of TGFBI in several human cancers, where its role remains controversial. Current study reports for the first time on the function of TGFBI in colorectal carcinoma liver metastasis (CRC‐LM), a frequently encountered malignancy with no satisfactory treatment to date.We show that TGFBI is highly expressed in human CRC‐LM and in LM originating from breast, lung and pancreatic tumors. From functional perspective, silencing TGFBI in SW1222 human colorectal carcinoma cells lead to a decrease in migration (−70%) and proliferation (−20%). To understand how this effects translate in vivo we have developed a novel animal model of colorectal carcinoma based on chicken chorioallantoic membrane (CAM) that mimics human CRC‐LM. TGFBI silencing resulted in 50% reduction of tumor volume in vivo. Finally, we have demonstrated that TGFBI is systemically reachable target for homing fluorescently‐labeled antibodies injected in tumor bearing animals. Collectively the data show that TGFBI is essential for promoting the development of CRC‐LM and therefore a promising target for designing novel therapeutic approaches.This work is supported by FNRS, Belgium and FP7 EU program.

PDGFRB Promotes Liver Metastasis Formation of Mesenchymal-Like Colorectal Tumor Cells

In epithelial tumors, the platelet-derived growth factor receptor B (PDGFRB) is mainly expressed by stromal cells of mesenchymal origin. Tumor cells may also acquire PDGFRB expression following epithelial-to-mesenchymal transition (EMT), which occurs during metastasis formation. Little is known about PDGFRB signaling in colorectal tumor cells. We studied the relationship between PDGFRB expression, EMT, and metastasis in human colorectal cancer (CRC) cohorts by analysis of gene expression profiles. PDGFRB expression in primary CRC was correlated with short disease-free and overall survival. PDGFRB was co-expressed with genes involved in platelet activation, transforming growth factor beta (TGFB) signaling, and EMT in three CRC cohorts. PDGFRB was expressed in mesenchymal-like tumor cell lines in vitro and stimulated invasion and liver metastasis formation in mice. Platelets, a major source of PDGF, preferentially bound to tumor cells in a non-activated state. Platelet activation caused robust PDGFRB tyrosine phosphorylation on tumor cells in vitro and in liver sinusoids in vivo. Platelets also release TGFB, which is a potent inducer of EMT. Inhibition of TGFB signaling in tumor cells caused partial reversion of the mesenchymal phenotype and strongly reduced PDGFRB expression and PDGF-stimulated tumor cell invasion. These results suggest that PDGFRB may contribute to the aggressive phenotype of colorectal tumors with mesenchymal properties, most likely downstream of platelet activation and TGFB signaling.

Inhibition of Metastatic Potential in Breast CarcinomaIn VivoandIn Vitrothrough Targeting VEGFRs and FGFRs

Angiogenesis and lymphangiogenesis are considered to play key roles in tumor metastasis. Targeting receptor tyrosine kinases essentially involved in the angiogenesis and lymphangiogenesis would theoretically prevent cancer metastasis. However, the optimal multikinase inhibitor for metastasis suppression has yet to be developed. In this study, we evaluated the effect of NSTPBP 0100194-A (194-A), a multikinase inhibitor of vascular endothelial growth factor receptors (VEGFRs)/fibroblast growth factor receptors (FGFRs), on lymphangiogenesis and angiogenesis in a mammary fat pad xenograft model of the highly invasive breast cancer cell line 4T1-Luc+. We investigated the biologic effect of 194-A on various invasive breast cancer cell lines as well as endothelial and lymphatic endothelial cells. Intriguingly, we found that 194-A drastically reduced the formation of lung, liver, and lymph node metastasis of 4T1-Luc+ and decreased primary tumor growth. This was associated with significant reductions in intratumoral lymphatic vessel length (LVL) and microvessel density (MVD). 194-A blocked VEGFRs mediated signaling on both endothelial and lymphatic endothelial cells. Moreover, 194-A significantly inhibited the invasive capacity induced by VEGF-C or FGF-2 in vitro in both 4T1 and MDA-MB231 cells. In conclusion, these experimental results demonstrate that simultaneous inhibition of VEGFRs/FGFRs kinases may be a promising strategy to prevent breast cancer metastasis.

Abstract PR9: Granulocyte macrophage colony stimulating factor (GM-CSF) pancreas tumor vaccine in combination with blockade of PD-1 in a preclinical model of pancreatic cancer.

Abstract Introduction: Our phase I/II human clinical trials utilizing a GM-CSF secreting allogeneic pancreas tumor vaccine (GVAX) have been shown to be safe and effective in inducing anti-tumor immune response in pancreatic adenocarcinoma patients. GVAX treated patients have demonstrated infiltration of PD-1+ T cells which is a major immunosuppressive mechanism in the tumor microenvironment. We hypothesized that the use of a PD-1 blocking antibody and GVAX will improve vaccine therapy and pancreatic cancer survival. Methods: Mice were orthotopically transplanted with 2 x 106 Panc02 pancreatic tumor cells to form liver metastases by a hemisplenectomy technique on day 0. Following tumor transplantation, wild-type or PD-1 knockout mice were treated subcutaneously with a mouse GM-CSF secreting pancreatic tumor vaccine (mouse GVAX) in combination with anti-PD-1 antibodies or IgG isotype control. GVAX was given subcutaneously to mice on days 4, 7, 14, and 21 following tumor transplantation, together with a single low-dose of Cytoxan on day 3. Anti-PD-1 antibodies or IgG were administered twice weekly starting post-operative day 3. Results: PD-1 knockout mice challenged with pancreatic liver metastases had a significant survival advantage over wild-type mice (p=0.0002). Wild-type mice showed improved survival both with anti-PD-1 antibody alone (p=0.008) or in combination with vaccine (p<0.0001) versus IgG controls. GVAX in combination with anti-PD-1 antibodies versus anti-PD-1 antibodies alone shows a statistically significant improved survival (p<0.05). Memory T cell analysis demonstrated a strong trend toward increased tumor infiltration of CD8+ effector memory T cells in the combinatorial treatment group versus vaccine alone (66.67% vs 26.47%, p= 0.1). Tumor antigen (mesothelin)-specific cytotoxic CD8+ tumor infiltrating lymphocytes were increased in the combination treatment group (GVAX and anti-PD1 therapy) versus GVAX alone (13.4% vs 5.29%). Conclusions: PD-1 blockade through monoclonal antibodies or genetic knockout in combination with vaccine result in a synergistic anti-tumor effect in a preclinical model of pancreatic cancer versus treatment with anti-PD1 alone. Additionally, anti-PD1 treatment facilitates tumor infiltration of functionally activated mesothelin specific CD8+ T cells. Anti-PD-1 blockade antibody is currently being tested in phase II clinical trials for treating chemotherapy-refractory solid tumor patients. Our study provides a strong rationale for combining PD-1 antibody with GVAX therapy for pancreatic cancer treatment. This abstract is also presented as Poster B28. Citation Format: Kevin C. Soares, Kelly Olino, Barish Edil, Donger Zhou, Anthony Wamwea, Ashley Leubner, Richard Schulick, Drew Pardoll, Elizabeth Jaffee, Lei Zheng. Granulocyte macrophage colony stimulating factor (GM-CSF) pancreas tumor vaccine in combination with blockade of PD-1 in a preclinical model of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR9.