RICERCA ITALIANA     

Research program

Innovative strategies for the diagnosis and therapy of malignant pleural mesothelioma

University Co-ordinator

Università Politecnica delle MARCHE - PATOLOGIA MOLECOLARE E TERAPIE INNOVATIVE - ANCONA(AN)

Research Unit Leader

Antonio Domenico PROCOPIO

Description

This study is divided in 4 phases and will cover a 2 year-period. First, it will be identified the presence of tumour-associated fibroblast and it will be characterised the nature of infiltrated inflammatory and immune cells in MM biopsies. Later, we will evaluate the role of CAFs and of immune cells in MM. We will focus our attention on the mechanisms by which the stromal environment supports tumour growth and progression through the stimulation of MM cell proliferation, migration and invasion, cell death resistance and the immunosuppression. Moreover, we will investigate whether tumour stroma have a more direct role in tumorigenesis, by acting as a mutagen. Finally, on the basis of the finding obtained in the previous part of the study, we expect to develop and test innovative strategies to be rapidly transferred into clinic. The main objectives of this project proposal are:

Phase 1. Identification and characterisation of CAFs and immune cells in MM biopsies and pleural effusions (Time work: 6 months).
We will identify CAFs and immune cells immunocytochemically into human MM biopsies. For CAFs, we will utilize a combination of different markers, including the expression of alpha-smooth-muscle actin (a-SMA), vimentin, fibroblast activation protein (FAP), and beta-galactosidase activity. Immunohistochemistry for a-SMA, vimentin and FAP will be also performed on serial sections taken from reactive mesothelium (n = 20) and infiltrating lung adenocarcinomas (n = 25). Sections will be stained with the specific primary antibodies that are commercially available and beta-galactosidase-positive sections will be scored in accordance with the manufacturer's specification (Senescence detection kit; Oncogene). The immunohistochemistry will be performed by using a standard indirect avidin-biotin horseradish peroxidase method. Sections will be also counterstained with haematoxylin. For each case, the sections will be stained with anti-CD34 and anti-CD31 antibodies to identify non-senescent and endothelial cells, respectively.
For immune cells, we will identify different DCs and T cells subpopulations. The presence of immature myeloid DCs (iDCs), which induce either suppressive Treg cells or T-cell unresponsiveness, and mature DCs (mDCs) will be studied by specific anti-CD83, -CD80 and -CD86 antibodies. Since factors within the tumor microenvironment can stimulate B7-H1 expression in myeloid DCs, affecting their capacity to activate TAA-specific immunity, B7-H1+ DCs will be checked in MM biopsies. Furthermore indoleamine 2,3-dioxygenase (IDO) positive DCs that block the cell cycle progression of T cells, and consequently promote T cell death by apoptosis or anergy, will be studied by IDO specific antibodies. The presence of angiogenesis-stimulatory plasmacytoid DCs and vascular DCs will be looked at. Since tumor-bearing patients have increased numbers of both peripherally circulating and tumor-associated CTLA4+CD4+CD25+ Treg cells, the presence of Treg cells will be studied in MM biopsies, pleural exudates and blood samples of MM patients using anti-CTLA4, CD25 and CD4 specific antibodies. CD4+T cells will be isolated from exudates and blood samples using CD4 MACS beads (Miltenyi Biotec). PD-1+Treg subpopulations will be also studied. Most of the samples needed are already available for this study. We have collected 65 human MM biopsies, which were diagnosed as MM at our Institution. Additionally, unit II of this project, will provide samples and clinical data from about 40 MM patients. Histopathological MM diagnosis will be confirmed for each specimen. The study will be approved by the local ethics committee.

Phase 2. Definition of the CAFs-mediated pathways promoting MM progression. (Time work: 12 months).
a) Migratory response of MM cells to CAFs and CAFs-derived conditioned media. CAFs express a range of growth factors and cytokines which may promote tumor cell survival as well as migration and invasion. Consistently, the effect of CAFs or CAFs-derived conditioned media on cell motility will be examined. Migration and invasion will be assayed as previously described (Saiki et al., 1990. Jpn Cancer Res 81, 1003-11). CAFs will be used as chemotactic factor. We will also test the conditioned media (CM) from human CAFs as well as primary mesothelial cells, for their chemotactic activity on lung and MM cancer cells. Human mesothelium-derived primary myofibroblasts and fibroblasts will be derived from tumour tissue and adjacent normal-appearing mucosa, respectively, obtained in 10 MM patients, submitted to surgical resection. The stromal cell lines will be considered as myofibroblasts when >95% positive for vimentin, prolyl 4-hydroxylase, and a-SMA; <5% positive for smooth muscle myosin; and negative for cytokeratin. They will be considered as fibroblasts when >95% positive for vimentin and prolyl 4-hydroxylase; <5% positive for a-SMA and smooth muscle myosin; and negative for cytokeratin. Myofibroblast and fibroblasts will be used untill passage 10 and 5, respectively. As control, we will also prepare myofibroblasts from fibroblasts in vitro, as previously described (De Wever et al., 2004. FASEB J 18, 1016-18). CM will be obtained from mesothelial cells or myofibroblasts after 48 h of conditioning. Then, neutralization studies will be performed to confirm the specificity of the chemotactic response. Cells will be pre-incubated with various concentrations of either anti-human VEGF, bFGF, Sema-3A, SCF monoclonal antibody (goat IgG; all R&D Systems) as well as with specific inhibitors of the lipoxygenases or ciclooxygenases pathways.
b) Cocultures CAFs-MM cells. We will also determine if CAFs stimulate MM cell growth. MM cells will be seeded onto previously starved nondividing CAFs cells. Five MM cell lines, previously described (28), will be preincubated and then seeded onto fibroblast lawns in a growth factor-deficient medium. We will also use primary mesothelial cells derived from donors. Like fibroblasts, these cells have a finite replicative capacity and no known mutations that predispose to malignancy. Co-cultures will be fixed and stained with Rhodanile blue or DAPI to visualize preferentially MM colonies. Alternatively, we will express EGFP in MM cells and measure EGFP fluorescence by standard procedures.
c) Influence of CAFs or CAFs-derived CM on DC maturation and Treg induction and differentiation. Myeloid DCs maturation is suppressed by factors present in the tumor microenvironment. The effect of CAFs or CAFs-derived CM on DCs maturation and regulation will be analysed. Briefly, PBMCs will be isolated by Ficoll density gradient centrifugation and monocytes isolated by standard protocols. CM of CAFs will be added at different steps of the DC maturation process or, alternatively, monocytes will be co-cultured with CAFs. Immature and mature DCs will be stained by immunfluorescence and analysed by cytofluorimeter assays. The effects of CAFs, DCs or CAFs+DCs conditioned media on the induction and differentiation of Treg cells will be also studied.
d) Effect of MM cells on DC maturation and Treg induction and differentiation. Monocytes will be plated onto 50-80% confluent MM cells and then treated for DCs differentiation. In parallel, it will be studied the effects on MM cells or their CM on Treg cells differentiation. Naïve CD4+T cells will be positive selected from the peripheral blood of healthy donors using a CD4/CD45RA Mulisort kit (Myltenyi Biotec).
e) Effect of CAFs and MM cells on T cell apoptosis. The hypothesis that tumor microenvironment and particularly CAFs and MM cells itself, may sensitise T cells to programmed cell death and induce apoptosis, will be considered. To this purpose, human T cell lines (Jurkat and HuT78) or CD4+ or CD8+ T cells, freshly isolated and activated with IL-2 and anti-CD3 (OKT3), will be co-cultured with CAFs, MM or CAFs plus MM and the effect on T cell apoptosis will be investigated by annexin-V and propidium iodide staining.
f) Tumorigenic Assays. To test the idea that CAFs create a microenvironment that promotes the growth of potentially or frankly MM cells in vivo, we will inject MM cells or immortalized mesothelial cells that are non-tumorigenic but express the SV40-T antigen (Met-5A), alone or with CAFs, into immunocompromised (nu/nu) mice. We will also use EGFP-expressing MM or Met-5A cells to confirm that tumours originated from injected MM or Met-5A cells. The animals will be sacrificed at different times to study the effects of CAFs by macroscopic, microscopic and ultrastructural analysis. The survival of groups treated with mesothelial cells plus CAFs will be compared with groups injected with mesothelial cells alone. On day 90 after transplantation, mice will be killed and weighed and autopsy will be done carefully. Macroscopic liver metastasis and peritoneal dissemination will be also evaluated.

Phase 3. Genetic analysis of target genes in malignant and nonneoplastic mesothelial cells induced by CAFs (Time work: 8 months).
Tumor cells acquire various defense mechanisms during tumorigenesis to invade the host. Among these, tumor cells acquire resistance to programmed cell death (apoptosis) and can counterattack the immune system, inducing T cell apoptosis. This resistance can be caused in many ways. One mechanism of chemoresistance is caused by overexpression MDR1, a transmembrane protein that functions to export chemotherapeutic compounds from cells (Baldini N, 1997. Nat Med 3, 378-380). Other mechanisms involved mutations in p53 tumor suppressor or overexpression of antiapoptotic proteins such as Bcl-2. Induction of activated T cell apoptosis, on the other hand, is mediated by the expression on tumor cell membranes of pro-apoptotic cytokines, such as FasL.
a) Analysis of ability of CAFs to promote both the resistance to cell death and the pro-apoptotic activity of tumor cells against effector T cells. The amount of MDR1 and FasL mRNA in MM cells and nonneoplastic mesothelial cells cocultured with CAFs will be assessed by northern blots. In parallel, the MDR1 and FasL protein expression will be examined by cytometric analysis. Moreover, we will correlate the presence of CAFs-induced MDR1 with the susceptibility to apoptosis induced by different stimuli. Inactivation of MDR1 gene by the specific small interfering RNA (siRNA) could restore the normal apoptotic pathway rendering CAFs-exposed tumor cells susceptible to therapy. Apoptosis will be induced by two different stimuli, immunological, such as Fas-L and TRAIL, or chemical. Apoptosis will be quantified as above described. We will assess the ability of CAFs-exposed MM cells to induce T cell apoptosis by co-cultures experiments. Cytotoxicity will be evaluated by use commercially available assay. To verify the ability of FasL to kill effector cells in co-cultures, FasL blocking antibodies or FasL specific siRNA will be used. In the same way, siRNA specific for Fas will be introduced in a leukemia cell line (Jurkat) to protect them from killing by MM cells. Furthermore, the presence of soluble FasL will be analyzed in MM cell lines and NM cell cultures co-cultured with stroma-derived cells as well as in serum and exudate samples from patients affected by MM.
b) Study the secreted semaphorin signaling. Several studies have demonstrated that the signalling mediated by secreted semaphorins regulates cell invasion and migration. In particular, Semaphorin 3A (Sema-3A), the ligand of the transmembrane receptor complex Np-1/plexin-A1, has been implicated in the regulation of VEGF biological effects. We will test the possibility that the CAFs regulates tumor dissemination by modulating the expression of Sema-3 family members. To this end, MM or Met-5A cell lines cocultured with CAFs will be compared to uncultured control cells for the capability to express members of secreted semaphorin family, including Sema-3A, -3B, -3F and -3C. This expression will be studied by real-time RT-PCR (MJ Research) and by DNA sequence analysis. The identified Sema-3 proteins will be analyzed also by Western Blot using specific monoclonal antibodies.
c) Study the effect of soluble factors on MM cell death resistance and on immune cells regulation. We will evaluate whether some of the factors present in the tumor microenvironment, such as VEGF, FGF-2, PDGF, TGF-beta, PGE2 and Semaphorins may influence i) the expression of anti-apoptotic genes in MM cells (i.e. FLIP); ii) DC differentiation, maturation and functions and iii) the imbalance between Treg cells and effector T cells. Single or combined recombinant proteins will be added to serum free cell colture medium and anti-apoptotic proteins levels and differentiation markers expression analysed by real-time RT-PCR, western blot analysis and cytofluorimetric analysis. Immune cells population and T-cell subsets will be studied using antibodies to specific markers and then examined by cytofluorimetric analysis.

Phase 4. Effect of "Normalization" of the stromal environment on preclinical models of MM (Time work: 6-8 months).
Taken together, our observations could indicate that the tumour microenvironment may be a potential therapeutic target for MM. Therefore, we will combine drugs that target different aspects of the activated stroma with cytotoxic therapies that are directed against MM cells. We will examine the effect of inhibitors of either lipoxygenase signalling (by zileuton), angiogenesis (by bevacizumab), inflammatory cells and cytokines (by celecoxib) as well as immuno-modulatory drugs (by AMD3100) on tumor growth and metastasis of MM cells orthotopically implanted into nude mice. These drugs will be combined to the treatment with pemetrexed, a promising new multi-targeted antifolate agent for the treatment of MM or with Vitamin E derivatives, which will be provided by unit III of this project.
a) In vitro studies. We will focus first on five types of microenvironment-based drugs: Zileuton (a lipoxygenase inhibitor), bevacizumab (a VEGF-blocking antibody), transtuzumab (a ERBB2-blocking antibody), Celecoxib (a Cox-2 inhibitor) and AMD3100 (a CXCR4 antagonist). Several of these agents are already utilized in clinical trials on solid malignancy. MM cells will be treated at increasing concentration of drugs and monitored daily for cell proliferation. Cells exposed to varying concentrations of drugs, will be fixed, washed and stained by MTT. The IC-50 (dose that gives 50% inhibition of cell growth) will be calculated for each cell line. The mechanisms of cell growth inhibition and other possible effects by these agents will be further investigated by FACS and fluorescence microscopy after Hoechst 33258 staining.
b) In vivo studies. Then, the microenvironment-based drugs will be administered to experimental animals. Balb/c athymic nude mice will be injected i.p. with 3 million MM cells each and treated after one week from the inoculum. The drug concentration should be adjusted so that mice will be injected i.p. with 25 microL of the specific drug plus pemetrexed. The animals will be sacrificed at different time intervals to study the effects of the agents by macroscopic, microscopic and ultrastructural analysis. The survival of groups treated with microenvironment-based drug alone will be also compared with control untreated groups. Analogous strategies may be taken in consideration for testing other drugs with potential stroma "normalization" properties which derived during the project. Two human cancer will be used in this study: i) an poorly differentiated MM-B1 mesothelioma cell line, and ii) a epithelioid H-Meso mesothelioma cell line. On day 60 after transplantation, mice will be killed and weighed and autopsy will be done carefully. Macroscopic liver metastasis and peritoneal/pleural dissemination will be evaluated.