Special Issue "Tumor Stroma"

Guest Editor
Prof. Dr. Kristian Pietras
Karolinska Institute, Department of Medical Biochemistry and Biophysics, Division of Vascular Biology, Scheeles Väg 2, House A4, level 4, SE-171 77, Stockholm, Sweden
Website: http://ki.se/ki/jsp/polopoly.jsp?d=30531&l=en
E-Mail: kristian.pietras@ki.se
Phone: +46 8524 87891
Interests: molecular studies of the endothelial cell-pericyte interplay; carcinoma-associated fibroblasts: recruitment into tumors, phenotype and therapeutic opportunities; TGF-beta signaling in the tumor microenvironment

Dear Colleagues,

The process of tumorigenic conversion of normal cells by genetic events is paralleled by co-evolution of an activated stromal compartment. It is increasingly evident that the microenvironment in which a neoplastic lesion persists holds critical influence over cancer initiation, maintenance, growth and progression into a metastatic and lethal disease. The tumor stroma is populated by endothelial cells and pericytes in the angiogenic vasculature, various types of inflammatory cells of the innate and adaptive immune system, as well as by fibroblasts and a rich connective tissue. Collectively, the stromal components sustain tumor proliferation, survival, stem cell maintenance, angiogenesis, invasiveness and distant spread. As such, the tumor microenvironment is an attractive target for novel anti-cancer therapies. The special issue of Cancers on “Tumor Stroma” will include original research articles and review articles on the influence of the microenvironment on all aspects of tumor biology.

We are looking forward to your contributions.

Prof. Dr. Kristian Pietras
Guest Editor

Submission

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• tumor stroma
• microenvironment
• angiogenesis
• cancer-associated fibroblast
• inflammation
• cancer stem cell niche
• extra-cellular matrix
• metastasis
• targeted therapy
• paracrine growth factor signaling

Type of Paper: Review
Title: P190B RhoGAP Regulates Centrosome Amplification and Chromosome Segregation in Cancer Cells
Authors: Melissa Hwang, Sirisha Peddibhotla, Peter McHenry, Peggy Chang, Zachary Yochum, Ko Un Park, James Cooper Sears, and Tracy Vargo-Gogola
Affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, South Bend, IN 46617, USA; E-Mail: vargo-gogola.1@nd.edu (T.V.-G.)
Abstract: Rho GTPases are overexpressed and hyperactivated in many cancers, including breast cancer.  Rho proteins, as well as their regulators and effectors, have been implicated in mitosis, and their altered expression promotes mitotic defects and aneuploidy. Previously, we demonstrated that p190B Rho GTPase activating protein (RhoGAP) deficiency of inhibits ErbB2-induced mammary tumor formation in mice. Here we describe a novel role for p190B as a regulator of mitosis. We found that p190B localized to centrosomes during interphase and spindle microtubules during mitosis. Knockdown of p190B expression in MCF-7 and Hela cells increased the incidence of aberrant microtuble-kinetochore attachments at metaphase, lagging chromosomes at anaphase, and micronucleation, all of which are indicative of aneuploidy.  Interestingly, p190B deficiency also suppressed centrosome amplification, a hallmark of many breast cancers that contributes to aneuploidy. Cell cycle analysis of p190B deficient MCF-7 cells revealed a significant increase in apoptotic cells with a concomitant decrease in cells in G1 and S phase, suggesting that p190B deficient cells die at the G1 to S transition.  Chemical inhibition of the Rac GTPase during mitosis reduced the incidence of lagging chromosomes in p190B knockdown cells to levels detected in control cells, suggesting that aberrant Rac activity in the absence of p190B promotes chromosome segregation defects. Taken together, these data have lead us to conclude that p190B regulates centrosome amplification, chromosome segregation, and apoptosis in cancer cells. We propose that disruption of mitosis may be one mechanism by which p190B deficiency inhibits tumorigenesis.

Type of Paper: Review
Title: Stroma-directed Therapy in Gastric Cancer
Author: Yasuhiko Kitadai
Affiliation: Department of Gastroenterology and Metabolism, Hiroshima University Graduate School of Biomedical Sciences, Japan; E-Mail: kitadai@hiroshima-u.ac.jp
Abstract: Recent studies in molecular and cellular biology have shown that tumor growth and metastasis are not determined by cancer cells alone but also by a variety of stromal cells. Tumor stroma contains several types of cells including carcinoma-associated fibroblasts (CAFs), endothelial cells, and inflammatory cells including macrophages.  In gastric cancer tissues, tumor cells express platelet-derived growth factor (PDGF)-B. PDGF receptor (PDGFR)-β is expressed by stromal cells, including CAFs, pericytes, and lymphatic endothelial cells. Administration of PDGFR tyrosine kinase inhibitor, significantly decreases the stromal reaction, microvessel area and pericyte coverage of tumor microvessels. Administration of PDGFR inhibitor in combination with cytotoxic chemotherapeutic drugs impairs the progressive growth and metastasis of gastric cancer. Activated stroma might serve as novel therapeutic targets in cancer.

Type of Paper: Article
Title: Tumor-endothelial Cell Crosstalk in Hepatocellular Carcinoma
Author: Rob Griffin
Affiliation: Department of Radiation Oncology, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205, USA; E-Mail: RJGriffin@uams.edu
Abstract: Communication between the tumor cell and its microenvironment is critical for tumor progression. One of the key hallmarks of cancer is ‘angiogenesis’ where tumor cells recruit blood vessels for nutrients, oxygen supply and removal of waste. Recent evidence indicates that, in addition to communication via cell-cell contact and gradients of soluble mediators, tumor cells communicate with the body and the surrounding normal tissue via the production of microvesicles, or exosomes, which contain biologically active protein and RNA species. Hepatocellular carcinoma (HCC) is a highly vascularized tumor. Unlike the normal liver tissue, which derives its blood supply predominantly from the portal vein, HCC preferentially recruits new blood vessels from the hepatic artery to fuel its growth, through a mechanism not well understood. Recent studies have shown that antiangiogenic agents like Sorafenib, a tyrosine kinase inhibitor (TKI) and Bevacizumab, a monoclonal antibody to VEGF are somewhat effective in the treatment of HCC. However, the mechanisms of tumor-endothelial cell communication and the corresponding pathways involved in HCC remain elusive. Deciphering the molecular mechanism in a co-culture experimental model that accommodates microenvironmental conditions would accelerate the drug discovery pipeline for effective treatment of HCC. Such an approach is also likely to elucidate the mechanisms of action for the success of antiangiogenic therapy (Sorafenib, Bevacizumab). We have successfully demonstrated tumor-endothelial cell crosstalk in HCC in vitro using a transwell co-culture experimental model using HEPG2, (HCC cells) and Endothelial Progenitor cells (EPCs). More importantly, we have shown that (i) HepG2 cells are able to secrete factors to induce an ‘activated’ phenotype (survival, growth, proliferation and migration) of EPCs, (ii) Activated EPCs in turn improve the survival of HepG2 cells, (iii) Factors involved in initiation of angiogenesis that were believed to be activated by cell-cell contact and within cells like Ephrin B2 and Dll-4 were found as soluble factors produced only after co-culture, (iv) elevated expression of Ephrin B2 induced in endothelial cells by the presence of HCC cells, a critical event in the initiation of an arterial phenotype, offers a new target that has never been explored in this cancer. Efforts are underway to further elucidate upon the specific extracellular signaling molecules involved in facilitating the intercellular crosstalk in the HCC tumor microenvironment.

Type of paper: Article
Title: Discriminating Different Cancer Cells using Zebrafish in vivo Assay
Authors: Karni S. Moshal, Karine F. Ferri-Lagneau, Jamil Haider, Pooja Pardhanani and TinChung Leung
Affiliation: Julius L. Chambers Biomedical Biotechnology Research Institute, North Carolina Central University, North Carolina Research Campus, Rm 4302, Nutrition Research Center, 500 Laureate Way, Kannapolis, NC 28081, USA; E-Mail: tleung@nccu.edu
Abstract: Despite the expanded understanding of tumor angiogenesis phenomenon and how it impacts cancer treatment outcomes; we have yet to develop a robust assay that can quickly, easily, and quantitatively measure tumor-induced angiogenesis. Since the zebrafish/tumor xenograft represents an emerging tool in this regard, the present study strives to capitalize on the ease, effectiveness, and the adaptability of this model to quantify the tumor angiogenesis. The assay scores angiogenic activity by measuring the neovascular response originating from the developing subintestinal vessels (SIV) induced by cancer cell implantation near to the perivitelline space of zebrafish embryos. In order to test a range of responses, we chose two different tumorigenic cell lines, the human non-small cell lung carcinoma (H1299) and the mouse lung adenocarcinoma (CL13). We also used the non-tumorigenic 3T3-L1 cells as a negative control. The cells were grafted into zebrafish embryos at 1 day-post-fertilization (dpf) and the angiogenic response induced by cancer cell implantation was analyzed using whole-mount alkaline phosphatase (AP) vessel staining and fluorescence microscopy. Angiogenic activity was scored reliably based on the length and number of the newly formed ectopic vessels. At 2 dpf, we detected significantly a greater neovascular response of the length and number of ectopic vessels with human H1299 carcinoma implantation compared to mouse CL13 carcinoma. We also observed a high percentage of embryos with ectopic vessels with both H1299 and CL13 transplantation compared to the 3T3-L1 control. Furthermore, the systemic exposure of zebrafish embryos to anti-angiogenesis drug (PTK 787, inhibitor of vascular endothelial growth factor receptor 2) inhibited tumor-induced angiogenesis suggesting that the assay can be used to evaluate anti-angiogenic drugs. This study implicates the feasibility of using zebrafish xenotransplantation to perform quantitative measurement of the angiogenic activity of cancer cells which can be further extended to measure cancer cell metastatic potential for patient diagnosis and have the potential for evaluating anti-cancer drugs treatment.

Type of Paper: Article
Title: Characterizing the Tumor Microenvironment in Patient-derived Cervix Xenografts (OCICx)
Authors: Naz Chaudary ¹, Shawn Stapleton ², Michael Milosevic ^3,4, Anthony Fyles ^3,4, Melania Pintilie ⁵, Warren Foltz ⁴, David Green ⁴, Douglas Vines ^3,4, and Richard P. Hill ^1,2,3
Affiliation: ¹ Ontario Cancer Institute/Princess Margaret Hospital/Campbell Family Institute for Cancer Research; E-Mail: hill@uhnres.utoronto.ca
² Department of Medical Biophysics University of Toronto
³ Department of Radiation Oncology, University of Toronto
⁴ Radiation Medicine Program, Princess Margaret Hospital/University Health Network
⁵ Department of Biostatistics, Princess Margaret Hospital, 610 University Ave, Toronto, Ontario, Canada M5G 2M9 
Abstract: The tumor microenvironment (TME) is heterogeneous. There are regions of hypoxia, elevated interstitial fluid pressure (IFP) and poor nutrient supply that relate to the weakly organized vascular network that exists in many tumors. We are currently studying various microenvironmental parameters in early (orthotopic) xenografts of cervix cancers and relating these to measurements made in the primary tumour in the patient. Cervix cancer biopsies from patients in clinical studies at Princess Margaret Hospital were used for implantation into the cervix of mice for the generation of orthotopic cervix xenografts (OCICx). IFP measurements were recorded in each xenograft prior to sacrifice and para-aortic lymph nodes are excised based on size (>1-2mm) and assessed histologically for metastases. The OCICx xenografts are imaged with a series of multimodalities (F18-FDG-PET, DCE-CT, DCE-MRI) using a series of small animal imagers.  IFP levels in the xenografts were similar to those in the primary tumour in the patient and there was a strong correlation between IFP and hypoxia (CA9-EF5) in the xenografts. There were also correlations between CA9and Ki67, EF5 and LYVE1, and KI67 and EF5 labelling. The Ki67 and CD31 labelling levels were also correlated with the SUVmean for PET-FDG imaging but the hypoxic fraction was not correlated with the SUVmean. Evaluation of tumor stroma of the OCICx and patient biopsies is being investigated. In addition expression levels of metastasis-related genes will be evaluated in these tumors. The current study will allow selection of xenografts which most closely model clinical features of the microenvironment of cervix cancers to allow testing of new therapeutic approaches associated with microenvironmental conditions in the tumor.

Type of Paper: Article
Title: T cell Mediated Immune Responses Induced in Ret Transgenic Mouse Model of Malignant Melanoma
Author: Oliver Abschuetz ¹, Wolfram Osen ¹, Kathrin Frank ¹, Dirk Schadendorf ², and Viktor Umansky ¹
Affiliation: ¹ Skin Cancer Unit, German Cancer Research Center and University Hospital Mannheim, 69120 Heidelberg, Germany; E-Mail: V.Umansky@dkfz-heidelberg.de
² Department of Dermatology, University Hospital Essen, 45122 Essen, Germany
Abstract: Poor response of human malignant melanoma to currently available treatments requires a development of innovative therapeutic strategies. Their evaluation should be based on animal models that resemble human melanoma with respect to genetics, histopathology and clinical features. Here we used a transgenic mouse model of spontaneous skin melanoma, in which ret transgene is expressed in melanocytes under the control of metallothionein-I promoter. After a short latency, around 30% mice develop macroscopic skin melanoma metastasizing to lymph nodes, bone marrow, lungs and brain, whereas other transgenic mice showed only metastatic lesions without visible skin tumors. We found that tumor lesions expressed melanoma associated antigens (MAA) tyrosinase, tyrosinase related protein (TRP)-1, TRP-2 and gp100, which could be applied as targets for the immunotherapy. Upon peptide vaccination, ret transgenic mice without macroscopic melanomas were able to generate T cell responses not only against strong model antigen ovalbumin but also against MAA TRP-2. Although mice bearing macroscopic primary tumors could also display an antigen-specific T cell reactivity, it was significantly down-regulated as compared to tumor-free transgenic mice or non-transgenic littermates. We suggest that ret transgenic mice could be used as a pre-clinical model for the evaluation of novel strategies of melanoma immunotherapy.

Type of Paper: Review
Title: Modulation of Tumor Stroma and Response to Therapy
Authors: Anna Johansson and Ruth Ganss
Affiliation: Western Australian Institute for Medical Research, University of Western Australia, Centre for Medical Research, Perth, Australia; E-Mail: ganss@waimr.uwa.edu.au
Abstract: Solid tumors are intrinsically resistant to therapy. Cancer growth is initially fostered by tumor cells, but later orchestrated by a variety of cell types within the tumor stroma such as inflammatory cells, fibroblasts, blood vessels and their support cells; these cells collectively form the tumor microenvironment and provide direct support for tumor growth but also evasion from chemo-, immune and radiation therapies. Moreover, high interstitial fluid pressure in solid tumors is an indirect result of abnormal and leaky blood vessels and reduces penetration of drugs; it also creates a hypoxic environment which further augments tumor cell growth and metastatic spread. Importantly, however, research during the last decade has shown that the tumor stroma, including the vasculature, can be modulated, or re-educated, to allow better delivery of chemotherapeutic drugs or enhance the efficiency of active immune therapy. Such remodeling of the tumor stroma using genetic, pharmacological and other therapeutic approaches not only enhances access into tumors but also reduces toxic side effects. This review focuses on recent concepts to modulate tumor stroma to locally increase therapeutic efficacy.