transforming-growth-factor-beta and Sarcoma

Two polysaccharides from Crocus sativus petals (PCSPs), PCSPA and PCSPB have been previously reported to possess the immunopotentiation activity and improve innate immunity in mice. In this study, PCSPB was evaluated for the anti-tumor activity and explored its immunological mechanisms based on tumor microenvironment (TME) using S180 sarcoma-bearing mice. Although PCSPB showed the lower toxicity to a series of tumor cells, it significantly and dose-dependently suppressed the growth of S180 sarcomas transplanted in mice. HE staining, immunohistochemical analysis, and TUNEL assay revealed that PCSPB significantly induced tumor cell necrosis, apoptosis, and vessel disruption in sarcoma tissues. Meanwhile, PCSPB markedly decreased the levels of inflammatory factors TGF-β, IFN-γ, IL-10 and TNF-α and down-regulated the mRNA expression levels of TGF-β and TNF-α in tumor tissues. Flow cytometric analysis showed that PCSPB significantly increased the proportion of CD8

Topics: Animals; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Crocus; Immunity, Innate; Mice; Polysaccharides; Sarcoma; Transforming Growth Factor beta; Tumor Microenvironment; Tumor Necrosis Factor-alpha

Topics: Adaptor Proteins, Signal Transducing; Animals; Animals, Genetically Modified; Cell Line, Tumor; Extracellular Matrix Proteins; Fibrosarcoma; HCT116 Cells; HEK293 Cells; Hippo Signaling Pathway; Humans; Hyaluronan Receptors; Mice; Mice, Nude; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Sarcoma; Transcription Factors; Transforming Growth Factor beta; YAP-Signaling Proteins; Zebrafish

Pulmonary sarcomatoid carcinoma (PSC) is a rare and aggressive form of NSCLC. Rarity and poor characterization have limited the development of PSC-tailored treatment protocols, leaving patients with inadequate therapeutic options. In this study, we investigated the gene expression profile of PSCs, with the aim to characterize the molecular mechanisms responsible for their evolution and to identify new drugs for their treatment.. A training set of 17 biphasic PSCs was selected and tested for the expression of a large panel of 770 genes related to cancer progression using NanoString technology. Computational analyses were used to characterize a PSCs-gene specific signature from which pathways and drivers of PSC evolution were identified and validated using functional assays in vitro. This signature was validated in a separate set of 15 PSCs and 8 differentiated NSCLC and used to interrogate the cMAP database searching for FDA-approved small molecules able to counteract PSC phenotype.. We demonstrated that the transcriptional activation of an epithelial mesenchymal transition (EMT) program drives PSC phylogeny. Our data provide new insights into PSC evolution and provide the rationale for further clinical studies with dasatinib.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Transformation, Neoplastic; Computational Biology; Dasatinib; Drug Substitution; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Immunohistochemistry; Lung Neoplasms; Models, Biological; Phenotype; Protein Kinase Inhibitors; Sarcoma; Transcription, Genetic; Transforming Growth Factor beta

TGF-β induces epithelial-mesenchymal transition (EMT), which is involved in tumour progression. This study aims to identify and characterise novel factors potentially related to TGF-β-mediated tumour aggression in breast cancer. We treated the human mammary epithelial cell line MCF10A with TGF-β and observed TGF-β-dependent upregulation of FBN1, involving demethylation of CpG sites, in MCF10A cells undergoing EMT. The biological importance of fibrillin-1, encoded by FBN1, was evaluated through immunohistochemistry on 225 breast cancer specimens of various subtypes. Fibrillin-1 expression was observed only in metaplastic carcinoma of the breast (MCB) (51.7%), and the expression was observed in spindle sarcomatous metaplasia (SSM), but not in other metaplasia, including matrix-producing, pleomorphic, and squamous metaplasia, and carcinomatous components of both MCB and non-MCB. Fibrillin-1 expression was also restricted to the SSM of non-mammary carcinosarcomas of various organs. Overall, fibrillin-1 expression was enriched in MCB and non-mammary carcinosarcoma with SSM (93.7% and 93.3%, respectively), but not in MCBs and non-mammary carcinosarcoma without SSM. FBN1 knockdown in MDA-MB-231 cells with high FBN1 expression did not compromise migration, invasion, and tumourigenesis, and did not alter the expression of other EMT-related markers. In conclusion, fibrillin-1 is a novel TGF-β-induced marker. Fibrillin-1 expression in SSM, but not in other metaplasia and carcinomatous components, in both MCBs and non-mammary carcinosarcomas, together with the inability of FBN1-knockdown to compromise migration and invasion, indicates that fibrillin-1 is a marker induced solely in spindle metaplasia during EMT and does not induce EMT nor lead to tumour aggressiveness.

Topics: Breast; Breast Neoplasms; Carcinosarcoma; Cell Line, Tumor; Cell Transformation, Neoplastic; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Fibrillin-1; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Immunohistochemistry; Metaplasia; Sarcoma; Transforming Growth Factor beta; Up-Regulation

Fusion proteins, comprising peptides deriving from the translation of two parental genes, are produced in cancer by chromosomal aberrations. The expressed fusion protein incorporates domains of both parental proteins. Using a methodology that treats discrete protein domains as binding sites for specific domains of interacting proteins, we have cataloged the protein interaction networks for 11 528 cancer fusions (ChiTaRS-3.1). Here, we present our novel method, chimeric protein-protein interactions (ChiPPI) that uses the domain-domain co-occurrence scores in order to identify preserved interactors of chimeric proteins. Mapping the influence of fusion proteins on cell metabolism and pathways reveals that ChiPPI networks often lose tumor suppressor proteins and gain oncoproteins. Furthermore, fusions often induce novel connections between non-interactors skewing interaction networks and signaling pathways. We compared fusion protein PPI networks in leukemia/lymphoma, sarcoma and solid tumors finding distinct enrichment patterns for each disease type. While certain pathways are enriched in all three diseases (Wnt, Notch and TGF β), there are distinct patterns for leukemia (EGFR signaling, DNA replication and CCKR signaling), for sarcoma (p53 pathway and CCKR signaling) and solid tumors (FGFR and EGFR signaling). Thus, the ChiPPI method represents a comprehensive tool for studying the anomaly of skewed cellular networks produced by fusion proteins in cancer.

Topics: Gene Expression Regulation, Neoplastic; Humans; Metabolic Networks and Pathways; Oncogene Proteins, Fusion; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Protein Interaction Maps; Receptors, Notch; Sarcoma; Signal Transduction; Software; Transforming Growth Factor beta; Wnt Proteins

The activated tumor stroma participates in many processes that control tumorigenesis, including tumor cell growth, invasion and metastasis. Cancer-associated fibroblasts (CAFs) represent the major cellular component of the stroma and are the main source for connective tissue components of the extracellular matrix and various classes of proteolytic enzymes. The signaling pathways involved in the interactions between tumor and stromal cells and the molecular characteristics that distinguish normal 'resting' fibroblasts from cancer-associated or '-activated' fibroblasts remain poorly defined. Recent studies emphasized the prognostic and therapeutic significance of CAF-related molecular signatures and a number of those genes have been shown to serve as putative therapeutic targets. We have used immuno-laser capture microdissection and whole-genome Affymetrix GeneChip analysis to obtain transcriptional signatures from the activated tumor stroma of colon carcinomas that were compared with normal resting colonic fibroblasts. Several members of the Wnt-signaling pathway and gene sets related to hypoxia, epithelial-to-mesenchymal transition (EMT) and transforming growth factor-β (TGFβ) pathway activation were induced in CAFs. The putative TGFβ-target IGFBP7 was identified as a tumor stroma marker of epithelial cancers and as a tumor antigen in mesenchyme-derived sarcomas. We show here that in contrast to its tumor-suppressor function in epithelial cells, IGFPB7 can promote anchorage-independent growth in malignant mesenchymal cells and in epithelial cells with an EMT phenotype when IGFBP7 is expressed by the tumor cells themselves and can induce colony formation in colon cancer cells co-cultured with IGFBP7-expressing CAFs by a paracrine tumor-stroma interaction.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Extracellular Matrix; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Proteins; Male; Neoplasm Invasiveness; Neoplasm Proteins; Paracrine Communication; Sarcoma; Transcription, Genetic; Transforming Growth Factor beta; Wnt Signaling Pathway

Methionine enkephalin (MENK), an endogenous neuropeptide, plays an crucial role in both neuroendocrine and immune systems. CD4+Foxp3+ regulatory T cells (Tregs) are identified as a major subpopulation of T lymphocytes in suppressing immune system to keep balanced immunity. The aim of this research work was to elucidate the mechanisms via which MENK interacts with Tregs in cancer situation. The influence of MENK on transforming growth factor-β (TGF-β) mediated conversion from naïve CD4+CD25- T cells to CD4+CD25+ Tregs was determined and the data from flow cytometry (FCM) analysis indicated that MENK effectively inhibited the expression of Foxp3 during the process of TGF-βinduction. Furthermore, this inhibiting process was accompanied by diminishing phosphorylation and nuclear translocation of Smad2/3, confirmed by western blot (WB) analysis and immunofluorescence (IF) at molecular level. We established sarcoma mice model with S180 to investigate whether MENK could modulate Tregs in tumor circumstance. Our findings showed that MENK delayed the development of tumor in S180 tumor bearing mice and down-regulated level of Tregs. Together, these novel findings reached a conclusion that MENK could inhibit Tregs activity directly and retard tumor development through down-regulating Tregs in mice. This work advances the deepening understanding of the influence of MENK on Tregs in cancer situation, and relation of MENK with immune system, supporting the implication of MENK as a new strategy for cancer immunotherapy.

Topics: Animals; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Enkephalin, Methionine; Flow Cytometry; Forkhead Transcription Factors; Humans; Immunotherapy; Mice; Sarcoma; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Epithelial ovarian cancer (EOC) is an immunogenic tumour and exploits many suppressive ways to escape immune eradication. EOC is known to spread primarily by tumour cell implantations in peritoneal cavity. Therefore, ascites may be an ideal fluid compartment to unravel the immune status of the peritoneal cavity. We analysed the expression of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IFN-γ, TNF-α, TNF-β, TGF-β and CCL22 in ovarian cancer ascites, representing immune activating and suppressing cytokines. We observed high expression of pro-inflammatory cytokines IL-6, IL-8 and immune suppressive cytokines IL-10, CCL22 and TGF-β in most samples whereas Th1 (IL-12p70, IFN-γ) and Th2 (IL-4, IL-5) cytokines were only detectable in 13% of the samples. TGF-β was only detected in latent form, questioning its immune suppressive role. CCL22 was in similar levels present in early stage compared to advanced stage tumours. At advanced stage, we observed a negative correlation with CCL22 levels and Th1/2 cytokine expression. We found a positive correlation between IL-6 concentration in ascites and residual disease after debulking. Additionally, IL-6 levels were remarkably higher at recurrence compared to primary advanced disease, which opens an opportunity for inhibition of IL-6 expression in the prevention of recurrence. Despite the heterogeneity of EOC and the complexity of cytokine functions, our results show that cytokine analysis in ascites may aid in understanding tumour-host interaction in EOC.

Topics: Adenocarcinoma, Mucinous; Adult; Aged; Aged, 80 and over; Ascites; Carcinoma, Ovarian Epithelial; Chemokine CCL22; Cystadenocarcinoma, Serous; Cytokines; Female; Gene Expression Regulation, Neoplastic; Humans; Interferon-gamma; Interleukins; Middle Aged; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Sarcoma; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Regulatory T cells (Tregs) are major immunosuppressors in tumor-bearing hosts. Although Treg-depletion therapy has been shown to induce a complete cure in tumor-bearing mice, this treatment is not always successful. Using 3-methylcholanthrene-induced primary mouse tumors, we examined the distinct regulation of Treg-mediated immunosuppression between carcinomas and sarcomas. We showed that the number of Tregs was greatly increased in squamous cell carcinoma (SCC)-bearing mice compared with sarcoma-bearing mice. This appeared to be because SCC produced higher levels of active transforming growth factor (TGF)-beta, which is essential for inducing Tregs, compared with sarcoma. Moreover, SCC, but not sarcomas, were refractory to Treg-depletion therapy by treatment with anti-CD25 mAb. The refractoriness of SCC against Treg-depletion therapy was due to the rapid recovery of Tregs in SCC-bearing mice compared with sarcoma-bearing mice. However, combination treatment of anti-TGF-beta mAb with anti-CD25 mAb caused a significant reduction in Treg recovery and induced a complete cure in SCC-bearing mice. Thus, we showed the refractoriness of mouse carcinoma against Treg-depletion therapy using anti-CD25 mAb treatment. We also proposed a novel Treg-blocking combination therapy using anti-CD25 mAb and anti-TGF-beta mAb to induce a complete cure of tumor-bearing hosts.

Topics: Animals; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Female; Interleukin-2 Receptor alpha Subunit; Lymphocyte Depletion; Methylcholanthrene; Mice; Mice, Inbred BALB C; Sarcoma; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Escape

Modulation of apoptosis may influence sarcoma pathogenesis and/or aggressiveness. The Fas death pathway, mediated by FasL or TGFbeta, is one of two apoptotic pathways. Recent studies report that EGF can modulate TGFbeta and/or FasL expression/activity; thus, EGF has the potential to influence activation of the Fas pathway. EGF is not always detectable in mesenchymal tumors; therefore, we hypothesized EGF would define which Fas ligand predominates. We assayed 57 surgically removed human sarcomas for 10 genes involved in the Fas pathway. Skeletal muscle biopsies from eight patients served as controls. Sample transcripts were detected by real-time RT-PCR. We attempted to identify relevant predictor variables. The 57 sarcomas were segregated into two categories defined by EGF mRNA content: (1) 23 tumors with EGF concentrations that approximated muscle EGF transcript levels (high-EGF tumors); and (2) 34 tumors that either lacked EGF mRNA, or whose mRNA levels were very low and frequently undetected by PCR (low-EGF tumors). TGFbeta1 expression best predicted Fas transcript concentrations in the 34 low-EGF sarcomas, while FasL predicted Fas mRNA levels in the remaining 23 high-EGF sarcomas. The results suggest ligand activity in the Fas death pathway correlates with EGF transcription in sarcomas.

Topics: Apoptosis; Bone Neoplasms; Caspase 10; Caspase 8; Death Domain Receptor Signaling Adaptor Proteins; Epidermal Growth Factor; Fas Ligand Protein; Fas-Associated Death Domain Protein; Humans; Muscle, Skeletal; Regression Analysis; RNA, Messenger; Sarcoma; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta

We have generated effector T cells from tumor-draining lymph nodes (TDLN) that are efficacious in adoptive immunotherapy. We now examine the effect of concomitant tumors on the generation of effector T cells. We inoculated methylcholanthrene (MCA) 205 in the flanks of normal mice and mice bearing MCA 205 lung metastases. TDLN cells from these mice were activated and expanded in vitro, and adoptively transferred to mice bearing lung metastases. Effector T cells generated from TDLN in mice with only flank tumor mediated potent antitumor activity. However, antitumor efficacy of the effector T cells generated from TDLN in mice with pre-existent lung tumor (cTDLN) was reduced. Phenotyping studies showed that dendritic cells in cTDLN expressed higher levels of B7-H1, whereas cTDLN T cells expressed higher levels of PD-1. The levels of IFNgamma were reduced, and the levels of CD4(+)Foxp3(+) regulatory T cells were increased in cTDLN versus TDLN. The in vitro activation of cTDLN was increased by blocking B7-H1 or transforming growth factor (TGF)-beta. Importantly, we found a synergistic up-regulation of IFNgamma with simultaneous blockade of B7-H1 and TGF-beta that was much greater than observed with TDLN. In vitro activation of cTDLN with anti-B7-H1 and anti-TGF-beta and in vivo administration of these antibodies after adoptive transfer resulted in the abrogation of the suppression associated with cTDLN. These results show a major role for the B7-H1/PD-1 axis and TGF-beta as synergistic suppressive mechanisms in cTDLN. Our data have clinical relevance in the generation of effector T cells in the tumor-bearing host.

Topics: Animals; Antigens, Surface; Apoptosis Regulatory Proteins; B7-1 Antigen; B7-H1 Antigen; Immune Tolerance; Intestinal Neoplasms; Lung Neoplasms; Lymphatic Metastasis; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Models, Biological; Neoplasm Transplantation; Neoplasms, Second Primary; Peptides; Programmed Cell Death 1 Receptor; Sarcoma; Signal Transduction; Skin Neoplasms; T-Lymphocytes; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Escape

The use of adenovirus serotype 2 or 5 (Ad2/5) E1A as therapy for human malignancy requires an understanding of the mechanisms involved in E1A-induced tumor suppression. The prevailing use of E1A in the treatment of human malignancy stresses the non-immunologically mediated, anti-tumorigenic activities of E1A. However, the capacity of E1A to elicit a NK-cell and T-cell anti-tumor immune response and to sensitize tumor cells to lysis by immune effector molecules utilized by NK cells and T cells is also an important component of the anti-tumorigenic activity of E1A. This immune-mediated anti-tumorigenic activity of E1A is not shared by functionally similar viral oncoproteins such as the human papillomavirus type 16 (HPV16) E7 oncoprotein and is dependent on the capacity of E1A to interact with transcriptional coadapter, p300. To further define the molecular mechanisms whereby E1A reduces tumorigenicity, we compared total cellular gene expression in H4 cells, a human fibrosarcoma cell line, to gene expression in H4 cells stably expressing E1A, E7, or mutant forms of E1A that do not bind p300. The expression of E1A, but not E7, in H4 cells modulated the expression of cellular genes that may promote apoptosis, enhance immunogenicity and reduce tumor cell metastasis. The difference in the ability of E1A and E7 to modulate the expression of cellular genes that may influence tumorigenicity was largely attributable to distinct interactions of E1A and E7 with p300. Results of this study will be useful in designing novel strategies to augment the anti-tumorigenic activities of E1A.

Topics: Adenovirus E1A Proteins; Animals; Blotting, Northern; Cell Line, Tumor; E1A-Associated p300 Protein; Enzyme-Linked Immunosorbent Assay; Fibrosarcoma; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred C57BL; Oligonucleotide Array Sequence Analysis; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Sarcoma; Transforming Growth Factor beta

In a recent study, we demonstrated that the ability of dermal fibroblasts, obtained from soft tissue sarcoma (STS) patients, to undergo initial division in vitro following radiation exposure correlated with the development of wound healing morbidity in the patients following their treatment with preoperative radiotherapy. Transforming growth factor beta (TGF-beta) is thought to play an important role in fibroblast proliferation and radiosensitivity both of which may impact on wound healing. Thus, in this study we examined the interrelationship between TGF-beta activity, radiosensitivity and proliferation of cultured fibroblasts and the wound healing response of STS patients after preoperative radiotherapy to provide a validation cohort for our previous study and to investigate mechanisms.. Skin fibroblasts were established from skin biopsies of 46 STS patients. The treatment group consisted of 28 patients who received preoperative radiotherapy. Eighteen patients constituted a control group who were either irradiated postoperatively or did not receive radiation treatment. Fibroblast cultures were subjected to the colony forming and cytokinesis-blocked binucleation assays (low dose rate: approximately 0.02 Gy/min) and TGF-beta assays (high dose-rate: approximately 1.06 Gy/min) following gamma-irradiation. Fibroblast radiosensitivity and initial proliferative ability were represented by the surviving fraction at 2.4 Gy (SF(2.4)) and binucleation index (BNI), respectively. Active and total TGF-beta levels in fibroblast cultures were determined using a biological assay. Wound healing complication (WHC), defined as the requirement for further surgery or prolonged deep wound packing, was the clinical endpoint examined.. Of the 28 patients treated with preoperative radiotherapy, 8 (29%) had wound healing difficulties. Fibroblasts from patients who developed WHC showed a trend to retain a significantly higher initial proliferative ability after irradiation compared with those from individuals in the treatment group with normal wound healing, consistent with the results of our previous study. No link was observed between fibroblast radiosensitivity and WHC. Neither active nor total TGF-beta levels in cultures were significantly affected by irradiation. Fibroblast proliferation in unirradiated and irradiated cultures, as well as radiosensitivity, was not influenced by TGF-beta content. TGF-beta expression in fibroblast cultures did not reflect wound healing morbidity.. These data are consistent with our previous study and combined the results suggest that in vitro fibroblast proliferation after irradiation may be a useful predictor of wound healing morbidity in STS patients treated with preoperative radiotherapy. TGF-beta levels in culture do not predict WHC, suggesting that the role of TGF-beta in wound healing is likely controlled by other in vivo factors.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Cell Nucleus; Cell Proliferation; Cells, Cultured; Female; Fibroblasts; Humans; Linear Models; Male; Middle Aged; Preoperative Care; Radiation Tolerance; Regression Analysis; Sarcoma; Skin; Soft Tissue Neoplasms; Transforming Growth Factor beta; Wound Healing

Endoglin (CD105, an accessory component of the TGF-beta receptor complex) expression and distribution on different human tumour cells and its role in cellular proliferation were evaluated. We examined: 1) sixteen human carcinoma cell lines, 2) eight human sarcoma cell lines, 3) five miscellaneous tumour cell lines. HECV (endothelial cells) were employed as a positive control for endoglin expression. Normal Human Dermal Fibroblasts (NHDF) and 293 cells (epithelial kidney cells) were used as normal controls for connective and epithelial tissues, respectively. The results showed that CD105 was poorly expressed in the majority of human carcinoma cells (10/16), whereas it was highly expressed in most human sarcoma cells (7/8), and differently expressed by miscellaneous tumour cell lines. These data reflect endoglin expression by the normal counterparts of tumour cell lines, i.e. NHDF and 293 cells. However, CD105 levels in sarcoma cell lines, even though consistently lower than in NHDF, were significantly higher than those observed in carcinoma cells. Interestingly, CD105 presented a strong expression in the cytoplasm of MDA-MB-453 (breast carcinoma), NPA (papillary thyroid carcinoma), COLO-853 (melanoma) and SaOS-2 (osteosarcoma), but was weakly expressed on their cell membrane. This differential expression in the cytoplasm and on the membrane of some tumour cells, suggests a complex mechanism of translocation for this protein. The analysis of clonal growth in soft agar of some cell lines, characterized by high CD105 expression, showed an increased colony formation potential that was antagonized by the addition of anti-CD105 blocking mAb. The results indicated that endoglin is differentially expressed in human carcinoma and sarcoma cells and its overexpression modulates the proliferative rate of human solid tumour cells. Moreover, these data suggest that CD105 is involved in the regulation of TGF-beta effects in human solid malignancies, and therefore it could play an important role in tumour diagnosis and treatment.

Topics: Antigens, CD; Carcinoma; Cell Membrane; Cell Proliferation; Cytoplasm; Endoglin; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Receptors, Cell Surface; Sarcoma; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation; Vascular Cell Adhesion Molecule-1

The progression of a lesion to a carcinoma is dependent on the engagement of 'reactive stroma' that provides structural and vascular support for tumour growth and also leads to tissue reorganization and invasiveness. The composition of reactive stroma closely resembles that of granulation tissue, and myofibroblasts are thought to play a critical role in driving the stromal reaction of invasive tumours as well as of physiological wound repair. In the present work, we established a myofibroblast-like cell line, named A17, from a mouse mammary carcinoma model in which tumourigenesis is triggered in a single step by the overexpression of HER-2/neu transgene in the epithelial compartment of mammary glands. We showed that although they derived from a tumour of epithelial origin and did not express HER-2/neu transgene, their subcutaneous injection into the backs of syngeneic mice gave rise to sarcomatoid tumours which expressed alpha-smooth muscle actin at the invasive edge. The expression of cytokeratin 14 suggested a myoepithelial origin but immunophenotypical profile, invasive and neoangiogenic potential of A17 cells and tumours showed many similarities with the reactive stroma that occurs in wound repair and in cancerogenesis. Our results suggest that epithelial tumours have the potential to develop highly tumourigenic and invasive reactive stromal cells and our cell line represents a novel, effective model for studying epithelial-stromal interaction and the role of myofibroblasts in tumour development.

Topics: Animals; Blotting, Western; Epithelial Cells; Female; Fibroblasts; Flow Cytometry; Fluorescent Antibody Technique; Immunophenotyping; Injections, Subcutaneous; Magnetic Resonance Spectroscopy; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Mice, Transgenic; Neoplasm Invasiveness; Polymerase Chain Reaction; Rats; Receptor, ErbB-2; Retroviridae Infections; Sarcoma; Stromal Cells; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Virus Infections

Macrophages and Meth A sarcoma cells spontaneously fuse and give rise to tumorigenic hybrid cell lines with a mixed phenotype. We report here that the hybrid tumors grow faster and have a strikingly better developed vasculature than the parent sarcoma. Thus, electron microscopy and immunohistochemical analysis revealed that in the most active areas of neovascularization, the tumors that emerged from inocula of monoclonal hybrid cell populations had a microvessel density nearly twice that of Meth A tumors after 1 week of growth. Moreover, the proportion of vessels associated with pericytes, detected by staining for smooth muscle alpha-actin, was 3 times higher in the hybrid tumors, attesting to the more advanced differentiation of their vasculature. The collagenous stroma component was also more extensive in the hybrid tumors. Concentration of the angiogenic proteins vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-beta) were significantly higher in supernatants of hybrid cell cultures compared with Meth A cultures. These observations indicate that the growth advantage of the hybrid tumors over the parental sarcoma is due to a higher angiogenic capacity. Because the malignant features of many tumors correlate with angiogenesis and because macrophages are known to be major producers of angiogenic factors, our data open the possibility that the intense neovascularization of highly aggressive cancers in some cases reflects the acquisition of macrophage traits by heterotypic cell fusion.

Topics: Animals; Antigens, Neoplasm; Cellular Senescence; Endothelial Growth Factors; Female; Histocompatibility Antigens; Hybrid Cells; Lymphokines; Macrophages; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; Peptides; Sarcoma; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

A transplantable tumor line (KB) was established in syngeneic rats from a naturally occurring sarcoma that had arisen in the thymus of a 24-month-old male F344 rat. Further, a cell line (KB-P) was induced from KB and a cloned cell line (KB-D8) was isolated from KB-P. The primary thymic tumor and KB tumors showed heterogeneous histological growth patterns such as sheet-like, ill-defined bundle, fascicular and interwoven fashions, consisting of spindle cells, oval cells and histiocytic large round cells. Immunohistochemically, neoplastic cells in KB tumors and KB-P and KB-D8 cultures reacted to vimentin and were labeled with antibodies of OX6 (for rat major histocompatibility complex class-II antigens), ED5 (for rat follicular dendritic cells; FDCs) and RED-1 (for interdigitating dendritic cells) in varying degrees, indicating that neoplastic cells exhibited the immunophenotypes of rat dendritic cells. In addition, neoplastic cells were immunoreactive to ED1 (for rat monocytes/macrophages) and ED2 (for rat tissue macrophages), and also showed positive reactions to histiocytic lysosomal enzymes such as acid phosphatase and non-specific esterase. Ultrastructurally, neoplastic cells had cell surface projections, cisterna-like structures and variously developed lysosomes in the cytoplasm. Based on these findings, the present tumor was regarded as dendritic cell-derived sarcoma capable of expressing macrophage-like and histiocytic nature. A reverse-transcription polymerase chain reaction method revealed that the addition of lipopolysaccharide dose dependently increased the expression of mRNA of transforming growth factor-beta1, a proinflammatory factor, in KB-D8 cells. The transplantable line (KB) and cell lines (KB-P and KB-D8) may become useful tools for studying the histogenesis and pathobiological functions of dendritic cells.

Topics: Animals; Dendritic Cells; Histocompatibility Antigens Class II; Immunophenotyping; Male; Neoplasm Transplantation; Rats; Rats, Inbred F344; Sarcoma; Thymus Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured; Vimentin

Human epithelioid sarcoma (ES) is an extremely aggressive soft tissue tumour of unknown histogenesis. Although growth factor-dependent signalling cascades significantly affect the biological behaviour of malignant tumours, little is known so far about their role in human ES. The present investigation, therefore, analyses the coexpression and function of different growth factors and their receptors in the human ES cell line GRU-1 and its clonal subpopulations (GRU-1A, GRU-1B and GRU-1C). As shown by Northern blot, flow cytometry, immunocytochemistry and MTT assay, all ES cell lines expressed transforming growth factor (TGF)-alpha and the epidermal growth factor receptor (EGF-R). Although no response to exogenous TGF-alpha was observed, antagonistic anti-EGF-R antibodies (at 20 microg/ml) induced significant (P<0.05) growth inhibition in all cell lines. All cell lines showed coexpression of platelet-derived growth factor (PDGF)-A and the corresponding receptors. Neutralisation of ES-derived PDGF by anti-hPDGF antibodies resulted in significant (P<0.05) growth inhibition of all clonal subpopulations. Although all cell lines expressed TGF-beta(1) as well as TGF-beta type I and type II receptors (TGF-BI-R and TGF-BII-R), growth inhibition (P<0.05) by exogenous TGF-beta(1) was achieved in the clonal subpopulations only and not in the parental cell line. No ES cell line expressed acidic fibroblast growth factor (FGF) but stimulation of FGF type 3 and type 4 receptors (FGF-3R and FGF-4R) by exogenous acidic FGF (aFGF) resulted in a marked (P<0.05) acceleration of proliferation in all cell lines. In conclusion, our investigation suggests an intricate network of autocrine, juxtacrine and paracrine signalling between ES tumour cells and adjacent non-neoplastic stromal cells.

Topics: Blotting, Northern; Cell Communication; Cell Division; Enzyme-Linked Immunosorbent Assay; ErbB Receptors; Fibroblast Growth Factors; Flow Cytometry; Humans; Immunohistochemistry; Neoplasms, Glandular and Epithelial; Platelet-Derived Growth Factor; Receptors, Fibroblast Growth Factor; Receptors, Platelet-Derived Growth Factor; RNA, Messenger; Sarcoma; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

The human transforming growth factor-beta3 (TGF-beta3) gene has a typical CpG island, the core of which is centered just upstream of its principle promoter. Activation of an alternative downstream promoter, leading to the production of a truncated mRNA lacking the portion of the 5' noncoding region responsible for translational inhibition of TGF-beta3 mRNA, is only evident in breast cancer cells. We compared the methylation status of genomic DNA isolated from a panel of breast (SKBR-3 and T47-D) and non-breast cancer (HT-1080, A673, and A375) cell lines by sequencing sodium metabisulfite-treated DNA. In all cell lines, the core of the TGF-beta3 CpG island was predominantly unmethylated, irrespective of promoter usage associated with that cell line. In contrast, we observed a marked difference in methylation at 19 CpG sites immediately flanking and downstream of the alternative promoter's transcription initiation site. Specifically, the non-breast cancer cell lines exhibited nearly complete methylation of these CpG sites, whereas in the breast cancer cell lines, these CpGs were predominantly unmethylated. Our data support the hypothesis that methylation of a limited number of CpGs at the periphery of an otherwise unmethylated CpG island underlies the transcriptional repression of the downstream promoter in non-breast cancer cells, thereby serving to regulate the use of alternative promoters for TGF-beta3.

Topics: Breast Neoplasms; Cell Line; CpG Islands; DNA Methylation; DNA, Neoplasm; Female; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Organ Specificity; Promoter Regions, Genetic; RNA, Messenger; Sarcoma; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Bone morphogenetic proteins, which are capable of inducing mesenchymal tissue to form bone in mammals, have been implicated as important in normal skeletal development. The expression of bone morphogenetic proteins and their receptors were studied in 36 osteosarcoma specimens, six Ewing's sarcomas, 20 synovial sarcomas, and 20 chondrosarcomas by reverse transcriptase-polymerase chain reaction, and the findings were correlated with clinical data. Bone morphogenetic protein-2, and -4 messages were detected in most sarcoma samples. Bone morphogenetic protein-6 expression was detected in 22 of 32 osteosarcomas and seven of eight chondrosarcomas. Bone morphogenetic protein-7 and receptor IB were not detected in sarcoma samples but were detected in three osteosarcoma cell lines and one malignant fibrous histiocytoma cell line. Expression of bone morphogenetic protein receptor II was found in 25 of 36 osteosarcomas, eight of 20 chondrosarcomas, four of six Ewing's sarcomas, and 15 of 20 synovial sarcoma samples. Expression of bone morphogenetic protein type II receptor was found to correlate with metastasis in osteosarcomas, which suggests that the bone morphogenetic protein pathway may participate in tumor aggressiveness or progression. The expression of bone morphogenetic protein receptor II in metastatic synovial sarcoma and dedifferentiated chondrosarcoma lesions also supports this hypothesis. The current study showed that the ligands for bone morphogenetic protein receptors, bone morphogenetic proteins-2, -4, and -6 also are expressed in osteosarcoma and other sarcoma tissues, indicating a potential for autocrine or paracrine growth stimulation in these tumors.

Topics: Adult; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 6; Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Cell Differentiation; Chondrosarcoma; Female; Gene Expression Regulation, Neoplastic; Histiocytoma, Benign Fibrous; Humans; Male; Mesoderm; Osteogenesis; Osteosarcoma; Protein Serine-Threonine Kinases; Receptors, Cell Surface; Receptors, Growth Factor; Sarcoma; Sarcoma, Ewing; Sarcoma, Synovial; Transforming Growth Factor beta; Tumor Cells, Cultured

Soft tissue sarcomas of the extremities contribute to only 0.9% of all neoplasms. Even in experienced hand radical surgical resection is followed by a recurrency rate of 7 to 35%. The reasons for this high percentage are not yet clear. Since a angiogenesis is a key factor in tumor growth we have investigated concentrations of the angiogenic peptides bFGF, TGF-beta 1, TGF-beta 2 and VEGF in systemic and tumor venous blood of 50 soft tissue sarcoma patients undergoing radical surgical tumor-resection. Systemic blood was obtained preoperatively, during surgery, 1 hour, 1 day and 1 week after surgery. 10cc of venous blood was collected from tumor veins during surgery. Levels of cytokines were measured by Sandwich-ELISA. Preoperative and intraoperative levels were significantly elevated compared to values postoperative. Concentrations of bFGF determined in tumor blood were significantly higher than serum levels but lower than controls obtained from large soft tissue wounds. VEGF and TGF were not elevated.

Topics: Endothelial Growth Factors; Fibroblast Growth Factor 2; Humans; Intercellular Signaling Peptides and Proteins; Lymphokines; Neovascularization, Pathologic; Reference Values; Sarcoma; Soft Tissue Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The biological activity of transforming growth factor-beta 1 (TGF-beta) is governed by dissociation from its latent complex. Immunohistochemical discrimination of active and latent TGF-beta could provide insight into TGF-beta activation in physiological and pathological processes. However, evaluation of immunoreactivity specificity in situ has been hindered by the lack of tissue in which TGF-beta status is known. To provide in situ analysis of antibodies to differentiate between these functional forms, we used xenografts of human tumor cells modified by transfection to overexpress latent TGF-beta or constitutively active TGF-beta. This comparison revealed that, whereas most antibodies did not differentiate between TGF-beta activation status, the immunoreactivity of some antibodies was activation dependent. Two widely used peptide antibodies to the amino-terminus of TGF-beta, LC(1-30) and CC(1-30) showed marked preferential immunoreactivity with active TGF-beta versus latent TGF-beta in cryosections. However, in formalin-fixed, paraffin-embedded tissue, discrimination of active TGF-beta by CC(1-30) was lost and immunoreactivity was distinctly extracellular, as previously reported for this antibody. Similar processing-dependent extracellular localization was found with a neutralizing antibody raised to recombinant TGF-beta. Antigen retrieval recovered cell-associated immunoreactivity of both antibodies. Two antibodies to peptides 78-109 showed mild to moderate preferential immunoreactivity with active TGF-beta only in paraffin sections. LC(1-30) was the only antibody tested that discriminated active from latent TGF-beta in both frozen and paraffin-embedded tissue. Thus, in situ discrimination of active versus latent TGF-beta depends on both the antibody and tissue preparation. We propose that tissues engineered to express a specific form of a given protein provide a physiological setting in which to evaluate antibody reactivity with specific functional forms of a protein.

Topics: Antibodies, Monoclonal; Humans; Immunohistochemistry; Kidney Neoplasms; Mutagenesis, Site-Directed; Sarcoma; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Production of metalloproteinases such as collagenases has been reported to be involved in the metastasis of cancer cells. Granulocytic sarcoma in extramedullary sites can be formed by similar steps to other cancers. In this study, we have examined the secretion of type IV collagenases and a tissue inhibitor of metalloproteinase-1 (TIMP-1) in several human leukemia cell lines, including a granulocytic sarcoma-derived cell line established from a patient with granulocytic sarcomas in dermal tissues. We have also examined the invasive capacity of these leukemia cell lines into reconstituted basement membrane, Matrigel, which was used for in vitro invasion assay. Among the human leukemia cell lines used in this study, only the granulocytic sarcoma cell line was found to secrete type IV collagenase constitutively. Other myeloid leukemia cell lines such as HL-60 and U-937 produced type IV collagenase only after treatment with 12-O-tetradecanoylphorbol-13-acetate. All the cell lines secreted similar amounts of the tissue inhibitor of metalloproteinases. In vitro invasion assay revealed that the granulocytic sarcoma cell line showed higher invasive capacity than the other cell lines. These results suggest that the secretion of 92 kDa type IV collagenase plays a role in the leukemia cells' invasion of extramedullary tissues.

Topics: Collagenases; Glycoproteins; Humans; Interferon-alpha; Interleukin-1; Interleukin-6; Leukemia; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; Sarcoma; Tetradecanoylphorbol Acetate; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Tenascin, a novel six-armed extracellular-matrix glycoprotein, is expressed in a temporally and spatially restricted pattern during carcinogenesis in association with stromal-epithelial interactions. In this study, we have tested the hypothesis that tenascin expression depends upon the change of the cellular environment from in vitro to in vivo. The distribution and alterations in the expression of tenascin were compared between in vitro and in vivo studies in a variety of human epithelial- and nonepithelial-derived cell lines. When cell lines were transplanted into nude mice, all xenografts induced host-mouse-stroma-derived tenascin. Four carcinoma-derived cell lines and all sarcoma-derived lines, which secreted tenascin in vitro, were found to produce human tenascin after transplantation. Furthermore, three carcinoma-derived cell lines, A431, HEp-2, and MCF7, which did not synthesize tenascin in vitro, did synthesize human tenascin after transplantation. These tenascin nonproducing carcinoma cell lines did not express tenascin mRNA in vitro. The addition of TGF-beta 1 to the culture medium induced the synthesis and secretion of tenascin, but TGF-beta 2 and bFGF were less effective. TGF-beta 1 also induced other extracellular-matrix components, fibronectin and laminin. TGF-beta 1 did not induce tenascin in tenascin nonproducing carcinoma cell lines, such as WiDr and A549, in which human tenascin was not induced after transplantation. We have established an in vitro system in which tenascin is induced by the diffusible factor TGF-beta 1. This system could shed light on the mechanism of induction of human tenascin observed in vivo in tenascin nonproducing carcinoma cell lines.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Carcinoma; Cell Adhesion Molecules, Neuronal; Cell Line; Extracellular Matrix Proteins; Fetus; Humans; Immunoblotting; Mice; Mice, Inbred C3H; Mice, Nude; Neoplasm Proteins; Sarcoma; Tenascin; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured