- The study examines the expression of COL11A1/procollagen 11A1 in human mesenchymal cells, colon adenocarcinoma cells, and cancer-associated stromal cells.
- Immunostaining showed procollagen 11A1 expression in human bone marrow mesenchymal cells and colon cancer stromal cells, but not in normal colon cells.
- In colon cancers, high procollagen 11A1 expression in stromal cells was associated with nodal involvement, distant metastases, and advanced disease stage.
2011 - Cellular inhibitor of apoptosis protein-1 (cIAP1) can regulate E2F1 tr...Simon Gemble
Cellular inhibitor of apoptosis protein-1 (cIAP1) can directly interact with the transcription factor E2F1 and increase its transcriptional activity. cIAP1 is recruited to E2F1 binding sites on cyclin E and cyclin A promoters in a cell cycle-dependent manner. Silencing cIAP1 inhibits E2F1 DNA binding and transcriptional activation of cyclin E, reducing cell proliferation. Thus, one function of nuclear cIAP1 is to regulate E2F1 transcriptional activity and control cell cycle progression.
The document summarizes a study that investigated the cytotoxic effects and cell death mechanisms of the naturally occurring coumarin A/AA in various cancer cell lines and normal cells. The results showed that coumarin A/AA was cytotoxic to four cancer cell lines tested, including HeLa cells, and was significantly less toxic to normal peripheral blood cells. In HeLa cells, coumarin A/AA induced cell death through DNA fragmentation but not cell cycle disruption. It was found to induce the early release of the apoptosis-inducing factor AIF from mitochondria, suggesting a caspase-independent cell death pathway. This indicates coumarin A/AA may have potential applications in cancer treatment.
This study found that transforming growth factor beta 1 (TGF-β1) induces desmoplasia, the proliferation of fibrotic tissue, in experimental models of human pancreatic carcinoma. The researchers transfected pancreatic tumor cells (PANC-1) that do not normally induce desmoplasia or express TGF-β1 with the TGF-β1 gene. They found that the TGF-β1-transfected cells gained the ability to induce fibroblast growth in culture and in vivo in nude mice. This effect was due both to the direct effects of TGF-β1 and its upregulation of other growth factors and matrix proteins. Therefore, TGF-β1 plays an important role in the desm
JTM-Functional characterization of human Cd33+ And Cd11b+ myeloid-derived sup...Karolina Megiel
This study examined the ability of human tumor cell lines to induce myeloid-derived suppressor cells (MDSC) from healthy donor peripheral blood mononuclear cells (PBMC) using in vitro co-cultures. Two distinct MDSC subsets were identified and characterized: CD33+ HLA-DRlow HIF1a+/STAT3+ MDSC and CD11b+ HLA-DRlow C/EBPb+ MDSC. The induction of CD33+ MDSC depended on overexpression of IL-1b, IL-6, TNFa, VEGF, and GM-CSF by tumor cells, while CD11b+ MDSC induction correlated with FLT3L and TGF
- Ovarian cancer cell growth is markedly reduced by inhibitors of the TAK1-IKK pathway, suggesting TAK1 signaling drives ovarian cancer progression.
- Matrix Metalloproteinase-9 (MMP9) secretion is stimulated by TGF-β and TNF-α in some ovarian cancer cell lines, and this secretion may depend on TAK1 signaling.
- Motility of ovarian cancer cells is increased by TNF-α but decreased by TAK1-IKK pathway inhibitors, indicating TAK1-IKK signaling plays a role in ovarian cancer cell invasion.
1. LAM cells alter monocyte activation and maturation by secreting growth factors that skew macrophages toward a more immature phenotype. Mouse LAM cells decrease nitric oxide production in mouse monocytes and increase expression of genes associated with alternative activation.
2. Human LAM cells also induce an immature phenotype in human monocytes and increase expression of macrophage activation markers. They stimulate chemotaxis of human monocytes through growth factor secretion like VEGF-D.
3. LAM cells likely contribute to inflammatory lung damage in LAM patients by altering monocyte recruitment and maturation through paracrine signaling of growth factors.
Hesca-2 is a monoclonal antibody that was generated against the human embryonic stem cell line BG-01v. Hesca-2 binds with high affinity to a glycan epitope containing the disaccharide Galb1-3GlcNAc, which is commonly found on the surface of undifferentiated hESCs and certain carcinomas. Hesca-2 staining shows this epitope is present on some adult human tissues as well as several human ovarian cancer cell lines, and Hesca-2 is cytotoxic to these cancer cell lines. Immunohistochemistry also showed staining of tissue samples of common human tumor types including ovarian, breast, colon, and esophageal cancers.
1. The document presents the discovery of JSI-124, a selective inhibitor of the JAK/STAT3 pathway, which was identified from screening a library of compounds for inhibition of STAT3 phosphorylation in cancer cells.
2. JSI-124 was found to reduce phospho-STAT3 levels in multiple human cancer cell lines and block STAT3 DNA binding and gene transcription, inhibiting tumor growth.
3. JSI-124 selectively targeted STAT3 signaling over other pathways and showed potent antitumor activity against human tumor xenografts in mouse models, demonstrating its potential as an anticancer therapeutic targeting the STAT3 pathway.
2011 - Cellular inhibitor of apoptosis protein-1 (cIAP1) can regulate E2F1 tr...Simon Gemble
Cellular inhibitor of apoptosis protein-1 (cIAP1) can directly interact with the transcription factor E2F1 and increase its transcriptional activity. cIAP1 is recruited to E2F1 binding sites on cyclin E and cyclin A promoters in a cell cycle-dependent manner. Silencing cIAP1 inhibits E2F1 DNA binding and transcriptional activation of cyclin E, reducing cell proliferation. Thus, one function of nuclear cIAP1 is to regulate E2F1 transcriptional activity and control cell cycle progression.
The document summarizes a study that investigated the cytotoxic effects and cell death mechanisms of the naturally occurring coumarin A/AA in various cancer cell lines and normal cells. The results showed that coumarin A/AA was cytotoxic to four cancer cell lines tested, including HeLa cells, and was significantly less toxic to normal peripheral blood cells. In HeLa cells, coumarin A/AA induced cell death through DNA fragmentation but not cell cycle disruption. It was found to induce the early release of the apoptosis-inducing factor AIF from mitochondria, suggesting a caspase-independent cell death pathway. This indicates coumarin A/AA may have potential applications in cancer treatment.
This study found that transforming growth factor beta 1 (TGF-β1) induces desmoplasia, the proliferation of fibrotic tissue, in experimental models of human pancreatic carcinoma. The researchers transfected pancreatic tumor cells (PANC-1) that do not normally induce desmoplasia or express TGF-β1 with the TGF-β1 gene. They found that the TGF-β1-transfected cells gained the ability to induce fibroblast growth in culture and in vivo in nude mice. This effect was due both to the direct effects of TGF-β1 and its upregulation of other growth factors and matrix proteins. Therefore, TGF-β1 plays an important role in the desm
JTM-Functional characterization of human Cd33+ And Cd11b+ myeloid-derived sup...Karolina Megiel
This study examined the ability of human tumor cell lines to induce myeloid-derived suppressor cells (MDSC) from healthy donor peripheral blood mononuclear cells (PBMC) using in vitro co-cultures. Two distinct MDSC subsets were identified and characterized: CD33+ HLA-DRlow HIF1a+/STAT3+ MDSC and CD11b+ HLA-DRlow C/EBPb+ MDSC. The induction of CD33+ MDSC depended on overexpression of IL-1b, IL-6, TNFa, VEGF, and GM-CSF by tumor cells, while CD11b+ MDSC induction correlated with FLT3L and TGF
- Ovarian cancer cell growth is markedly reduced by inhibitors of the TAK1-IKK pathway, suggesting TAK1 signaling drives ovarian cancer progression.
- Matrix Metalloproteinase-9 (MMP9) secretion is stimulated by TGF-β and TNF-α in some ovarian cancer cell lines, and this secretion may depend on TAK1 signaling.
- Motility of ovarian cancer cells is increased by TNF-α but decreased by TAK1-IKK pathway inhibitors, indicating TAK1-IKK signaling plays a role in ovarian cancer cell invasion.
1. LAM cells alter monocyte activation and maturation by secreting growth factors that skew macrophages toward a more immature phenotype. Mouse LAM cells decrease nitric oxide production in mouse monocytes and increase expression of genes associated with alternative activation.
2. Human LAM cells also induce an immature phenotype in human monocytes and increase expression of macrophage activation markers. They stimulate chemotaxis of human monocytes through growth factor secretion like VEGF-D.
3. LAM cells likely contribute to inflammatory lung damage in LAM patients by altering monocyte recruitment and maturation through paracrine signaling of growth factors.
Hesca-2 is a monoclonal antibody that was generated against the human embryonic stem cell line BG-01v. Hesca-2 binds with high affinity to a glycan epitope containing the disaccharide Galb1-3GlcNAc, which is commonly found on the surface of undifferentiated hESCs and certain carcinomas. Hesca-2 staining shows this epitope is present on some adult human tissues as well as several human ovarian cancer cell lines, and Hesca-2 is cytotoxic to these cancer cell lines. Immunohistochemistry also showed staining of tissue samples of common human tumor types including ovarian, breast, colon, and esophageal cancers.
1. The document presents the discovery of JSI-124, a selective inhibitor of the JAK/STAT3 pathway, which was identified from screening a library of compounds for inhibition of STAT3 phosphorylation in cancer cells.
2. JSI-124 was found to reduce phospho-STAT3 levels in multiple human cancer cell lines and block STAT3 DNA binding and gene transcription, inhibiting tumor growth.
3. JSI-124 selectively targeted STAT3 signaling over other pathways and showed potent antitumor activity against human tumor xenografts in mouse models, demonstrating its potential as an anticancer therapeutic targeting the STAT3 pathway.
Normal tissues and tumors arise from a population of cells termed stem cells. In vivo experiments have provided evidence of the presence of stem cells throughout the mouse mammary gland. Premalignant mammary outgrowths that faithfully recapitulate the mammary epithelial cell lineage upon transplantation contain cells with tumor-forming potential. Cell sorting techniques have identified putative mouse mammary stem cell surface markers and human breast cancer stem cell surface markers. These markers do not identify only stem cells but in fact distinguish a mixed population of cells containing stem cell activity. Previous studies have demonstrated that clones arising from single cells in vitro can be categorized into three types based on the clone morphology. Here, we report the characterization, both in vitro and in vivo, of clonogenic cells from a non-tumorigenic mammary epithelial population and those from an erbB2-induced mammary tumor. We found that clones arising from normal mammary cells expressed different patterns of stem and developmental marker between the clone types and compared to the expression patterns observed on clones that developed from tumorigenic mammary cells.
This document summarizes research finding that elevated activity of the Akt protein protects prostate cancer LNCaP cells from apoptosis induced by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). The researchers found that LNCaP cells have high constitutive Akt activity due to lack of the PTEN lipid phosphatase. Inhibiting PI3-kinase, which activates Akt, sensitized LNCaP cells to TRAIL-induced apoptosis. TRAIL alone activated caspases 8 and XIAP in LNCaP cells but failed to induce full apoptosis. Combining TRAIL with Akt inhibitors allowed cleavage of BID and subsequent mitochondrial apoptosis steps. Akt inhibition of BID cleavage appears to mediate the protective effect
Resveratrol induces apoptotic death in human glioma U251 cells through multiple signaling pathways. Resveratrol treatment leads to dose- and time-dependent cell death, as measured by lactate dehydrogenase release and DNA fragmentation assays. It activates caspase-3 and increases cleavage of poly(ADP-ribose) polymerase. Resveratrol also induces cytochrome c release from mitochondria, caspase-9 activation, Bax expression/translocation, and G0/G1 cell cycle arrest. These findings suggest resveratrol may be an attractive anticancer agent for treating gliomas.
This document discusses a study examining the expression of cancer testis antigen (CTA) genes in glioma stem cells. The key findings are:
1) CTA genes were highly and frequently expressed in cancer stem cells isolated from glioma cell lines and tissues, compared to differentiated tumor cells.
2) Histone acetylation levels in promoter regions of CTA genes were high in cancer stem cells and low in differentiated cells, indicating epigenetic regulation of CTA gene expression.
3) CTA genes may be attractive targets for cancer vaccine therapy against glioma cancer stem cells due to their expression and ability to be presented as surface antigens on cancer stem cells.
This document describes a study investigating the role of the protein Morgana in breast cancer metastasis. The study found that knocking down Morgana impaired migration, invasion, and metastasis of breast cancer cells in vitro and in vivo. Mechanistically, Morgana was found to increase the transcriptional activity of NF-κB, leading to increased expression of metastasis-promoting genes like MMP-9. Overexpressing Morgana had the opposite effect of increasing NF-κB target gene expression. Therefore, Morgana appears to promote breast cancer metastasis by activating the NF-κB pathway and increasing expression of pro-metastatic genes.
Fluorescence activated cell sorted assay for Gaucher's diseaseMayank Sagar
Gaucher disease results from mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GC) (D-glucosyl-acylsphingosine glucohydrolase, and can be divided into three clinical types on the basis of the presence and severity of neurological involvement. Gaucher disease results from mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GC) (D-glucosyl-acylsphingosine glucohydrolase, and can be divided into three clinical types on the basis of the presence and severity of neurological involvement.
In the case of type 1 Gaucher disease, deficient GC activity leads to an accumulation of the catabolic intermediate glucocerebroside, primarily in macrophages of the reticuloendothelial.
This journal article summarizes a study investigating the cytotoxic effects and molecular mechanisms of action of coumarins isolated from Calophyllum brasiliense (mammea A/BA and A/BB) in K562 leukemia cells. The study found that the coumarin mixture induced cytotoxicity in the cancer cells and apoptosis, as shown by TUNEL staining and caspase-3 activation. Genotoxic effects were also observed. Additionally, an in silico analysis found the coumarins complied with criteria for drug-likeness. The results support further development of these natural compounds as potential anticancer agents.
1) The expression of the long non-coding RNA NEAT1 is higher in temozolomide (TMZ)-resistant glioblastoma multiforme (GBM) tissues and cells compared to TMZ-sensitive ones.
2) Knockdown of NEAT1 in TMZ-resistant GBM cells decreases their viability and increases their sensitivity to TMZ-induced apoptosis.
3) NEAT1 regulates MGMT, a gene involved in DNA repair and TMZ resistance, at both the mRNA and protein levels in GBM cells. Modulating MGMT expression also impacts TMZ resistance.
This study examined the potential of using the drug ribavirin to target the oncogene eIF4E in breast cancer cells. eIF4E is overexpressed in over 50% of breast cancers and promotes tumor growth. Ribavirin inhibits eIF4E function and reduced proliferation, clonogenic survival, and levels of eIF4E targets like cyclins in several breast cancer cell lines, with varying sensitivity. Ribavirin treatment was also associated with decreased Akt phosphorylation. Analysis of breast cancer biopsies found elevated eIF4E levels compared to normal tissue, supporting further study of ribavirin as a potential breast cancer therapeutic.
This document summarizes a study investigating the role of microRNA-122 (miR-122) in regulating intrahepatic metastasis of hepatocellular carcinoma (HCC). The study found that miR-122 expression is significantly downregulated in HCC tumors with intrahepatic metastasis. Restoring miR-122 expression in metastatic HCC cell lines reduced in vitro migration, invasion, and tumor growth in vivo. Computational analysis identified multiple target genes of miR-122, including ADAM17, which was shown to be involved in metastasis. Silencing ADAM17 had similar effects as restoring miR-122, reducing in vitro and in vivo measures of metastasis. The study suggests that miR-122 acts as a tumor suppressor
Hepatitis C virus (HCV) infection of primary human hepatocytes induces an epithelial-mesenchymal transition (EMT), as evidenced by changes in cell morphology and marker expression. Infected hepatocytes take on a fibroblast-like, spindle shape and have an extended lifespan compared to uninfected controls. Analysis of EMT markers showed increased expression of mesenchymal markers like vimentin and decreased expression of the epithelial marker E-cadherin. HCV was found to induce EMT through activation of the Akt/β-catenin signaling pathway. This suggests HCV infection may contribute to liver fibrosis progression and pathogenesis.
This document describes a study examining gene expression changes in breast epithelial cells and fibroblasts when co-cultured together. Non-malignant (MCF10A) and breast cancer (MDA-MB231) epithelial cell lines were co-cultured with fibroblasts isolated from either benign breast tissues (NAF) or breast carcinoma tissues (CAF) using a transwell system. Gene expression profiles of each cell type were compared between co-culture and monoculture conditions. While epithelial cells showed large changes in gene expression when co-cultured, fibroblasts were less affected. Specific gene expression changes were observed for each epithelial cell - fibroblast combination that could impact processes like cell polarity, membrane biogenesis, growth control
This study found that colon cancer cells express the chemokine receptor CCR4, which mediates migration of the cells in response to its ligand CCL17 (TARC) through the RhoA/Rho kinase signaling pathway. Quantitative RT-PCR and flow cytometry showed that the colon cancer cell lines HT-29 and AZ-97 expressed CCR4 at both the mRNA and protein levels. Stimulation with CCL17 induced dose-dependent migration of the colon cancer cells, which was inhibited by blocking CCR4 with an antibody or antagonist. CCL17 also increased mRNA levels of RhoA proteins and RhoA activation in the cells. Inhibition of Rho kinase or isoprenylation blocked CCL17-induced cell migration
This document summarizes research on cellular transforming genes in cancer. Experiments found that genes from normal cells, when abnormally expressed, can transform cells at high efficiencies. High molecular weight DNA from cancer cells also transforms cells at high efficiencies, suggesting the genes are no longer properly controlled. Various carcinogens were found to activate the same transforming genes within cancers of particular cell types. Cloning of these genes revealed they are evolutionarily conserved between species. The research aims to identify transforming genes activated at different stages of immune cell differentiation to examine genetic events in cancer development.
Generation of Clonal CRISPR/Cas9-edited Human iPSC Derived Cellular Models an...Thermo Fisher Scientific
This document describes a workflow for generating clonal CRISPR-edited human induced pluripotent stem cell (hiPSC) lines. Key aspects of the workflow include:
1) Developing a hiPSC line that stably expresses Cas9 to facilitate efficient genome editing.
2) Optimizing delivery methods for CRISPR/Cas9 editing tools and improving single cell clone survival and isolation using laminin-521 and StemFlex medium.
3) Applying the workflow to generate hiPSC lines carrying disease-relevant mutations and testing the cell models in assays, finding increased sensitivity to stress in models of Parkinson's disease.
This study examined the role of miR-138-5p in regulating human melanoma cell proliferation. The study found that miR-138-5p expression was significantly higher in melanoma tissues compared to controls. Overexpression of miR-138-5p promoted proliferation of Me45 melanoma cells, while inhibition of miR-138-5p suppressed proliferation. Bioinformatics analysis predicted that miR-138-5p targets the 3'-UTR of hTERT, and luciferase assays confirmed this. Knockdown of hTERT reversed the promotive effects of miR-138-5p on cell proliferation, indicating miR-138-5p regulates proliferation by directly targeting hTERT. Therefore, this study demonstrates that miR-138-5
The expression of ITPK in normal colon and colorectal cancer cells - Postermaldjuan
This document summarizes a student's summer research project investigating expression levels of inositol trisphosphate kinase (ITPK) isoforms in normal colon cells and colorectal cancer cells. Preliminary studies found ITPKC overexpression inhibits cancer cell binding to liver cells, suggesting it is anti-metastatic. The student aimed to measure ITPK mRNA and protein levels to test if ITPKC is downregulated in cancer versus normal cells. Initial results showed ITPKA protein levels varied between cancer cell lines, and an additional higher molecular weight protein was lower in cancer cells. RNA extraction was initially unsuccessful but a second attempt yielded intact RNA for further analysis.
The expression of ITPK in normal colon and colorectal cancer cells - Presenta...maldjuan
The document discusses research into the expression of the inositol 1,4,5-trisphosphate 3'-kinase (ITPK) enzyme in normal colon cells and colorectal cancer cells. The researchers aim to determine mRNA and protein levels of the three ITPK isoforms in colorectal cancer cell lines compared to normal colon epithelial cells. Preliminary results show they successfully extracted RNA from both normal and cancer cells. Western blot analysis detected ITPK isoform A protein levels across several colon cancer cell lines with varying metastatic potential. Future work will measure RNA expression levels and further characterize antibodies to study all three ITPK isoforms. The researchers hypothesize that ITPK levels may be downregulated in colon
The expression of ITPK in normal colon and colorectal cancer cells - Papermaldjuan
This document summarizes research into the expression of inositol 1,4,5-trisphosphate kinase (ITPK) isoforms in normal colon cells and colorectal cancer cells. The researchers hypothesize that ITPK isoform C (ITPKC) is downregulated in colorectal carcinoma cells compared to normal colon cells. They extracted RNA from normal colon and colorectal cancer cell lines and found the RNA to be intact. Quantitative PCR will be used to analyze expression levels of the three ITPK isoforms in the cell lines. Western blot analysis validated antibodies for detecting ITPK isoforms A and B. Preliminary results showed ITPK isoform A expression varied across colorectal cancer
1) Prodigiosin, a bacterial metabolite, induces apoptosis in human breast cancer cells. Gene expression profiling found that prodigiosin strongly increased expression of the NAG-1 gene.
2) Experiments showed that prodigiosin triggers accumulation of the tumor suppressor protein p53, but induction of NAG-1 was independent of p53.
3) Prodigiosin causes inhibition of AKT and activation of glycogen synthase kinase-3B (GSK-3B). Induction of NAG-1 and apoptosis correlated with GSK-3B activation. Inhibiting GSK-3B reduced apoptosis, suggesting GSK-3B plays a key role in the proap
Radiotherapy promotes the polarization of tumor-associated macrophages (TAMs) in mice with Lewis lung cancer into anti-tumor M1 macrophages. This is accompanied by increased expression of the long non-coding RNA lincRNA-p21 in the TAMs. TAMs exposed to radiation therapy suppress the viability and invasion of Lewis lung cancer cells in culture. Overexpression of lincRNA-p21 in TAMs enhances their anti-tumor effects, while decreasing lincRNA-p21 reduces the effects of radiation therapy, suggesting lincRNA-p21 plays a key role in the anti-tumor actions of radiotherapy in lung cancer.
Normal tissues and tumors arise from a population of cells termed stem cells. In vivo experiments have provided evidence of the presence of stem cells throughout the mouse mammary gland. Premalignant mammary outgrowths that faithfully recapitulate the mammary epithelial cell lineage upon transplantation contain cells with tumor-forming potential. Cell sorting techniques have identified putative mouse mammary stem cell surface markers and human breast cancer stem cell surface markers. These markers do not identify only stem cells but in fact distinguish a mixed population of cells containing stem cell activity. Previous studies have demonstrated that clones arising from single cells in vitro can be categorized into three types based on the clone morphology. Here, we report the characterization, both in vitro and in vivo, of clonogenic cells from a non-tumorigenic mammary epithelial population and those from an erbB2-induced mammary tumor. We found that clones arising from normal mammary cells expressed different patterns of stem and developmental marker between the clone types and compared to the expression patterns observed on clones that developed from tumorigenic mammary cells.
This document summarizes research finding that elevated activity of the Akt protein protects prostate cancer LNCaP cells from apoptosis induced by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). The researchers found that LNCaP cells have high constitutive Akt activity due to lack of the PTEN lipid phosphatase. Inhibiting PI3-kinase, which activates Akt, sensitized LNCaP cells to TRAIL-induced apoptosis. TRAIL alone activated caspases 8 and XIAP in LNCaP cells but failed to induce full apoptosis. Combining TRAIL with Akt inhibitors allowed cleavage of BID and subsequent mitochondrial apoptosis steps. Akt inhibition of BID cleavage appears to mediate the protective effect
Resveratrol induces apoptotic death in human glioma U251 cells through multiple signaling pathways. Resveratrol treatment leads to dose- and time-dependent cell death, as measured by lactate dehydrogenase release and DNA fragmentation assays. It activates caspase-3 and increases cleavage of poly(ADP-ribose) polymerase. Resveratrol also induces cytochrome c release from mitochondria, caspase-9 activation, Bax expression/translocation, and G0/G1 cell cycle arrest. These findings suggest resveratrol may be an attractive anticancer agent for treating gliomas.
This document discusses a study examining the expression of cancer testis antigen (CTA) genes in glioma stem cells. The key findings are:
1) CTA genes were highly and frequently expressed in cancer stem cells isolated from glioma cell lines and tissues, compared to differentiated tumor cells.
2) Histone acetylation levels in promoter regions of CTA genes were high in cancer stem cells and low in differentiated cells, indicating epigenetic regulation of CTA gene expression.
3) CTA genes may be attractive targets for cancer vaccine therapy against glioma cancer stem cells due to their expression and ability to be presented as surface antigens on cancer stem cells.
This document describes a study investigating the role of the protein Morgana in breast cancer metastasis. The study found that knocking down Morgana impaired migration, invasion, and metastasis of breast cancer cells in vitro and in vivo. Mechanistically, Morgana was found to increase the transcriptional activity of NF-κB, leading to increased expression of metastasis-promoting genes like MMP-9. Overexpressing Morgana had the opposite effect of increasing NF-κB target gene expression. Therefore, Morgana appears to promote breast cancer metastasis by activating the NF-κB pathway and increasing expression of pro-metastatic genes.
Fluorescence activated cell sorted assay for Gaucher's diseaseMayank Sagar
Gaucher disease results from mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GC) (D-glucosyl-acylsphingosine glucohydrolase, and can be divided into three clinical types on the basis of the presence and severity of neurological involvement. Gaucher disease results from mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GC) (D-glucosyl-acylsphingosine glucohydrolase, and can be divided into three clinical types on the basis of the presence and severity of neurological involvement.
In the case of type 1 Gaucher disease, deficient GC activity leads to an accumulation of the catabolic intermediate glucocerebroside, primarily in macrophages of the reticuloendothelial.
This journal article summarizes a study investigating the cytotoxic effects and molecular mechanisms of action of coumarins isolated from Calophyllum brasiliense (mammea A/BA and A/BB) in K562 leukemia cells. The study found that the coumarin mixture induced cytotoxicity in the cancer cells and apoptosis, as shown by TUNEL staining and caspase-3 activation. Genotoxic effects were also observed. Additionally, an in silico analysis found the coumarins complied with criteria for drug-likeness. The results support further development of these natural compounds as potential anticancer agents.
1) The expression of the long non-coding RNA NEAT1 is higher in temozolomide (TMZ)-resistant glioblastoma multiforme (GBM) tissues and cells compared to TMZ-sensitive ones.
2) Knockdown of NEAT1 in TMZ-resistant GBM cells decreases their viability and increases their sensitivity to TMZ-induced apoptosis.
3) NEAT1 regulates MGMT, a gene involved in DNA repair and TMZ resistance, at both the mRNA and protein levels in GBM cells. Modulating MGMT expression also impacts TMZ resistance.
This study examined the potential of using the drug ribavirin to target the oncogene eIF4E in breast cancer cells. eIF4E is overexpressed in over 50% of breast cancers and promotes tumor growth. Ribavirin inhibits eIF4E function and reduced proliferation, clonogenic survival, and levels of eIF4E targets like cyclins in several breast cancer cell lines, with varying sensitivity. Ribavirin treatment was also associated with decreased Akt phosphorylation. Analysis of breast cancer biopsies found elevated eIF4E levels compared to normal tissue, supporting further study of ribavirin as a potential breast cancer therapeutic.
This document summarizes a study investigating the role of microRNA-122 (miR-122) in regulating intrahepatic metastasis of hepatocellular carcinoma (HCC). The study found that miR-122 expression is significantly downregulated in HCC tumors with intrahepatic metastasis. Restoring miR-122 expression in metastatic HCC cell lines reduced in vitro migration, invasion, and tumor growth in vivo. Computational analysis identified multiple target genes of miR-122, including ADAM17, which was shown to be involved in metastasis. Silencing ADAM17 had similar effects as restoring miR-122, reducing in vitro and in vivo measures of metastasis. The study suggests that miR-122 acts as a tumor suppressor
Hepatitis C virus (HCV) infection of primary human hepatocytes induces an epithelial-mesenchymal transition (EMT), as evidenced by changes in cell morphology and marker expression. Infected hepatocytes take on a fibroblast-like, spindle shape and have an extended lifespan compared to uninfected controls. Analysis of EMT markers showed increased expression of mesenchymal markers like vimentin and decreased expression of the epithelial marker E-cadherin. HCV was found to induce EMT through activation of the Akt/β-catenin signaling pathway. This suggests HCV infection may contribute to liver fibrosis progression and pathogenesis.
This document describes a study examining gene expression changes in breast epithelial cells and fibroblasts when co-cultured together. Non-malignant (MCF10A) and breast cancer (MDA-MB231) epithelial cell lines were co-cultured with fibroblasts isolated from either benign breast tissues (NAF) or breast carcinoma tissues (CAF) using a transwell system. Gene expression profiles of each cell type were compared between co-culture and monoculture conditions. While epithelial cells showed large changes in gene expression when co-cultured, fibroblasts were less affected. Specific gene expression changes were observed for each epithelial cell - fibroblast combination that could impact processes like cell polarity, membrane biogenesis, growth control
This study found that colon cancer cells express the chemokine receptor CCR4, which mediates migration of the cells in response to its ligand CCL17 (TARC) through the RhoA/Rho kinase signaling pathway. Quantitative RT-PCR and flow cytometry showed that the colon cancer cell lines HT-29 and AZ-97 expressed CCR4 at both the mRNA and protein levels. Stimulation with CCL17 induced dose-dependent migration of the colon cancer cells, which was inhibited by blocking CCR4 with an antibody or antagonist. CCL17 also increased mRNA levels of RhoA proteins and RhoA activation in the cells. Inhibition of Rho kinase or isoprenylation blocked CCL17-induced cell migration
This document summarizes research on cellular transforming genes in cancer. Experiments found that genes from normal cells, when abnormally expressed, can transform cells at high efficiencies. High molecular weight DNA from cancer cells also transforms cells at high efficiencies, suggesting the genes are no longer properly controlled. Various carcinogens were found to activate the same transforming genes within cancers of particular cell types. Cloning of these genes revealed they are evolutionarily conserved between species. The research aims to identify transforming genes activated at different stages of immune cell differentiation to examine genetic events in cancer development.
Generation of Clonal CRISPR/Cas9-edited Human iPSC Derived Cellular Models an...Thermo Fisher Scientific
This document describes a workflow for generating clonal CRISPR-edited human induced pluripotent stem cell (hiPSC) lines. Key aspects of the workflow include:
1) Developing a hiPSC line that stably expresses Cas9 to facilitate efficient genome editing.
2) Optimizing delivery methods for CRISPR/Cas9 editing tools and improving single cell clone survival and isolation using laminin-521 and StemFlex medium.
3) Applying the workflow to generate hiPSC lines carrying disease-relevant mutations and testing the cell models in assays, finding increased sensitivity to stress in models of Parkinson's disease.
This study examined the role of miR-138-5p in regulating human melanoma cell proliferation. The study found that miR-138-5p expression was significantly higher in melanoma tissues compared to controls. Overexpression of miR-138-5p promoted proliferation of Me45 melanoma cells, while inhibition of miR-138-5p suppressed proliferation. Bioinformatics analysis predicted that miR-138-5p targets the 3'-UTR of hTERT, and luciferase assays confirmed this. Knockdown of hTERT reversed the promotive effects of miR-138-5p on cell proliferation, indicating miR-138-5p regulates proliferation by directly targeting hTERT. Therefore, this study demonstrates that miR-138-5
The expression of ITPK in normal colon and colorectal cancer cells - Postermaldjuan
This document summarizes a student's summer research project investigating expression levels of inositol trisphosphate kinase (ITPK) isoforms in normal colon cells and colorectal cancer cells. Preliminary studies found ITPKC overexpression inhibits cancer cell binding to liver cells, suggesting it is anti-metastatic. The student aimed to measure ITPK mRNA and protein levels to test if ITPKC is downregulated in cancer versus normal cells. Initial results showed ITPKA protein levels varied between cancer cell lines, and an additional higher molecular weight protein was lower in cancer cells. RNA extraction was initially unsuccessful but a second attempt yielded intact RNA for further analysis.
The expression of ITPK in normal colon and colorectal cancer cells - Presenta...maldjuan
The document discusses research into the expression of the inositol 1,4,5-trisphosphate 3'-kinase (ITPK) enzyme in normal colon cells and colorectal cancer cells. The researchers aim to determine mRNA and protein levels of the three ITPK isoforms in colorectal cancer cell lines compared to normal colon epithelial cells. Preliminary results show they successfully extracted RNA from both normal and cancer cells. Western blot analysis detected ITPK isoform A protein levels across several colon cancer cell lines with varying metastatic potential. Future work will measure RNA expression levels and further characterize antibodies to study all three ITPK isoforms. The researchers hypothesize that ITPK levels may be downregulated in colon
The expression of ITPK in normal colon and colorectal cancer cells - Papermaldjuan
This document summarizes research into the expression of inositol 1,4,5-trisphosphate kinase (ITPK) isoforms in normal colon cells and colorectal cancer cells. The researchers hypothesize that ITPK isoform C (ITPKC) is downregulated in colorectal carcinoma cells compared to normal colon cells. They extracted RNA from normal colon and colorectal cancer cell lines and found the RNA to be intact. Quantitative PCR will be used to analyze expression levels of the three ITPK isoforms in the cell lines. Western blot analysis validated antibodies for detecting ITPK isoforms A and B. Preliminary results showed ITPK isoform A expression varied across colorectal cancer
1) Prodigiosin, a bacterial metabolite, induces apoptosis in human breast cancer cells. Gene expression profiling found that prodigiosin strongly increased expression of the NAG-1 gene.
2) Experiments showed that prodigiosin triggers accumulation of the tumor suppressor protein p53, but induction of NAG-1 was independent of p53.
3) Prodigiosin causes inhibition of AKT and activation of glycogen synthase kinase-3B (GSK-3B). Induction of NAG-1 and apoptosis correlated with GSK-3B activation. Inhibiting GSK-3B reduced apoptosis, suggesting GSK-3B plays a key role in the proap
Radiotherapy promotes the polarization of tumor-associated macrophages (TAMs) in mice with Lewis lung cancer into anti-tumor M1 macrophages. This is accompanied by increased expression of the long non-coding RNA lincRNA-p21 in the TAMs. TAMs exposed to radiation therapy suppress the viability and invasion of Lewis lung cancer cells in culture. Overexpression of lincRNA-p21 in TAMs enhances their anti-tumor effects, while decreasing lincRNA-p21 reduces the effects of radiation therapy, suggesting lincRNA-p21 plays a key role in the anti-tumor actions of radiotherapy in lung cancer.
This document summarizes research on anaplastic large cell lymphoma (ALCL) associated with breast implants. The researchers established three new cell lines from patient biopsy specimens to study the unique biology of this cancer. Characterization found chromosomal abnormalities but no common genetic translocations. The cancer cells showed markers of T-cells and antigen presentation. Studies revealed strong activation of STAT3 signaling related to increased interleukin-6 and decreased SHP-1 phosphatase, suggesting a mechanism of cell survival. Inhibiting STAT3 or treating with chemotherapy killed the cancer cells in vitro, indicating potential new therapies for this disease. The cell lines provide models to further understand breast implant-associated ALCL and identify effective treatments.
This document discusses a study that investigated how inhibiting the epidermal growth factor receptor (EGFR) signaling pathway with the anti-EGFR monoclonal antibody IMC-C225 affects nuclear factor-kappa B (NF-κB) activation and regulation of apoptosis genes in human pancreatic cancer cells. The study found that IMC-C225 treatment blocked EGFR activation in pancreatic cancer cells, leading to decreased NF-κB DNA binding activity. This downregulation of NF-κB by IMC-C225 resulted in decreased expression of the anti-apoptotic genes bcl-xl and bfl-1. Therefore, targeting the NF-κB pathway with an anti-EGFR antibody may help restore apoptosis in pancreatic cancer cells and
TNF-alpha and LPA promote synergistic expression of COX-2 in human colonic my...Enrique Moreno Gonzalez
Enhanced EGF receptor (EGFR) signaling is a hallmark of many human cancers, though the role of enhanced EGFR signaling within the surrounding tumor stroma has not been well studied. The myofibroblast is an important stromal cell that demonstrates enhanced EGFR expression in the setting of inflammation, though the functional relevance is not known. We
recently reported that TNF-α and the G protein-coupled receptor (GPCR) agonist lysophosphatidic acid (LPA) lead to synergistic cyclo-oxygenase-2 (COX-2) expression, an enzyme strongly associated with the development of colitis-associated cancer. Here, we investigate whether EGFR signaling plays a role in the synergistic COX-2 expression induced by LPA and TNF-α.
This document summarizes a study examining the expression of Thomsen-Friedenreich antigen (TF-antigen), a mucin-type glycoprotein, in human esophageal squamous cell carcinoma (ESCC) using peanut agglutinin (PNA) binding. The study found increased levels of TF-antigen in the serum and tissues of ESCC patients compared to normal individuals. TF-antigen levels did not differ between well, moderately, and poorly differentiated ESCC grades and did not decrease after therapy. Expression of TF-antigen increased with histological progression and was localized to the Golgi apparatus and cell membrane in ESCC tissues. The study establishes TF-antigen as a potential diagnostic marker for ES
This research article investigates the effects of the natural flavonoid luteolin on colon cancer cells. The researchers found that physiological concentrations of luteolin induce apoptosis in colon cancer cells by increasing levels of the sphingolipid ceramide. Luteolin inhibits the conversion of ceramide to more complex sphingolipids and disrupts the transport of ceramide between organelles. These effects are mediated by luteolin's ability to inhibit the enzymes sphingosine kinase 2 and Akt, thereby reducing levels of the molecule sphingosine-1-phosphate which normally promotes cell survival. Overall, the study reveals that luteolin exerts anticancer effects by targeting the balance between ceramide and sphingosine-1-
This document reports on changes in expression of trk family neurotrophin receptors (trkA, trkB, trkC) during development and progression of medullary thyroid carcinoma (MTC). In normal thyroid C cells, a subset express trkB but not trkA or trkC. In C cell hyperplasia, cells consistently express trkB with variable expression of trkA and trkC. Later stage MTC tumors show substantially reduced trkB expression but increased trkC expression. Exogenous trkB expression in MTC cell cultures resulted in impaired tumorigenicity and lower levels of angiogenesis factor VEGF, suggesting trkB limits MTC growth. Changes in trk receptor expression are involved in M
Inhibition of glutathione by buthionine sulfoximine enhanced the anti-cancer ...Ashujit
Multiple myeloma (MM) is an incurable blood cancer. Melphalan is an alkylating agent given prior to stem cell transplantation to MM patients. Increased glutathione confers resistance to melphalan. This study investigate the effect of inhibition of glutathione by BSO in preclinical models of MM. Pretreatment with BSO enhanced the anti-cancer effect of melphalan in cell lines and animal models. BSO and melphalan combination was well tolerated by animals and enhanced the survival as compared to controls, BSO and melphalan alone. BSO enhanced depth and duration of responses induced by melphalan. In the combination group, majority of treated animals achieved complete response (CR) and more than 20% had maintained CR. Also, the survival of animals was doubled after combination treatment as compared to BSO or melphalan alone. Mechanistic investigation demonstrated that BSO enhanced melphalan induced DNA damage, caspase cleavage and apoptosis. The combination also achieved multi-logs of cells kills in nine human multiple myeloma cell lines and primary MM cells isolated from blood and bone marrows. Interestingly, the effect of BSO and melphalan combination was abolished when cells were treated with N-acetyl cysteine and sodium thiosulfate but not with vitamin C and vitamin E. This observation suggests that effect of BSO is primarily driven by its ability to deplete glutathione and therefore preventing melphalan detoxification. Together, this study provides framework for testing the combination in a Phase I trial.
CD34+ cells were isolated from the PLC/PRF/5 hepatoma cell line. These CD34+ cells appeared to function as liver cancer stem cells (LCSCs) based on their ability to form human liver carcinomas (HLCs) in immunodeficient mice with as few as 100 cells injected. When various subpopulations of these CD34+ PLC cells were injected into mice, they generated three types of HLCs - hepatocellular carcinomas (HCCs), cholangiocarcinomas (CCs), and combined hepatocellular cholangiocarcinomas (CHCs). The expression of different cell surface antigens like OV6 and CD31 on CD
Lenalidomide inhibits the proliferation of chronic lymphocytic leukemia (CLL) cells through a mechanism dependent on cereblon and p21, but independent of p53. Treatment with lenalidomide upregulates the expression of the cyclin-dependent kinase inhibitor p21 in CLL cells, leading to inhibition of proliferation. Silencing of either cereblon or p21 impairs the ability of lenalidomide to inhibit CLL cell proliferation. Lenalidomide also induces p21 expression in CLL cells isolated from patients, indicating it can directly inhibit CLL cell proliferation through cereblon and p21 in vivo.
Gene expression profiling in apoptotic mcf 7 cells infected with newcastle di...Mohamed Khalid Ali Xundhur
The document summarizes a study that examined gene expression profiling in MCF-7 breast cancer cells infected with Newcastle disease virus (NDV). The study found that NDV infection changed the expression levels of many genes involved in tumor progression, cell cycle regulation, apoptosis, and other cellular processes. Specifically, the study used a gene expression kit to measure changes in 21 genes related to these processes in MCF-7 cells infected with NDV. It found that NDV infection altered the expression of genes like PUMA, Bcl-2, ESR1, and MYBL2. The results provide insight into the gene regulation mechanisms by which NDV selectively kills cancer cells.
Gene expression profiling in apoptotic mcf 7 cells infected with newcastle di...Mohamed Khalid Ali Xundhur
1) The document analyzes gene expression profiling in MCF-7 breast cancer cells infected with Newcastle disease virus (NDV). It finds that NDV infection changes the expression level of many genes involved in tumor progression, cell cycle regulation, apoptosis, and other processes.
2) Specifically, it finds that NDV down-regulates expression of genes like estrogen receptor 1 (ESR1) and up-regulates genes like Bcl-2 binding component 3 (BBC3), both of which are involved in apoptosis.
3) Cell cycle analysis shows that 11% and 41% of MCF-7 cells underwent apoptosis at 3 and 6 hours post-infection respectively, indicating NDV induces apoptosis in the cancer cells
This document summarizes a study on the overexpression of COL11A1 in pancreatic cancer. The study found that:
1) COL11A1 gene expression is significantly higher in pancreatic cancer tissue samples compared to normal and chronic pancreatitis tissues.
2) A newly developed monoclonal antibody that detects proCOL11A1 protein stained cancer-associated fibroblasts in pancreatic cancer but not in chronic pancreatitis or normal tissues, accurately distinguishing between pancreatic cancer and chronic pancreatitis.
3) ProCOL11A1-positive cells expressed markers of mesenchymal, epithelial and stellate cells, indicating an epithelial-to-mesenchymal transition phenotype.
Expression profile of BRCA1 and BRCA2 genes in premenopausal Mexican women wi...Erwin Chiquete, MD, PhD
Gloria Loredo-Pozos, Erwin Chiquete,
Antonio Oceguera-Villanueva, Arturo Panduro,
Fernando Siller-Lo´pez, Martha E. Ramos-Márquez
Low BRCA1 gene expression is associated with
increased invasiveness and influences the response of
breast carcinoma (BC) to chemotherapeutics. However,
expression of BRCA1 and BRCA2 genes has not been
completely characterized in premenopausal BC. We analyzed
the clinical and immunohistochemical correlates of
BRCA1 and BRCA2 expression in young BC women. We
studied 62 women (mean age 38.8 years) who developed
BC before the age of 45 years. BRCA1 and BRCA2 mRNA
expression was assessed by reverse transcriptase-polymerase
chain reaction (RT-PCR) and that of HER-2 and
p53 proteins by immunohistochemistry. Body mass index
(BMI) C27 (52%) and a declared family history of BC
(26%) were the main risk factors. Ductal infiltrative adenocarcinoma
was found in 86% of the cases (tumor size
[5 cm in 48%). Disease stages I–IV occurred in 2, 40, 55,
and 3%, respectively (73% implicating lymph nodes).
Women aged B35 years (24%) had more family history of
cervical cancer, stage III/IV disease, HER-2 positivity, and
lower BRCA1 expression than older women (P-.05).
BRCA1 and BRCA2 expression correlated in healthy, but
not in tumor tissues (TT). Neither BRCA1 nor BRCA2
expression was associated with tumor histology, differentiation,
nodal metastasis or p53 and HER-2 expression.
After multivariate analysis, only disease stage explained
BRCA1 mRNA levels in the lowest quartile. Premenopausal
BC has aggressive clinical and molecular
characteristics. Low BRCA1 mRNA expression is associated
mainly with younger ages and advanced clinical stage
of premenopausal BC. BRCA2 expression is not associated
with disease severity in young BC women.
The document discusses strategies for xenotransplantation using genetically modified pigs. It notes that over 18 patients die each day waiting for organ transplants. Pigs are a promising donor species due to anatomical similarities to humans. However, immunological barriers like hyperacute and acute cellular rejection must be overcome. Methods discussed include genetically knocking out GGTA1 and CMAH genes to eliminate antigen epitopes and introducing human complement regulatory genes. Genome editing tools like ZFNs, CRISPR-Cas9, and SCNT are used to generate multitransgenic pigs lacking immunogenic antigens and capable of preventing rejection. Studies produced GGTA1 knockout piglets and fetuses lacking the alpha-Gal epitope using these techniques.
Ruta graveolens extract induces dna damage pathways and blocks akt activation...Tiensae Teshome
This study examined the anticancer properties of Ruta graveolens extract using several cancer cell lines. The extract dose-dependently decreased cancer cell viability and clonogenicity, induced G2/M cell cycle arrest and caspase activation, and induced the DNA damage response proteins p53, 53BP1 and γ-H2AX. The extract also reduced levels of phospho-Akt and cyclin B1 in cancer cells but only reduced cyclin B1 in normal cells. The results suggest the extract contains bioactive compounds that potently inhibit cancer cell proliferation and survival through multiple targets, including the p53 pathway and DNA damage response, without affecting normal cells.
As an uncommon malignant tumor, hypopharyngeal cancer accounts for 3–5% of head and neck tumors [1]. Most pathological types of hypopharyngeal cancer are squamous cell carcinoma. Due to the occult anatomical location of hypopharyngeal cancer and poor surgical effect, local recurrence or distant metastasis often occurs in patients with hypopharyngeal cancer following surgery.
Similar to Galvan et al. - COL11A1 BMC Cancer 2014 (20)
2. Background
A wealth of studies have reported that the COL11A1 hu-
man gene, coding for the α1 chain of procollagen and
mature collagen of type XI, which is an extracellular
minor fibrillar collagen, is up-regulated in some human
tumours and in mesenchymal-derived tumour cell lines
[1-32], as well as in mesenchymal stem cells and osteo-
blasts [33-35].
Collagen polypeptides are synthesized as procollagens,
with the N- and C-propeptides at the ends of the proto-
typical collagen triple helix. Upon secretion, the propep-
tides are excised and then the mature collagen molecules
assemble in fibrils.
In tumours, the expression of the COL11A1 gene is
currently associated to a fibroblast-like stromal pheno-
type [12,19] but the origin and nature of the cells which
produce procollagen and collagen 11A1 remain contro-
versial to some extent [26].
The so-called cancer-associated stromal cells, resulting
from the desmoplastic reaction which accompanies the
development of human invasive carcinomas, comprise
cells of different types, and are at least in part derived
from mesenchymal progenitors and local resident cells. It
is also well-established that TGF-β1 in cancer promotes
the activation of cancer-associated stromal cells [36].
For the present study, we set out to verify the expression
of the COL11A1 gene, by quantitative RT-PCR in TGF-
β1-exposed epithelial human colorectal HCT 116 cells
and Immortalised Human Bone Marrow Mesenchymal
Cells (hTERT-HMCs); and the expression of procollagen
11A1 by immunocytochemistry (ICC)/immunohisto-
chemistry (IHC), using the DMTX1/1E8.33 monoclo-
nal antibody (mAb) [37], on those cell cultures as well
as on biopsies of human colon adenocarcinomas. Concur-
rently, we studied the expression of DES/desmin, VIM/
vimentin and ACTA2/αSMA (alpha smooth muscle actin)
as mesenchymal (myofibroblast)/stromal markers.
Within the N-propeptide of human procollagen 11A1,
it is the so-called “variable region”, the most divergent
amino acid sequence stretch among different procollagens.
The DMTX1/1E8.33 mAb recognises an epitope in the
YNYGTMESYQTEAPR amino acid stretch within the
variable region of human procollagen 11A1 [37].
Methods
Cell cultures
Ascorbate is a well-known inducer of the synthesis of some
collagens [38,39]; thus, to favour the expression of procol-
lagen 11A1, cells were habitually cultured with this supple-
ment. Since TGF-β1 levels are increased in the serum of
patients with invasive carcinomas [40], we chose to analyse
its effects after continued and protracted exposure of cell
cultures to this cytokine.
The human colorectal adenocarcinoma HCT 116
(CCL-247) cell line, derived from a primary tumour,
was obtained from the American Type Culture Collec-
tion (ATCC) and cultured in DMEM, supplemented with
1 mM sodium pyruvate (Biochrom), 2 mM L-glutamine
(Biochrom), 1X non-essential amino acids (Biochrom),
10% foetal bovine serum (Biochrom), and ascorbate 2-
phosphate (37.5 μg/ml) (Wako Chemicals).
Immortalised Human Bone Marrow Mesenchymal
Cells-hTERT (hTERT-HMCs) were obtained from Applied
Biological Materials (ABM) Inc., Richmond, BC, Canada
(Cat. No. T0523), and grown in T25 ECM-coated flasks in
Prigrow II medium (ABM, Cat. No. TM002), with the
addition of 10% foetal bovine serum, 1 μM hydrocortisone
(Sigma) and ascorbate 2-phosphate (37.5 μg/ml) (Wako
Chemicals).
For TGF-β1 induction, media were further supplemented
with 10 ng/ml of recombinant TGF-β1 (Peprotech). The
medium was replaced every 3–4 days and the cells were
cultured for at least 15 days.
All the cultures were carried out in a humidified atmos-
phere of 5% CO2 in air at 37°C.
Culture passages and cell collections were done with
trypsin/EDTA 0.05%/0.02% (Biochrom). Three different
harvests from each cell culture type were obtained; for
Q-RT-PCR, fresh cell pellets were kept at −80°C.
Colon adenocarcinoma stromal cells isolation and culture
Fresh human tissue samples were procured after written
informed consent of the patients and approval by the
Principality of Asturias Ethics Committee of Clinical
Research, Oviedo, Spain.
Short-term cultures of colon adenocarcinoma stromal
cells were carried out, as previously described [41], from
samples of tumoral sites, avoiding necrotic areas. A sam-
ple from the operating theatre was directly transferred
to a sterile tube containing DMEM culture medium (Gibco,
Invitrogen), supplemented with vancomycin (40 μg/ml) and
amikacin (40 μg/ml) (Normon Laboratories, Madrid,
Spain), and stored for 24 hours at 4°C.
After three washings with phosphate buffer saline
(PBS), the sample was cut into several small fragments.
These fragments were first incubated with collagenases
(Type I 2 mg/ml, Sigma) for 1.5 hours and then centri-
fuged to eliminate supernatant; subsequently, the pellet
was subjected to a second incubation in trypsin/EDTA
for 30 min. After digestion, the cells were again collected
in a pellet, resuspended in DMEM culture medium, sup-
plemented with 10% foetal bovine serum, L-glutamine and
penicillin/streptomycin, transferred to T-flasks and culti-
vated in 5% CO2 at 37°C.
Stromal cell cultures were stable up to 5–6 passages
before going into senescence. The purity of these stromal
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3. cell cultures was assessed by morphology and by immuno-
staining for vimentin.
Q-RT-PCR
For normalisation of data, quantitative RT-PCR of DES,
VIM, ACTA2 and COL11A1 mRNA, and PUM1, RPL10,
and GAPDH mRNA was performed using the BioMark™
HD System of the Fluidigm technology (Fluidigm, San
Francisco, USA).
Briefly, total RNA was isolated from pooled cell cul-
tures, kept at −80°C, with the RNeasy Mini kit (Qiagen).
cDNA was synthesized from 100 ng of RNA from each
sample, using the AffinityScript Multiple Temperature
cDNA Synthesis kit (Agilent Technologies). A pre-
amplification was carried out, applying the QIAGEN®
Multiplex PCR Kit and the pool of all the 20x TaqMan®
Gene Expression Assays. Real time Q-PCR reactions
were carried out with the TaqMan Universal PCR Master
Mix kit (Applied Biosystems). Further details, according
to Applied Biosystems’ recommendations, are in Table 1.
Data were normalised by applying the ΔCt method,
after PCR efficiency corrections. These analyses were
performed by Progenika Biopharma, S.A., Derio, Spain.
Three independent samples (n =3) of different cell har-
vests of each cell type were studied. Data are presented
as mean and SEM. For each gene, differences between
cell culture expressions were analysed by a two-tailed
unpaired t-test. A P value <0.01 was considered statistically
significant.
Immunohistochemistry (IHC)
For immunohistochemical techniques, a cohort of 51 pa-
tients with colon adenocarcinoma and 6 patients diagnosed
with incipient bowel infarction were collected from the
Archive of the Pathology Department, Asturias Central
University Hospital, with the Principality of Asturias
Ethics Committee of Clinical Research, Oviedo, Spain,
approval for guidelines on ethical procedures. The sam-
ples had been fixed with 10% formaldehyde for 24 h and
embedded in paraffin.
Three-μm thick tissue sections were stained with
Hematoxylin and Eosin (H&E) for histological examin-
ation. Antigen retrieval was performed by heating in
PTLink (DakoCytomation, Denmark) in buffer solution
at high pH for 20 minutes. Endogenous peroxidase ac-
tivity was blocked with Peroxidase Blocking Reagent
(DakoCytomation, Denmark) for 5 minutes. After that,
samples were first incubated at 37°C with the primary
antibodies described in Table 2. Subsequently, the EnVision
system (HRP Flex) (DakoCytomation) was applied for
30 minutes at room temperature. Then, the samples were
stained with DAB (3-3′-Diaminobenzidine) (DakoCytoma-
tion, Denmark) for 10 minutes, counterstained for 10 mi-
nutes with hematoxylin (DakoCytomation), dehydrated and
mounted in Entellan® (Merck, Germany). Finally, the
stained tissue sections were studied and photographed
(40× objective) under a light microscope (Nikon -
Eclipse 80i).
Immunocytochemistry (ICC)
Cells were fixed in 10% formaldehyde for 10 minutes in
the chamber slide (BD Falcon™, ref. 354114). Endogen-
ous peroxidase activity was blocked with Peroxidase
Blocking Reagent (DakoCytomation, Denmark) for 5 minutes.
Permeabilization step was performed adding wash buffer
1× (DakoCytomation, Denmark) which contains 0.05 mol/L
Tris/HCl, 0.15 mol/L NaCl, 0.05% Tween-20 [41]. Primary
antibodies were applied, as described in Table 2, at room
temperature. After that, slides were incubated with the
EnVision system (HRP Flex) for 10 minutes at room
temperature. Then, the samples were visualised with DAB
for 5 minutes, and counterstained with hematoxylin for
5 minutes. Finally, the stained slides were dehydrated,
mounted, studied and photographed as above.
Immunohistochemistry assessment
Specimens were assessed by three observers (JAG, CGP
and CGR), following these criteria: procollagen 11A1 im-
munostaining was evaluated according to the cytoplasmatic
signal as the product of two parameters: extent of immuno-
reactivity, which was evaluated in the most densely stained
area (hot spot) under the 20× objective and scored on a
0–3 scale, according to the proportion of positive fibro-
blasts: (0) 0%; (1) <10%; (2) 10-50% and (3) >50%; and
granularity in the cytoplasm, evaluated as dispersed vs.
confluent (1 and 2 points, respectively), with the 40×
objective. Immunoscore values ranged from 0 to 6.
Adjacent non-malignant tissue was used as a negative
control.
Statistical analysis
The experimental results were tested for significance
employing the χ2
test (with Yates’ correction, when ap-
propriate). The statistical analysis was carried out with
Table 1 Assays selected and PCR conditions for Q-RT-PCR
of mRNA analysis
Gene Assay ID
COL11A1 Hs01097664_m1
GAPDH Hs02758991_g1
PUM1 Hs004472881_m1
RPL10 Hs00749196_s1
DES Hs00157258_m1
ACTA2 Hs00426835_g1
VIM Hs00185584_m1
PCR conditions were: 50°C – 2 min; 95°C – 10 min; and 40 amplification cycles:
95°C – 15 sec and 60°C – 1 min.
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4. the IBM SPSS 20.0 software package (SPSS, Inc., Chicago,
IL). All tests were two-sided and p < 0.05 values were con-
sidered statistically significant.
Results
Study of human cell cultures
So far, we are not aware of any human colorectal cell line
in which the expression of the COL11A1 gene has been re-
ported, but we had observed that primary cultures of bone
marrow-derived human mesenchymal cells, expressed
COL11A1/procollagen 11A1, especially after long ex-
posure (≥15 days) to TGF-β1 (data not shown).
We have presently studied the well-known epithelial
human colorectal HCT 116 cell line as a negative con-
trol for the expression of the mesenchymal DES, VIM,
ACTA2 and COL11A1 genes in relation to their expres-
sion by cultured immortalised hTERT-HMCs. The ex-
pression of the DES, VIM, ACTA2 and COL11A1 genes
was analysed by Q-RT-PCR; the immunodetection of
desmin, vimentin, αSMA and procollagen 11A1 was per-
formed by ICC.
According to the normalised Q-RT-PCR data we have
obtained (Figure 1), TGF-β1-activated hTERT-HMCs did
not express DES mRNA, but noticeable amounts of VIM,
ACTA2 and COL11A1 mRNA. The corresponding protein
expression was confirmed by ICC (Figure 2). An average
of 20% of these cells in cultures exposed to TGF-β1
showed a granular pattern of intracytoplasmic immu-
nostaining of procollagen 11A1. None of these markers
was expressed by the HCT 116 cells, but certain levels
of DES.
Examination of human tissues
Fifty one paraffin-embedded archival samples of human
colon adenocarcinomas were examined by IHC with the
DMTX1/1E8.33 mAb; all cases presented adjacent non-
malignant tissue as control. Six cases of incipient bowel
infarction were similarly studied. Table 3 shows the
characteristics of patients and samples, and their anti-
procollagen 11A1 immunoscores, evaluated as described
in Methods; as shown, these immunoscores ranged from
0 to 6. A more detailed description of these characteristics
Figure 1 Q-RT-PCR data of DES, VIM, ACTA2 and COL11A1 mRNA expression in cell cultures of the HCT 116 cell line and in
immortalised hTERT-HMCs, both after long exposure to ascorbate 2-phosphate and TGF-β1. The data were normalised in relation to
PUM1, RPL10, and GAPDH mRNA expression (n =3; mean ± SEM; *P <0.05, **P <0.01).
Table 2 Antibodies used in IHC/ICC analysis
Primary antibodies (species) Clone Commercial reference Dilution Incubation time (min)
Procollagen 11A1 (mAb) 1E8.33 DMTX1/Oncomatrix, Spain 1:400 30
Desmin (mAb) D33 Dako, Denmark R-t-U 20
α-SMA (mAb) 1A4 Dako, Denmark R-t-U 20
Vimentin (pAb) C-20 Santa Cruz Biotech, Germany 1:600 10
mAb: Mouse monoclonal antibody.
pAb: Rabbit polyclonal antibodies.
R-t-U: Ready-to-Use.
Galván et al. BMC Cancer 2014, 14:867 Page 4 of 12
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5. is in Additional file 1. In three of these 51 diagnosed adeno-
carcinoma cases, no procollagen 11A1 staining could be
detected, in spite of extensive re-examination. Procollagen
11A1 was neither immunodetected in the adjacent non-
malignant tissues nor in the infarction cases.
Figure 3 shows representative procollagen 11A1 im-
munostaining patterns (panels A, B, C and D); only a
granular cytoplasmic staining of peritumoral spindle-
shaped fibroblast-like stromal cells was observed. This
granularity was either dispersed, with a few granules in
the cytoplasm of stromal cells (panel G) or frankly con-
fluent (panel H). No staining was observed on specimens
of bowel ischemia (panel E) or on non-malignant tissues
(panel F).
Figure 4 shows a representative immunostaining of an
adenocarcinoma specimen with a procollagen 11A1
immunoscore of 6. As shown on panel A, only peritu-
moral stromal cells were stained with the anti-procollagen
11A1 mAb; besides, these cells seemed to be positive for
αSMA (C) and vimentin (D), but negative for desmin (B).
This immunostaining pattern was reproduced (panels E, F,
G and H) in stromal cells cultured from fresh specimens
of the same patient.
Association between procollagen 11A1 expression and
clinicopathological features
For statistical purposes, some variables (age at diagnosis,
tumour size, anti-procollagen 11A1 immunostaining) were
divided into 2 groups, taking the median score value as a
cut-off point (Table 4). Patients diagnosed with ischemia
were excluded from this analysis.
9/12 patients that had developed distant metastases at
diagnosis and 15/27 patients with advanced Dukes stages
were associated with high procollagen 11A1 expression
(p = 0.017 and p = 0.047, respectively). The same can be
observed for 15/26 patients with nodes affected, how-
ever, only with a trend towards significance (p = 0.059).
Discussion
We have presently shown, extending our previous obser-
vations [42], that procollagen 11A1, as a protein expres-
sion product of the COL11A1 gene, is immunodetected
in stromal cells of human colon adenocarcinoma. By
contrast, and contrary to a previous report [15], we have
never observed immunodetection of procollagen 11A1
in epithelial cells of normal colon tissue or colon adeno-
carcinoma with the DMTX1/1E8.33 mAb. This immu-
nodetection was observed in 48 of the 51 cases studied.
Three cases (5.9%), which were classified under the same
criteria as the rest of the cases examined as conventional
adenocarcinomas with desmoplastic reaction, did not
stain; so far, we have not identified in them any charac-
teristics to which this negative immunostaining could be
associated. Except for the report of Fischer et al. [1] who
did not find either the expression of COL11A in 5 out of
15 (33.3%) colonic carcinomas analysed, we are not aware
Figure 2 Representative immunostaining of cultured immortalised hTERT-HMCs after long exposure to ascorbate 2-phosphate and
TGF-β1. A) Procollagen 11A1 B) Desmin, C) αSMA and D) Vimentin. Scale bar 50 μm (400×).
Galván et al. BMC Cancer 2014, 14:867 Page 5 of 12
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6. of any more studies reporting the percentage of colon
adenocarcinomas expressing the COL11A1 gene; this as-
pect should be studied in detail.
We have very recently reported that procollagen 11A1+
cancer-associated stromal cells of pancreatic ductal adeno-
carcinoma co-express αSMA, and/or vimentin, and/or des-
min in different proportions [41]. We have now confirmed,
by Q-RT-PCR and IHC/ICC, the stromal expression of hu-
man procollagen 11A1 in colon adenocarcinoma. Although
not formally proven, procollagen 11A1+
colon adenocar-
cinoma stromal cells, being spindle-shaped, seem to sim-
ultaneously express alpha smooth muscle actin and
vimentin, but no desmin; these traits confer to them a
myofibroblast-like phenotype rather than a pericyte one
[43]. While in the desmoplastic component of hepatocel-
lular carcinomas and pancreatic ductal adenocarcinomas
there is a significant contribution of desmin + stellate cells,
this is not the case in colon adenocarcinomas. As normal
resident intestinal myofibroblasts are not immunoreactive
to the DMTX1/1E8.33 mAb, these procollagen 11A1+
desmin−
colon adenocarcinoma stromal cells could be a
type of “activated myofibroblasts”.
We have also shown that a fraction of cultured immor-
talised HMCs, after long exposure to TGF-β1, exhibit a
very similar phenotype to the described above for cancer-
associated stromal cells. It is intriguing that only as much
as 20% of these cultured cells express procollagen 11A1;
this aspect warrants further analysis as well as the global
genotype and phenotype of procollagen 11A1+
cells.
It has been reported that human bone marrow-derived
mesenchymal cells may differentiate in vitro to fibroblast/
myofibroblast-like cells under certain conditions, such as
coculture with human colon carcinoma cells and TGF-β1,
or prolonged exposure to conditioned medium from
MDA-MB-231 breast cancer cells; these fibroblast/
myofibroblast-like cells are able to promote tumour
growth both in vitro and in vivo [36,44-47]. As the
phenotype of procollagen 11A1+
“myofibroblasts” from
colon adenocarcinoma resembles that of cultured TGF-
β1-activated human bone marrow mesenchymal cells, all
these observations add support to the tenet that at least
some cancer-associated stromal cells, such as the procolla-
gen 11A1+
ones, could be bone marrow-derived mesen-
chymal cells. Altogether, we may suggest that procollagen
11A1 could be expressed by a more specialized subpopu-
lation among “activated myofibroblasts”.
Halsted et al. [14] reported the cytoplasmic, stromal
and vascular immunostaining of both normal and malig-
nant human breast tissues with polyclonal antisera to spe-
cific regions of N-terminal domains of human procollagen
11A1. Vargas et al. [30] immunodetected collagen 11A1
in the normal epithelium of human breast and Wu et al.
[31] performed it in some human ovarian cancer cell lines,
after applying another antibody preparation. Moreover,
Table 3 Patient characteristics (N = 51)*
Frecuency N (%)
Gender Female 19 37.3
Male 32 62.7
Age (years) Median (range) 70 (31–85)
Tumor size (cm) Median (range) 3.7 (0.5 -
11)
Localization Ascending
colon
21 41.2
Descending
colon
8 15.7
Sigmoid 22 43.1
Differentiation Well
differentiated
19 37.3
Moderately
differentiated
28 54,9
Poorly
differentiated
4 7.8
T T1 3 5,9
T2 7 13.7
T3 28 54.9
T4 13 25.5
N pN0 25 49.0
pN1 26 51.0
M M0 39 76.5
M1 12 23.5
TNM staging I 8 15,7
IIA 12 23,5
IIB 4 7,8
IIIA 1 2,0
IIIB 9 17,6
IIIC 5 9,8
IV 12 23,5
Dukes staging A 8 15,7
B 16 31,4
C 15 29,4
D 12 23,5
Anti-procollagen 11A1
immunostaining by score
0 3 5.9
1 12 23.5
2 13 25.5
3 4 7.8
4 6 11.8
6 13 25.5
Anti-procollagen 11A1
immunostaining
Low (≤2) 28 54.9
(Median =2) High (>2) 23 45.1
(*) Patients diagnosed with colon adenocarcinoma.
Patients diagnosed with ischemia were excluded.
Galván et al. BMC Cancer 2014, 14:867 Page 6 of 12
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7. Figure 3 Representative procollagen 11A1 immunostaining in colon adenocarcinoma. A) Score 1, B) Score 2; C) Score 4; D) Score 6. Arrow
heads point to stained peritumoral stromal cells. E) Bowel ischemia; F) Non-malignant tissue. Scale bar 50 μm (400X). G) Dispersed granularity
and H) Confluent granularity. Scale bar 20 μm (1000×).
Galván et al. BMC Cancer 2014, 14:867 Page 7 of 12
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8. Figure 4 Representative immunostaining of a colon adenocarcinoma: A) Procollagen 11A1 (immunoscore 6), B) Desmin, C) αSMA and
D) Vimentin (these images were taken from the same area of serial sections; and of cultured stromal cells from the same case:
E) Procollagen 11A1, F) Desmin, G) αSMA and H) Vimentin. Scale bar 50 μm (400×).
Galván et al. BMC Cancer 2014, 14:867 Page 8 of 12
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9. rather contradictory observations have been reported
in relation to the kind of cells in which COL11A1
mRNA has been detected. While in situ hybridization
studies have spotted its detection only in stromal cells
[1,17], another study, based on differentially expressed
gene analyses by GeneChip hybridization, has pointed
to the over-expression of COL11A1 mRNA in tumour
epithelia [13]; very recently, Cheon et al. [48] have re-
ported, also through in situ hybridization and immu-
nohistochemistry with the DMTX1/1E8.33 mAb of
serous ovarian cancer, that “COL11A1 expression was
confined to intra/peritumoral stromal cells and rare
foci of tumor epithelial cells”.
In our experience, the immunodetection of procolla-
gen 11A1 with the DMTX1/1E8.33 mAb has never been
observed in normal epithelial, vascular or stromal cells
but in cancer-associated stromal cells; immunochemistry
discrepancies between our observations and those above
mentioned may be attributed to the different fine specifi-
city of the applied antibody preparations. Besides this,
transcription profiling studies of human colon biopsies
obtained from active and inactive areas of ulcerative col-
itis and Crohn’s disease, compared with samples from
infectious colitis and healthy controls, have shown that
there are no differences in the expression levels of the
COL11A1 gene between any of the above referred to
conditions [49-53]. COL11A1/procollagen 11A1 expres-
sion is mostly absent in benign inflammatory processes
such as breast sclerosing adenosis [16,54], chronic pan-
creatitis [41], and diverticulitis (our own observations;
data not shown), and is rather low in familial adenomatosis
polyposis adenomas [1,2]. Thus, the in vivo up-regulation
Table 4 Association between procollagen 11A1 expression and clinicopathological features
Anti-procollagen 11A1 immunostaining (Median =2)
Low (≤2) High (>2) p
Age (Median 70 years) ≤ 70 years 13 14 0.304
> 70 years 15 9
Gender Female 18 14 0.802
Male 10 9
Localization Ascending colon 9 12 0.260
Descending colon 6 2
Sigmoid 13 9 0.200
Tumor size (Median = 3.7 cm) Small ≤3.7 cm 12 14
Large >3.7 cm 16 9 0.370
Differentiation Well differentiated 12 7
Moderately differentiated 15 13
Poorly differentiated 1 3
T T1-T2 8 2 0.075
T3-T4 20 21
N Absent 17 8
Present 11 15 0.059
M Absent 25 14
Present 3 9 0.017
Stage grouping I 7 1 0.141
IIA 7 5
IIB 2 2
IIIA 1 0
IIIB 6 3
IIIC 2 3
IV 3 9
Dukes staging A 7 1
B 9 7
C 9 6 0.047
D 3 9
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10. of the COL11A1 gene may be considered as a biomarker
of cancer-associated stromal cells.
In this study, high procollagen 11A1 immunostaining
was associated with clinicopathological variables such as
lymph node involvement, advanced Dukes stages and
presence of distant metastases. These results go according
to the role of COL11A1 in promoting carcinoma aggres-
siveness and progression [11,17,20,30,31,48,55-57].
Conclusions
Based on its high specificity, our observations stress once
more the usefulness of the DMTX1/1E8.33 mAb for can-
cer research, and the clinical significance of procollagen
11A1 as a very valuable biomarker to characterise cancer-
associated stromal cells and to evaluate human colon
adenocarcinomas.
Additional file
Additional file 1: Detailed description of patients and their
clinicopathological characteristics.
Abbreviations
HMCs: Human mesenchymal cells; hTERT-HMCs: Immortalised hTERT- human
bone marrow mesenchymal cells; ICC: Immunocytochemistry;
IHC: Immunohistochemistry; mAb: Monoclonal antibody; pAb: Polyclonal
antibodies; H&E: Hematoxylin and eosin; PBS: Phosphate-buffered saline;
DAB: 3-3′-Diaminobenzidine.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JAG carried out immunostainings, photographs and statistical analyses. The
immunostainings were evaluated by JAG, CGP and CGR, and supervised by
CGP and PMR. JGM isolated and cultured tumour stromal cells. FVV and
MGO carried out other cell cultures. All authors discussed, read and
approved the final manuscript. CGP, PMR, LBS, and JRT contributed equally
to this study as senior authors.
Acknowledgements
The authors thank Inti Zlobec for the critical reading of the manuscript and
helpful comments. The excellent technical assistance of Laura Suárez-
Fernández is greatly acknowledged.
This research has been co-financed by European Union ERDF Funds; by the
INNPACTO-ONCOPAN IPT-010000-2010-31 Project; by the FISS-09-PS09/01911
Project, Ministry of Science and Innovation, Spain; by the FC-11-PC10-23,
FICYT Project, Axe 1 of the 2007–2013 ERDF Operational Framework
Programme of the Principality of Asturias, Spain; and by Oncomatrix, S.L.
Derio, Spain.
Author details
1
Surgery Department, School of Medicine and Health Sciences, University of
Oviedo, 33006 Oviedo, Spain. 2
Oncology University Institute of the
Principality of Asturias (IUOPA), 33006 Oviedo, Spain. 3
Preparative
Biotechnology Unit, Technical-Scientific Services, University of Oviedo, 33006
Oviedo, Spain. 4
Pathological Anatomy Service, Asturias Central University
Hospital (HUCA), 33006 Oviedo, Spain. 5
Immunology Department, School of
Medicine and Health Sciences, University of Oviedo, c/ Julián Clavería s/n,
33006 Oviedo, Spain. 6
Present address: Translational Research Unit (TRU),
Institute of Pathology, University of Bern, Bern, Switzerland.
Received: 29 April 2014 Accepted: 12 November 2014
Published: 23 November 2014
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Cite this article as: Galván et al.: Validation of COL11A1/procollagen
11A1 expression in TGF-β1-activated immortalised human
mesenchymal cells and in stromal cells of human colon
adenocarcinoma. BMC Cancer 2014 14:867.
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Galván et al. BMC Cancer 2014, 14:867 Page 12 of 12
http://www.biomedcentral.com/1471-2407/14/867