A FTIR Imaging Characterization of Fibroblasts Stimulated by Various Breast Cancer Cell Lines

It is well known that the microenvironment plays a major role in breast cancer progression. Yet, the mechanism explaining the transition from normal fibroblasts to cancer-stimulated fibroblasts remains to be elucidated. Here we report a FTIR imaging study of the effects of three different breast cancer cell lines on normal fibroblasts in culture. Fibroblast activation process was monitored by FTIR imaging and spectra compared by multivariate statistical analyses. Principal component analysis evidenced that the fibroblasts stimulated by these cancer cell lines grouped together and remained distinctly separated from normal fibroblasts indicating a modified different chemical composition in the cancer-stimulated fibroblasts. Similar changes in fibroblasts were induced by the various breast cancer cell lines belonging to different sub-types. Most significant changes were observed in the region of 2950 and 1230 cm⁻¹, possibly related to changes in lipids and in the 1230 cm⁻¹ area assigned to phosphate vibrations (nucleotides). Interestingly, the cancer-cell induced changes in the fibroblasts also occurred when there was no possible direct contact between the two cell lines in the co-culture. When contact was possible, the spectral changes were similar, suggesting that soluble factors but not direct cell-cell interactions were responsible for fibroblast activation. Overall, the results indicate that IR imaging could be used in the future for analyzing the microenvironment of breast tumors.     

小鼠胎肝基质细胞体外扩增骨髓来源的LTC-IC

基质细胞是胎肝造血微环境的主要成分,参与造血干/祖细胞的自我更新、增殖分化的调控。为了研究小鼠胎肝基质细胞在造血微环境中的功能,采用转染SV40大T抗原基因的方法建立了小鼠胚胎期第12.5天（Embryonic-day 12.5, E12.5d）胎肝基质细胞系A4、B3,并进一步鉴定基质细胞系的一般细胞生物学特性和造血支持功能。结果：A4、B3为细胞形态、生长行为以及表面分子表达不同细胞系,二者均可维持骨髓源长期培养启动细胞（Longterm cultureinitiating cell,LTC-IC）至少4周并且有不同程度的扩增LTC-IC能力,其中B3扩增LTC-IC的能力是A4的83倍。外源性细胞因子组合SCF+IL-3+IL6+Epo在本实验体系中不影响LTC-IC数量的维持和扩增。暗示E12.5d胎肝造血微环境中基质细胞的功能是不同的,其机制有待进一步研究。     

Stromal Fibroblasts Mediate Extracellular Matrix Remodeling and Invasion of Scirrhous Gastric Carcinoma Cells

Scirrhous gastric carcinoma (SGC) has the worst prognosis of all gastric cancers, owing to its rapid expansion by invasion and frequent peritoneal dissemination. Due to the increased proliferation of stromal fibroblasts (SFs) that occurs within SGC lesions and the peritoneal metastatic sites, SFs have been proposed to support the progression of this disease. However, the biological and molecular basis and the pathological role of the intercellular interaction between SGC cells and SFs remain largely unknown. In this study, we investigated the role of SFs in the invasion of the extracellular matrix (ECM) by SGC cells. When SGC cells were cocultured with SFs derived from SGC tissue on three-dimensional (3D) Matrigel, they were attracted together to form large cellular aggregates that invaded within the Matrigel. Time-lapse imaging revealed that this process was associated with extensive contraction and remodeling of the ECM. Immunofluorescence and biochemical analysis showed that SGC cells stimulate phosphorylation of myosin light chain and actomyosin-mediated mechanical remodeling of the ECM by SFs. By utilizing this assay system for inhibitor library screening, we have identified several inhibitors that potently suppress the cooperation between SGC cells and SFs to form the invasive structures. Among them, a Src inhibitor dasatinib impaired the interaction between SGC cells and SFs both in vitro and in vivo and effectively blocked peritoneal dissemination of SGC cells. These results indicate that SFs mediate mechanical remodeling of the ECM by SGC cells, thereby promoting invasion and peritoneal dissemination of SGC.     

Overexpression of COL11A1 by Cancer-Associated Fibroblasts: Clinical Relevance of a Stromal Marker in Pancreatic Cancer

Background

The collagen11A1 (COL11A1) gene is overexpressed in pancreatic cancer. The expression of COL11A1 protein could be involved in desmoplastic events in pancreatic cancer, but an antibody that specifically stains the COL11A1 protein is not currently available.

Methods and findings

A total of 54 pancreatic ductal adenocarcinomas (PDAC), 23 chronic pancreatitis (CP) samples, and cultured peritumoral stromal cells of PDAC (passages 3-6) were studied. Normal human pancreas tissue samples were obtained through a cadaveric organ donation program.1) Validation of COL11A1 gene overexpression by q-RT-PCR. Findings: the expression of COL11A1 gene is significantly increased in PDAC samples vs. normal and CP samples.2) Analysis of COL11A1 by immunohistochemistry using highly specific anti-proCOL11A1 antibodies. Findings: anti-proCOL11A1 stains stromal cells/cancer-associated fibroblasts (CAFs) of PDAC but it does not stain chronic benign condition (chronic pancreatitis) stromal cells, epithelial cells, or normal fibroblasts.3) Evaluation of the discrimination ability of the antibody. Findings: anti-proCOL11A1 immunostaining accurately discriminates between PDAC and CP (AUC 0.936, 95% CI 0.851, 0.981).4) Phenotypic characterization of proCOL11A1+ stromal cells co-staining with mesenchymal, epithelial and stellate cell markers on pancreatic tissue samples and cultured peritumoral pancreatic cancer stromal cells. Findings: ProCOL11A1+ cells present co-staining with mesenchymal, stellate and epithelial markers (EMT phenotype) in different proportions.

Conclusions/Significance

Detection of proCOL11A1 through immunostaining with this newly-developed antibody allows for a highly accurate distinction between PDAC and CP. Unlike other available antibodies commonly used to detect CAFs, anti-proCOL11A1 is negative in stromal cells of the normal pancreas and almost absent in benign inflammation. These results strongly suggest that proCOL11A1 is a specific marker for CAFs, and thus, anti-proCOL11A1 is a powerful new tool for cancer research and clinical diagnostics.     

Experimental Studies on Hemopoiesis in the Pronephros of Rana pipiens

Embryogenesis of hemopoietic cell populations in the pronephros of Rana pipiens was examined during embryonic and early larval development. Differential cell counts of Wright-Giemsa-stained cell suspensions demonstrated that granulopoiesis is the predominant hemopoietic activity in the pronephros, erythropoiesis accounts for a minor component of the hemopoietic activity (> 10%), and lymphopoiesis within the organ is negligible. Microdensitometric analysis of Feulgen-DNA stained granulocyte populations in pronephroses from larvae that had received chromosomally labeled pronephric anlagen transplants between 84 and 96 h of development demonstrated that hemopoiesis in this organ is dependent on colonization by an extrinsic hemopoietic stem cell. A similar analysis of pronephric hemopoiesis in larvae which had received chromosomally labeled, presumptive ventral blood island transplants between 62 and 67 h of development, indicates that granulopoietic cells are not derived from the embryonic blood islands. It is proposed that the pronephros may be the initial site of granulocyte differentiation during early embryogenesis. Although the embryonic origin of the hemopoietic stem cell is unknown, indirect evidence from this study indicates a dorsal stem cell compartment     

Selection of Morphologically Normal Cell Lines from Polyoma-transformed BHK21/13 Hamster Fibroblasts

A selective method was devised for the isolation of "revertants" from polyoma-transformed sublines derived from BHK21/13 Syrian hamster fibroblasts. A hybrid, polyploid subline was obtained by growing together, in mixed culture in the presence of aminopterin, two variant BHK21/13 sublines lacking either inosinic acid pyrophosphorylase or thymidine kinase. Whereas these variant sublines were resistant to 6-thioguanine or to 5-bromodeoxyuridine, the hybrid had regained sensitivity to both analogues. By plating a polyoma-transformed subline derived from this hybrid in the presence of 6-thioguanine, resistant clones were obtained with a frequency of about 10(-4). All of these surviving clones had a reduced chromosome complement and some of them had regained a normal phenotype.     

Hemopoiesis on Acetate Cellulose Membranes in the Presence of Lipopolysaccharide

The formation of hemopoietic colonies on acetate cellulose membranes in the peritoneal cavity of mice was markedly enhanced after the injection of bacterial lipopolysaccharide. In addition to granulocytic-macrophagal differentiation, the foci of erythropoiesis appeared. The stimulating effect of lipopolysaccharide was not expressed in nonirradiated mice and during the formation of hemopoietic foci on acetate cellulose membranes in the subcutaneous connective tissue.     

Studies on Biologically Active Substances Formed by Bacilli

The products of several Bacillus strains were investigated on rabbit serum calcium decreasing, oxytocic and toad heart function promoting activities. These products were obtained from the clear supernatant fluid of the culture medium after the cells were removed by centrifugation.

For the production of rabbit serum calcium decreasing substance, Bacillus subtilis K and Bacillus natto No. 8 were found to be usefull, Bacillus megaterium KM, Bacillus cereus var. mycoides and Bacillus subtilis K produced oxytocic principle. Bacillus subtilis K, Bacillus brevis and Bacillus megaterium KM also produced toad heart function promoting factor.

A procedure was developed to obtain the electrophoretically homogenous rabbit serum calcium decreasing substance from culture filtrate of Bacillus subtilis K. The crude substance was obtained as isoelectric precipitate by adjusting the culture filtrate to pH 3.0. The crude substance was purified by gel filtration on a Sephadex G-75 column, isoelectric fractionation and chromatography on DEAE-cellulose column. The purified preparation was shown to be homogenous by Tiselius electrophoresis but was separated into two bands by polyacrylamide electrophoresis. The chemical analysis of this biologically active substance indicated this substance to be a lipoprotein. The substance decreased rabbit serum calcium level about 12% at 6~8hr after intravenous injection (dose; 0.5 mg/kg body weight).     

Heteromorphic Change of Adult Fibroblasts by Ribonucleoproteins

Cultures of subcutaneous areolar fibroblasts from adult rats, when confronted in vitro with microsomes from rat liver or kidney, are changed heteromorphically so as to resemble cultures of nervous tissue. Similar effects follow exposure to the deoxycholate-insoluble fraction of microsomes, and to purified RNA from rat liver or from yeast. An equivalent ribonucleoside mixture has no heteromorphic effect. The degree of heteromorphosis can be related quantitatively to the amount of RNA administered, up to a maximum of 150 γ per slide, above which toxicity intervenes. Ribonuclease destroys in considerable degree the effectiveness of the active agents. Heteromorphosis cannot be induced in this adult tissue by a short exposure (1 to 3 hours) followed by removal to normal medium. A 24 hour exposure to microsome suspensions, however, is followed by partial change lasting for at least several days. Results are most clear cut when cultures of the explant type are maintained continuously in contact with the RNA-containing agents; nevertheless, cell suspensions exposed for 2 to 3 days to heteromorphic agents in suitable concentration appear to be permanently changed. Interspecies experiments between rat and mouse indicate that rat fibroblasts are more labile than mouse, and/or rat microsomes are more potent as agents of heteromorphosis. Mouse liver microsomes have no morphogenetic effect on homologous fibroblasts, but exert a slight action on rat fibroblasts. Rat microsomes have a growth-stimulating effect, but no heteromorphic action, on mouse fibroblasts. Purified protein from snake venom, which is highly active as a growth factor for avian nervous tissue, is growth-stimulating to rat fibroblasts but has no heteromorphic action on this material.     

Curcumin Suppresses Crosstalk between Colon Cancer Stem Cells and Stromal Fibroblasts in the Tumor Microenvironment: Potential Role of EMT

Objective

Interaction of stromal and tumor cells plays a dynamic role in initiating and enhancing carcinogenesis. In this study, we investigated the crosstalk between colorectal cancer (CRC) cells with stromal fibroblasts and the anti-cancer effects of curcumin and 5-Fluorouracil (5-FU), especially on cancer stem cell (CSC) survival in a 3D-co-culture model that mimics in vivo tumor microenvironment.

Methods

Colon carcinoma cells HCT116 and MRC-5 fibroblasts were co-cultured in a monolayer or high density tumor microenvironment model in vitro with/without curcumin and/or 5-FU.

Results

Monolayer tumor microenvironment co-cultures supported intensive crosstalk between cancer cells and fibroblasts and enhanced up-regulation of metastatic active adhesion molecules (β1-integrin, ICAM-1), transforming growth factor-β signaling molecules (TGF-β3, p-Smad2), proliferation associated proteins (cyclin D1, Ki-67) and epithelial-to-mesenchymal transition (EMT) factor (vimentin) in HCT116 compared with tumor mono-cultures. High density tumor microenvironment co-cultures synergistically increased tumor-promoting factors (NF-κB, MMP-13), TGF-β3, favored CSC survival (characterized by up-regulation of CD133, CD44, ALDH1) and EMT-factors (increased vimentin and Slug, decreased E-cadherin) in HCT116 compared with high density HCT116 mono-cultures. Interestingly, this synergistic crosstalk was even more pronounced in the presence of 5-FU, but dramatically decreased in the presence of curcumin, inducing biochemical changes to mesenchymal-epithelial transition (MET), thereby sensitizing CSCs to 5-FU treatment.

Conclusion

Enrichment of CSCs, remarkable activation of tumor-promoting factors and EMT in high density co-culture highlights that the crosstalk in the tumor microenvironment plays an essential role in tumor development and progression, and this interaction appears to be mediated at least in part by TGF-β and EMT. Modulation of this synergistic crosstalk by curcumin might be a potential therapy for CRC and suppress metastasis.     

Noggin Over-Expressing Mouse Embryonic Fibroblasts and MS5 Stromal Cells Enhance Directed Differentiation of Dopaminergic Neurons from Human Embryonic Stem Cells

Directed methods for differentiating human embryonic stem cells (hESCs) into dopaminergic (DA) precursor cells using stromal cells co-culture systems are already well established. However, not all of the hESCs differentiate into DA precursors using these methods. HSF6, H1, H7, and H9 cells differentiate well into DA precursors, but CHA13 and CHA15 cells hardly differentiate. To overcome this problem, we modified the differentiation system to include a co-culturing step that exposes the cells to noggin early in the differentiation process. This was done using γ-irradiated noggin-overexpressing CF1-mouse embryonic fibroblasts (MEF-noggin) and MS5 stromal cells (MS5-noggin and MS5-sonic hedgehog). After directed differentiation, RT-PCR analyses revealed that engrailed-1 (En-1), Lmx1b, and Nurr1, which are midbrain DA markers, were expressed regardless of differentiation stage. Moreover, tyrosine hydroxylase (Th) and an A9 midbrain-specific DA marker (Girk2) were expressed during differentiation, whereas levels of Oct3/4, an undifferentiated marker, decreased. Immunocytochemical analyses revealed that protein levels of the neuronal markers TH and TuJ1 increased during the final differentiation stage. These results demonstrate that early noggin exposure may play a specific role in the directed differentiation of DA cells from human embryonic stem cells.     

Products Formed by Photosensitized Oxidation of Tocopherols

Two isomers of primary products formed by méthylene blue sensitized photooxidation of δ-tocopherol were separated by thin layer chromatography. The products were characterized by UV, IR and NMR spectra and were identified to be 8aS- and 8aR-hydroperoxy δ-tocopheryl dienones, respectively. On decomposition of the photooxidized α-, γ- and δ-tocopherols, to-copheryl quinone and tocopheryl quinone epoxide were formed. The effect of hydroperoxy 5-tocopheryl dienone on methyl linoleate peroxidation was examined. The hydroperoxy dienone formed by the photooxidation of tocopherols did not accelerate lipid peroxidation.     

Transformation by Polyoma Virus affects Adhesion of Fibroblasts

WHEN freshly trypsinized hamster fibroblasts (BHK 21, Clone 13¹) are shaken in suspension, intercellular adhesions are rapidly formed and there is some support for the view that this process is the expression of an adhesive property of the cells in culture which has survived the procedures used to separate them and is not simply an artefact of the use of trypsin². Transformation of animal cells in culture by tumour viruses alters the cell surfaces, as various techniques have shown^3–9 and we have investigated whether intercellular adhesiveness, as defined by the particular assay we have been using, is also affected.     

Pleiotrophin,A Multifunctional Tumor Promoter Through Induction of Tumor Angiogenesis,Remodeling of the Tumor Microenvironment,and Activation of Stromal Fibroblasts

Pleiotrophin (PTN, Ptn) is a widely expressed, developmentally regulated 136 amino acid secreted heparin-binding cytokine. It signals through a unique signaling pathway; the PTN receptor is the transmembrane receptor protein tyrosine phosphatase (RPTP)β/ζ. RPTPβ/ζ is inactivated by PTN, which leads to increased tyrosine phosphorylation of the downstream targets of the PTN/RPTPβ/ζ signaling pathway. Pleiotrophin gene expression is found in cells in early differentiation during different developmental periods. It is upregulated in cells with an early differentiation phenotype in wound repair. The Ptn gene also is a proto-oncogene; PTN is expressed in human tumor cells, and, in cell lines derived from human tumors that express Ptn, Ptn expression is constitutive and thus "inappropriate". Importantly, properties of different cells induced by PTN in PTN-stimulated cells are strikingly similar to properties of highly malignant cells. Furthermore, transformed cells into which Ptn is introduced undergo "switches" to malignant cells of higher malignancy with properties that are strikingly similar to properties of PTN-stimulated cells. These unique features of PTN support the conclusion that constitutive PTN signaling in malignant cells that inappropriately express Ptn functions as a potent tumor promoter. Recently, in confirmation, Ptn targeted by the mouse mammary tumor virus (MMTV) promoter in a transgenic mouse model was found to promote breast cancers to a more aggressive breast cancer cell phenotype that morphologically closely resembles scirrhous carcinoma in human; in addition, it promoted a striking increase in tumor angiogenesis and a remarkable degree of remodeling of the micro-environment. Pleiotrophin thus regulates both different normal and pathological functions; collectively, the different studies have uncovered the unique ability of a single cytokine PTN, which signals through the unique PTN/RPTPβ/ζ signaling pathway, to induce the many properties associated with tumor promotion in the malignant cells that constitutively express Ptn and in their microenvironment.     

Entrainment of Spontaneously Hypertensive Rat Fibroblasts by Temperature Cycles

The functional state of the circadian system of spontaneously hypertensive rats (SHR) differs in several characteristics from the functional state of normotensive Wistar rats. Some of these changes might be due to the compromised ability of the central pacemaker to entrain the peripheral clocks. Daily body temperature cycles represent one of the important cues responsible for the integrity of the circadian system, because these cycles are driven by the central pacemaker and are able to entrain the peripheral clocks. This study tested the hypothesis that the aberrant peripheral clock entrainment of SHR results from a compromised peripheral clock sensitivity to the daily temperature cycle resetting. Using cultured Wistar rat and SHR fibroblasts transfected with the circadian luminescence reporter Bmal1-dLuc, we demonstrated that two consecutive square-wave temperature cycles with amplitudes of 2.5°C are necessary and sufficient to restart the dampened oscillations and entrain the circadian clocks in both Wistar rat and SHR fibroblasts. We also generated a phase response curve to temperature cycles for fibroblasts of both rat strains. Although some of the data suggested a slight resistance of SHR fibroblasts to temperature entrainment, we concluded that the overall effect it too weak to be responsible for the differences between the SHR and Wistar in vivo circadian phenotype.