1. The study developed a novel 2.5D in vitro dot migration assay to better understand the invasive behaviors of proneural and mesenchymal glioblastoma tumor cells.
2. The assay incorporated both a surface (glass coverslip) and extracellular matrix components (Matrigel and FBS) to model the in vivo tumor microenvironment.
3. Preliminary results showed that the inflammatory cytokine TNF-alpha promoted greater cellular adhesion and dissemination of proneural PBT003 tumor cells in the assay, supporting one of the study's hypotheses. Further testing of additional cell lines was needed to fully evaluate the hypotheses.
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.
Growth Kinetics of 2- and 3-D Cell Models as Influenced by the MicroenvironmentТатьяна Гергелюк
The noncontact cocultivation system was developed for the study of the paracrine interactions
between MCF-7 (breast carcinoma cells) and MT-4 (a line of human T-cell leukemia). Viability and proliferation
rates were determined in the adhesion and suspension fractions of MCF-7 cells sampled from two model
systems: monolayer culture and multicellular tumor spheroids (MTS). Cocultivation with MT-4 reduced the
number of MCF-7 cells in the adhesion fraction and had no effect upon the suspension fraction, despite an
increase in the total population of MCF-7 cells. The two model systems displayed a substantial difference in
cell viability, alone and in the presence of MT-4 cells – the fraction of viable cells in the monolayers was greater
than in the spheroids. It is suggested that cocultivation with MT-4 stimulates proliferation of MCF-7 cells via
a paracrine mechanism, reduces adhesion to the substrate, and leads to MTS formation.
This document reports on a study that identified cysteine-rich protein 2 (CRP2) as a new component of breast cancer cell invadopodia that promotes invasion and metastasis. The study found that CRP2 expression is higher in mesenchymal/invasive breast cancer cells and its expression level correlates with increased risk of metastasis in basal-like breast cancer patients. CRP2 was shown to localize to the actin core of invadopodia, where it bundles actin filaments. Knockdown of CRP2 reduced breast cancer cell invasion, matrix degradation, and MMP-9 expression/secretion. Ectopic expression of CRP2 in less invasive cells increased invasion. Depletion of CR
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.
1) Experiments compared the ability of eye and skin melanoma cells to stimulate T cell proliferation. Primary skin melanoma cells stimulated significant T cell proliferation, while primary eye melanoma cells failed to induce proliferation.
2) Eye melanoma cells expressed normal levels of HLA class I and II molecules but still failed to stimulate T cells. In contrast, eye melanoma cells inhibited T cell proliferation in mixed lymphocyte cultures through direct cell contact.
3) The ability of eye melanoma cells to inhibit T cell proliferation was lost when the cells metastasized from the eye to other sites like the liver, suggesting the unique eye microenvironment alters the immunogenicity of melanoma cells developing there.
This research article examines the expression of RhoA and Rac1 in fibroblasts found at primary breast tumor sites and corresponding lymph node metastases. Immunohistochemistry on tissue microarrays revealed that 59% of fibroblasts at primary tumors and 41% at lymph node metastases expressed RhoA. Similarly, 57.1% at primary tumors and 42.9% at lymph node metastases expressed Rac1. Since expression levels were similar between primary and metastatic sites, the researchers suggest that fibroblasts actively participate in cancer cell invasion to lymph nodes and that metastatic cells continue relying on their microenvironment. Primary cell cultures were used to validate differences in focal adhesion pathways between carcinoma-associated fibroblasts and normal fibroblasts previously found via genomic profiling.
1. The study developed a novel 2.5D in vitro dot migration assay to better understand the invasive behaviors of proneural and mesenchymal glioblastoma tumor cells.
2. The assay incorporated both a surface (glass coverslip) and extracellular matrix components (Matrigel and FBS) to model the in vivo tumor microenvironment.
3. Preliminary results showed that the inflammatory cytokine TNF-alpha promoted greater cellular adhesion and dissemination of proneural PBT003 tumor cells in the assay, supporting one of the study's hypotheses. Further testing of additional cell lines was needed to fully evaluate the hypotheses.
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.
Growth Kinetics of 2- and 3-D Cell Models as Influenced by the MicroenvironmentТатьяна Гергелюк
The noncontact cocultivation system was developed for the study of the paracrine interactions
between MCF-7 (breast carcinoma cells) and MT-4 (a line of human T-cell leukemia). Viability and proliferation
rates were determined in the adhesion and suspension fractions of MCF-7 cells sampled from two model
systems: monolayer culture and multicellular tumor spheroids (MTS). Cocultivation with MT-4 reduced the
number of MCF-7 cells in the adhesion fraction and had no effect upon the suspension fraction, despite an
increase in the total population of MCF-7 cells. The two model systems displayed a substantial difference in
cell viability, alone and in the presence of MT-4 cells – the fraction of viable cells in the monolayers was greater
than in the spheroids. It is suggested that cocultivation with MT-4 stimulates proliferation of MCF-7 cells via
a paracrine mechanism, reduces adhesion to the substrate, and leads to MTS formation.
This document reports on a study that identified cysteine-rich protein 2 (CRP2) as a new component of breast cancer cell invadopodia that promotes invasion and metastasis. The study found that CRP2 expression is higher in mesenchymal/invasive breast cancer cells and its expression level correlates with increased risk of metastasis in basal-like breast cancer patients. CRP2 was shown to localize to the actin core of invadopodia, where it bundles actin filaments. Knockdown of CRP2 reduced breast cancer cell invasion, matrix degradation, and MMP-9 expression/secretion. Ectopic expression of CRP2 in less invasive cells increased invasion. Depletion of CR
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.
1) Experiments compared the ability of eye and skin melanoma cells to stimulate T cell proliferation. Primary skin melanoma cells stimulated significant T cell proliferation, while primary eye melanoma cells failed to induce proliferation.
2) Eye melanoma cells expressed normal levels of HLA class I and II molecules but still failed to stimulate T cells. In contrast, eye melanoma cells inhibited T cell proliferation in mixed lymphocyte cultures through direct cell contact.
3) The ability of eye melanoma cells to inhibit T cell proliferation was lost when the cells metastasized from the eye to other sites like the liver, suggesting the unique eye microenvironment alters the immunogenicity of melanoma cells developing there.
This research article examines the expression of RhoA and Rac1 in fibroblasts found at primary breast tumor sites and corresponding lymph node metastases. Immunohistochemistry on tissue microarrays revealed that 59% of fibroblasts at primary tumors and 41% at lymph node metastases expressed RhoA. Similarly, 57.1% at primary tumors and 42.9% at lymph node metastases expressed Rac1. Since expression levels were similar between primary and metastatic sites, the researchers suggest that fibroblasts actively participate in cancer cell invasion to lymph nodes and that metastatic cells continue relying on their microenvironment. Primary cell cultures were used to validate differences in focal adhesion pathways between carcinoma-associated fibroblasts and normal fibroblasts previously found via genomic profiling.
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
Gastric cancer is one of the leading causes of cancer death worldwide. It is a heterogeneous cancer that has been useful for studying carcinogenesis and tumorigenesis. One idea discussed is how the bacterial pathogen H. pylori and the inflammatory cytokine IL-1α cooperate to promote α-catenin translocation, which is associated with epithelial-to-mesenchymal transition (EMT). Another idea is that the inflammatory microenvironment contributes to EMT in gastric cancer by altering cancer cells, potentially through specific immune cells. A third idea is that infection with H. pylori induces EMT markers and the emergence of cancer stem cell-like properties in gastric cells, and eradicating H. pylori reduces these
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 document describes a study examining gene expression changes in breast epithelial cells and fibroblasts when cocultured together. MCF10A non-cancerous breast epithelial cells and MDA-MB231 cancerous breast epithelial cells were cocultured with either normal adjacent fibroblasts (NAF) isolated from benign breast tissues or carcinoma-associated fibroblasts (CAF) isolated from breast cancer tissues, using a transwell system. Gene expression profiles of each cell type were analyzed after coculture and compared to monoculture controls. The results showed reciprocal changes in gene expression between the epithelial cells and fibroblasts. Specifically, CAFs induced genes related to cell proliferation in both epithelial cell lines, while NAFs downregulated genes involved in growth control and adhesion in
Odontogenic ameloblast-associated protein (ODAM) inhibits growth and migratio...Enrique Moreno Gonzalez
The Odontogenic Ameloblast-associated Protein (ODAM) is expressed in a wide range of
normal epithelial, and neoplastic tissues, and we have posited that ODAM serves as a novel
prognostic biomarker for breast cancer and melanoma. Transfection of ODAM into breast
cancer cells yields suppression of cellular growth, motility, and in vivo tumorigenicity.
Herein we have extended these studies to the effects of ODAM on cultured melanoma cell
lines.
Cytokine Immunotherapy: A Forthcoming Visible Feature in Cancer TherapeuticsSachin K. S. Chauhan
The document discusses cytokine immunotherapy as a promising approach for cancer treatment. It notes that cytokines can stimulate the immune system to fight tumors, but that mono-cytokine therapy has limitations. Combined cytokine therapy or cytokine therapy combined with other treatments may be more effective by creating a specific immune response. The document advocates focusing research on combination therapies to help overcome drawbacks of traditional cancer treatments.
Achieving High Yields in Scalable Xeno Free Culture Formats with Mesenchymal ...Merck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/3ryE5ST
Optimize your mesenchymal stem cell growth. Join our webinar to learn more about our GMP-compliant xeno free media formulation that supports high performance expansions and compatibility with scalable xeno free manufacturing conditions.
Optimizing ex vivo cell expansion processes in preparation for clinical use is a critical step in cell therapy manufacturing. Given the curative and lifesaving impacts these therapies can have on patients, overcoming roadblocks with scalability and supply chain, using high quality raw materials are essential for therapeutic access.
The GMP-compliant Stemline® XF MSC Medium and cocktail promotes expansion of human mesenchymal stromal/stem cells (hMSCs) to high densities while maintaining cell identity and quality. This product was designed for derivation and expansion of MSCs using xeno free conditions in planar and microcarrier-based culture platforms, easing the transfer between research, clinical, and manufacturing scale cultures.
In this webinar, you will:
• Explore the current landscape and future trends of cell culture media for adult mesenchymal stem cells
• Discover ways to derive MSC's from Bone Marrow in Xeno Free conditions from static to microcarrier-based suspension culture platforms.
• Learn how Stemline® XF MSC Media provides robust performance and reduces scalability roadblocks
Presented by: Kathleen Ongena, Ph.D., Head of Customer Applications and Mark Ventresco, Cell Therapy Product Manager
Achieving High Yields in Scalable Xeno Free Culture Formats with Mesenchymal ...MilliporeSigma
Watch the presentation of this webinar here: https://bit.ly/3ryE5ST
Optimize your mesenchymal stem cell growth. Join our webinar to learn more about our GMP-compliant xeno free media formulation that supports high performance expansions and compatibility with scalable xeno free manufacturing conditions.
Optimizing ex vivo cell expansion processes in preparation for clinical use is a critical step in cell therapy manufacturing. Given the curative and lifesaving impacts these therapies can have on patients, overcoming roadblocks with scalability and supply chain, using high quality raw materials are essential for therapeutic access.
The GMP-compliant Stemline® XF MSC Medium and cocktail promotes expansion of human mesenchymal stromal/stem cells (hMSCs) to high densities while maintaining cell identity and quality. This product was designed for derivation and expansion of MSCs using xeno free conditions in planar and microcarrier-based culture platforms, easing the transfer between research, clinical, and manufacturing scale cultures.
In this webinar, you will:
• Explore the current landscape and future trends of cell culture media for adult mesenchymal stem cells
• Discover ways to derive MSC's from Bone Marrow in Xeno Free conditions from static to microcarrier-based suspension culture platforms.
• Learn how Stemline® XF MSC Media provides robust performance and reduces scalability roadblocks
Presented by: Kathleen Ongena, Ph.D., Head of Customer Applications and Mark Ventresco, Cell Therapy Product Manager
Cord Blood Mesenchymal Stem Cells Conditioned Media Suppress Epithelial Ovari...ijtsrd
MSC CM suppresses epithelial ovarian cancer cells in vitro in a concentration-dependent manner. When ovarian cancer cells were treated with MSC CM at concentrations of 100%, 75%, 50%, and 25% for 72 hours, cell morphology changes were observed including cell shrinkage, debris and reduced cell numbers compared to control. MTT assays showed reduced proliferation and Annexin V testing demonstrated increased early and late apoptosis. Cell cycle analysis found an increased sub-G1 phase, indicating apoptosis. Expression of embryonic stemness genes was also progressively suppressed in cancer cells treated with MSC CM compared to control. Therefore, MSC CM has potential as an ovarian cancer inhibitor by creating new treatment modalities.
This document discusses tumor cell proliferation and immunotherapy for cancers. It provides details on the cell cycle phases (M, G1, S, G2, G0) and how they relate to tumor growth and response to treatment. It also discusses cell kinetics, the growth fraction, and cancer stem cells. Targeted therapies discussed include those that inhibit angiogenesis by targeting VEGF, as well as EGFR inhibitors. Bevacizumab is highlighted as an anti-angiogenic therapy shown to improve outcomes for ovarian cancer both as a single agent and in combination with other drugs.
CoH Summer Academy 2016 Poster (Lauren)Lauren T. Hui
This study examined how two patient-derived glioblastoma cell lines - one proneural (PBT003) and one mesenchymal (PBT030) - responded to tumor necrosis factor (TNF) under different growth conditions. The cell lines were cultured in stem-like or differentiating conditions with and without TNF, then analyzed using a dot migration assay and immunofluorescence staining. The results showed that the cell lines' expression of proteins like VCAM1 and CD44, and binding of chlorotoxin-Cy5.5, varied depending on molecular subtype and exposure to TNF. In particular, PBT030 cells grown in stem-like conditions with TNF had higher expression of the mesenchymal marker CD44.
- Physiologic concentrations of resistin and IL-6 were found to stimulate melanoma cell proliferation, with resistin and high-dose IL-6 increasing cell numbers compared to controls.
- Macrophages were shown to significantly increase melanoma cell migration in co-culture experiments, while individual cytokines had no effect on migration.
- Previous research from the authors' lab revealed that the adipokine leptin promotes melanoma tumor growth in obese mice. The current results further understanding of how obesity increases melanoma by identifying links between inflammation, melanoma cell proliferation and migration.
This document discusses cancer stem cells (CSCs). It introduces CSCs and their properties of self-renewal and differentiation. CSCs have been identified in many cancers using cell surface markers and can mediate metastasis, treatment resistance, and relapse. CSCs are regulated by the tumor microenvironment through growth factors and cytokines. Several signaling pathways important in CSCs are discussed, including Hedgehog, Notch, and WNT, which are potential therapeutic targets. The relationship between epithelial-mesenchymal transition (EMT) and CSCs is also covered.
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 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 document discusses cancer and its genetic basis. It begins by introducing cancer and how it is caused by the accumulation of genetic damage over time through mutations in genes that control cell growth. Key points include: cancers originate from mutations in somatic cells, not germ cells; carcinomas make up over 90% of cancers and originate from endodermal or ectodermal tissues; the ability of cancer cells to invade and metastasize distinguishes malignant from benign tumors. The document then covers specific genetic mutations and cellular processes involved in cancer development.
This document summarizes research optimizing the transfection of umbilical cord mesenchymal stem cells (UC-MSCs) with minicircle plasmid DNA using Lipofectamine LTX. The researchers tested different ratios of plasmid to Lipofectamine LTX, volumes of transfection complexes, cell densities, and presence or absence of medium. They found that a 1:2 ratio of 3μg plasmid to 6μl Lipofectamine LTX, transfecting 50,000 cells with 150μl of the transfection complex in the presence of medium, resulted in the highest expression of the GFP reporter gene as observed by fluorescence microscopy 72 hours after transfection. Flow cytometry was also used to quantitatively measure transfection efficiency
IRF5 Promotes the Progression of Hepatocellular Carcinoma and is Regulated by...NainaAnon
1. The study found that IRF5 was upregulated in hepatocellular carcinoma (HCC) tissues compared to normal tissues based on mRNA and protein levels.
2. Overexpression of IRF5 promoted the growth and colony formation of HCC cells in vitro, while silencing IRF5 inhibited HCC cell growth and proliferation.
3. TRIM35 was found to interact with and promote the degradation of IRF5. TRIM35 expression was negatively correlated with IRF5 levels in HCC clinical samples.
Trimodal Management of Locally Invasive Urinary Bladder CancerNainaAnon
To evaluate the response of the modern bladder-preservation treatment modality; Trimodal Therapy (TMT) in Muscle-Invasive Bladder Cancer (MIBC). Aiming at bladder preservation in MIBC, TMT was to offer a quality- of-life advantage and avoid potential morbidity and mortality of Radical Cystectomy (RC) without compromising oncologic outcomes.
Alterations of Gut Microbiota From Colorectal Adenoma to CarcinomaNainaAnon
The document analyzes differences in gut microbiota between healthy individuals, those with colorectal adenomas (CRA), and those with colorectal cancer (CRC). 16S rRNA sequencing was performed on tissue samples from 11 individuals in each group. Microbial diversity was lower in CRA patients and higher in CRC patients compared to healthy individuals. The relative abundance of Fusobacteria was lower in CRA and higher in CRC. This suggests alterations in gut microbiota occur from adenoma to carcinoma, with Fusobacteria potentially playing a role in colorectal cancer development.
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Similar to Functional Disparity of Carcinoma Associated Fibroblasts in Different Stages Influences Immune Response in Cancer
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
Gastric cancer is one of the leading causes of cancer death worldwide. It is a heterogeneous cancer that has been useful for studying carcinogenesis and tumorigenesis. One idea discussed is how the bacterial pathogen H. pylori and the inflammatory cytokine IL-1α cooperate to promote α-catenin translocation, which is associated with epithelial-to-mesenchymal transition (EMT). Another idea is that the inflammatory microenvironment contributes to EMT in gastric cancer by altering cancer cells, potentially through specific immune cells. A third idea is that infection with H. pylori induces EMT markers and the emergence of cancer stem cell-like properties in gastric cells, and eradicating H. pylori reduces these
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 document describes a study examining gene expression changes in breast epithelial cells and fibroblasts when cocultured together. MCF10A non-cancerous breast epithelial cells and MDA-MB231 cancerous breast epithelial cells were cocultured with either normal adjacent fibroblasts (NAF) isolated from benign breast tissues or carcinoma-associated fibroblasts (CAF) isolated from breast cancer tissues, using a transwell system. Gene expression profiles of each cell type were analyzed after coculture and compared to monoculture controls. The results showed reciprocal changes in gene expression between the epithelial cells and fibroblasts. Specifically, CAFs induced genes related to cell proliferation in both epithelial cell lines, while NAFs downregulated genes involved in growth control and adhesion in
Odontogenic ameloblast-associated protein (ODAM) inhibits growth and migratio...Enrique Moreno Gonzalez
The Odontogenic Ameloblast-associated Protein (ODAM) is expressed in a wide range of
normal epithelial, and neoplastic tissues, and we have posited that ODAM serves as a novel
prognostic biomarker for breast cancer and melanoma. Transfection of ODAM into breast
cancer cells yields suppression of cellular growth, motility, and in vivo tumorigenicity.
Herein we have extended these studies to the effects of ODAM on cultured melanoma cell
lines.
Cytokine Immunotherapy: A Forthcoming Visible Feature in Cancer TherapeuticsSachin K. S. Chauhan
The document discusses cytokine immunotherapy as a promising approach for cancer treatment. It notes that cytokines can stimulate the immune system to fight tumors, but that mono-cytokine therapy has limitations. Combined cytokine therapy or cytokine therapy combined with other treatments may be more effective by creating a specific immune response. The document advocates focusing research on combination therapies to help overcome drawbacks of traditional cancer treatments.
Achieving High Yields in Scalable Xeno Free Culture Formats with Mesenchymal ...Merck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/3ryE5ST
Optimize your mesenchymal stem cell growth. Join our webinar to learn more about our GMP-compliant xeno free media formulation that supports high performance expansions and compatibility with scalable xeno free manufacturing conditions.
Optimizing ex vivo cell expansion processes in preparation for clinical use is a critical step in cell therapy manufacturing. Given the curative and lifesaving impacts these therapies can have on patients, overcoming roadblocks with scalability and supply chain, using high quality raw materials are essential for therapeutic access.
The GMP-compliant Stemline® XF MSC Medium and cocktail promotes expansion of human mesenchymal stromal/stem cells (hMSCs) to high densities while maintaining cell identity and quality. This product was designed for derivation and expansion of MSCs using xeno free conditions in planar and microcarrier-based culture platforms, easing the transfer between research, clinical, and manufacturing scale cultures.
In this webinar, you will:
• Explore the current landscape and future trends of cell culture media for adult mesenchymal stem cells
• Discover ways to derive MSC's from Bone Marrow in Xeno Free conditions from static to microcarrier-based suspension culture platforms.
• Learn how Stemline® XF MSC Media provides robust performance and reduces scalability roadblocks
Presented by: Kathleen Ongena, Ph.D., Head of Customer Applications and Mark Ventresco, Cell Therapy Product Manager
Achieving High Yields in Scalable Xeno Free Culture Formats with Mesenchymal ...MilliporeSigma
Watch the presentation of this webinar here: https://bit.ly/3ryE5ST
Optimize your mesenchymal stem cell growth. Join our webinar to learn more about our GMP-compliant xeno free media formulation that supports high performance expansions and compatibility with scalable xeno free manufacturing conditions.
Optimizing ex vivo cell expansion processes in preparation for clinical use is a critical step in cell therapy manufacturing. Given the curative and lifesaving impacts these therapies can have on patients, overcoming roadblocks with scalability and supply chain, using high quality raw materials are essential for therapeutic access.
The GMP-compliant Stemline® XF MSC Medium and cocktail promotes expansion of human mesenchymal stromal/stem cells (hMSCs) to high densities while maintaining cell identity and quality. This product was designed for derivation and expansion of MSCs using xeno free conditions in planar and microcarrier-based culture platforms, easing the transfer between research, clinical, and manufacturing scale cultures.
In this webinar, you will:
• Explore the current landscape and future trends of cell culture media for adult mesenchymal stem cells
• Discover ways to derive MSC's from Bone Marrow in Xeno Free conditions from static to microcarrier-based suspension culture platforms.
• Learn how Stemline® XF MSC Media provides robust performance and reduces scalability roadblocks
Presented by: Kathleen Ongena, Ph.D., Head of Customer Applications and Mark Ventresco, Cell Therapy Product Manager
Cord Blood Mesenchymal Stem Cells Conditioned Media Suppress Epithelial Ovari...ijtsrd
MSC CM suppresses epithelial ovarian cancer cells in vitro in a concentration-dependent manner. When ovarian cancer cells were treated with MSC CM at concentrations of 100%, 75%, 50%, and 25% for 72 hours, cell morphology changes were observed including cell shrinkage, debris and reduced cell numbers compared to control. MTT assays showed reduced proliferation and Annexin V testing demonstrated increased early and late apoptosis. Cell cycle analysis found an increased sub-G1 phase, indicating apoptosis. Expression of embryonic stemness genes was also progressively suppressed in cancer cells treated with MSC CM compared to control. Therefore, MSC CM has potential as an ovarian cancer inhibitor by creating new treatment modalities.
This document discusses tumor cell proliferation and immunotherapy for cancers. It provides details on the cell cycle phases (M, G1, S, G2, G0) and how they relate to tumor growth and response to treatment. It also discusses cell kinetics, the growth fraction, and cancer stem cells. Targeted therapies discussed include those that inhibit angiogenesis by targeting VEGF, as well as EGFR inhibitors. Bevacizumab is highlighted as an anti-angiogenic therapy shown to improve outcomes for ovarian cancer both as a single agent and in combination with other drugs.
CoH Summer Academy 2016 Poster (Lauren)Lauren T. Hui
This study examined how two patient-derived glioblastoma cell lines - one proneural (PBT003) and one mesenchymal (PBT030) - responded to tumor necrosis factor (TNF) under different growth conditions. The cell lines were cultured in stem-like or differentiating conditions with and without TNF, then analyzed using a dot migration assay and immunofluorescence staining. The results showed that the cell lines' expression of proteins like VCAM1 and CD44, and binding of chlorotoxin-Cy5.5, varied depending on molecular subtype and exposure to TNF. In particular, PBT030 cells grown in stem-like conditions with TNF had higher expression of the mesenchymal marker CD44.
- Physiologic concentrations of resistin and IL-6 were found to stimulate melanoma cell proliferation, with resistin and high-dose IL-6 increasing cell numbers compared to controls.
- Macrophages were shown to significantly increase melanoma cell migration in co-culture experiments, while individual cytokines had no effect on migration.
- Previous research from the authors' lab revealed that the adipokine leptin promotes melanoma tumor growth in obese mice. The current results further understanding of how obesity increases melanoma by identifying links between inflammation, melanoma cell proliferation and migration.
This document discusses cancer stem cells (CSCs). It introduces CSCs and their properties of self-renewal and differentiation. CSCs have been identified in many cancers using cell surface markers and can mediate metastasis, treatment resistance, and relapse. CSCs are regulated by the tumor microenvironment through growth factors and cytokines. Several signaling pathways important in CSCs are discussed, including Hedgehog, Notch, and WNT, which are potential therapeutic targets. The relationship between epithelial-mesenchymal transition (EMT) and CSCs is also covered.
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 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
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2. the production of regulatory cytokine IL-10 and iNOS expression
compared to TGF-β and IDO. Whereas CAFs induce IL-17 and
PGE2 production in both stages. These findings place CAFs as key
regulators of cancer microenvironment and suggest new interven-
tion strategies to restore immune response in cancer and related
inflammation.
3. Material and Methods
3.1. Establishment of Stage 2 and 4 Model
In studies involving mouse models, terms such as “early” and
“late” stages is used to denote various stages of cancer. However,
this terminology is not applicable when translating animal mod-
els into human studies. Therefore, here, we employed the model
of human tumor growth kinetics [61, 62] and the metabolic rate
differences [63] to establish a stage II and stage IV model of tu-
mor growth in Balb/C mice. According to TNM staging of breast
cancer [64], a combination of tumor size, lymph node involvement
and metastasis are used to assign a breast cancer stage. Initially,
tumor size is the first parameter. In stage II a tumor size of 2-5 cm
with/out lymph node involvement and in stage IV, any tumor size
with distant metastasis are the chosen criteria. To translate these
sizes to mouse tumor growth, tumor size was transformed to num-
ber of cancer cells using equation 1.
Equation 1: transforming tumor size to the number of cells in a
given tumor size
Where D is the tumor diameter and d is the cancer cell diameter
which was assumed as 10 µm. Therefore, a human tumor with a
diameter of 2 cm, has a volume of 4186.67 mm3
and 8×109
number
of cells. Counting in the differences in mouse and human metabol-
ic rate (7:1), a stage II cancer of mouse would have approximately
109
cancer cells and 200 mm3
volume. A stage IV cancer in mouse
would have 2×109
cells and 600 mm3
volume and distant metas-
tasis.
The cell line of choice was MBL-6. isolated from a spontaneous
breast cancer of Balb/C mouse MBL-6 tumors possess similar pa-
thology to human invasive ductal carcinoma [15]. Ten 4-6 weeks
old female Balb/C mice (Pasture Institute, Tehran, Iran) were sub-
cutaneously inoculated with 5×105
MBL-6 cells. When the tumors
reached an approximate size of 200 mm3
(stage II) and 600 mm3
(stage IV) mice were sacrificed. Tissues including tumor, liver,
spleen, lung and lymph nodes were separated aseptically and fixed
in 10% formalin for metastasis analysis.
3.2. CAF Isolation
Tumor tissues were aseptically removed, washed in PBS and
minced with scalpel into 1-3 mm sized fragments. Fragments were
washed in PBS and incubated in an enzyme cocktail consisting
of 0.5% collagenase IV, 0.02% hyaluronidase, 0.25% trypsin and
0.002% DNaseI in DMEM/F12. After 4 hours in 37°C, cell sus-
pensions were fractioned using ficol separation technique. The in-
terface layer of ficol consisted of fibroblast and the pellet consisted
of mostly cancer cells. Fibroblasts were washed in PBS and plat-
ed in DMEM/F12 (GIBCO, USA) supplemented with 30% FBS,
(GIBCO, USA) and 1% penicillin/streptomycin (Biosera, UK) in
flasks coated with 0.1% gelatin (Sigma, Germany). Fibroblastic
colonies were passaged in 10% FBS DMEM/F12. The passage 2
and 3 cells were characterized and used for future evaluations.
3.3. CAF Characterization and Immunostaining
The cell surface expression of CAF markers were analyzed using
the following antibodies: PE conjugated anti-CD29, anti-CD105,
anti-HLA-DR, anti-CD45, anti-CD90 and anti-FAP; FITC con-
jugated anti-CD104a (PDGFRa), anti-Sca-1 (eBioscience). Cells
were divided into aliquots (5 × 105 each), stained with FITC- or
PE-conjugated antibodies. Results were analyzed by BD FACS
flow cytometry and Flowjo software (version 7.6.1). Additionally,
isolated CAFs were stained for expression of FAP-1 using rabbit
anti-FAP-1 and FITC conjugated goat anti-rabbit IgG.
3.4. Colony Forming Unit – Fibroblast
The CFU-F assay was performed using a modification of a de-
scribed protocol. Cells cultured were resuspended in the above
medium in three concentrations of 100, 500 and 1000 viable cells
in 10cm dish. The medium was changed 2 times per week. On
the 14th day, cultures were fixed with 4% PFA and stained with
crystal violet. Fibroblastic colonies with more than 50 cells and
or possessing a diameter greater than 2mm were counted under
an inverted microscope. Three CAFs were evaluated in triplicate.
3.5. Growth Curve
CAFs were cultured in DMEM at an initial density of 5000 per
well in 24 well plates. At 24-hour intervals, MTT assay was per-
formed and the optical density was corrected using a standard cell
curve. The growth curve was evaluated for 13 consecutive days.
3.6. CAFs and the Immune System in Vitro
3.6.1. Proliferation of Splenocytes: The ability of CAF to affect
immune cell proliferation was assessed using MTT proliferation
assay. Triplicates of mitomycin-inactivated CAFs or MBL-6 and
splenocytes were co-cultured in contact or with Conditioned Me-
dia (CM), in a 96-well plate (TPP) at four ratios of 1:10, 1:50,
1:100 and 1:250 in 200µL of RPMI supplemented with 10% FBS,
penicillin/streptomycin (100 mg/mL) and amphotericin B (2.5 ng/
mL) at 37°C in a humid atmosphere containing 5% CO2
. PHA
was added to stimulate splenocytes. After 72 hours, splenocyte
proliferation was assessed using MTT assay. As a positive con-
trol, splenocytes were activated by 5µg/ mL PHA (Baharafshan,
Teahran, Iran) and the negative control was untreated splenocytes.
clinicsofoncology.com 2
Volume 4 Issue 1 -2021 Research Article
3. Proliferation of treated splenocytes was calculated and expressed
as stimulation index.
3.6.2. Cytokine Production: Splenocytes were co-cultured in
contact or with conditioned medium of Mitomycin-inactivated
CAF and MBL-6 cell line in a 24-well plate (TPP) at 1:10 ratio
in 1 mL of RPMI supplemented with 10% FBS, (100 units/mL)
penicillin/(100 mg/mL) streptomycin at 37°C in a humid atmo-
sphere containing 5% CO2
. PHAwas added andAfter 72 hours, the
supernatant was collected and the level of IL-10, IL-17, TGF-β1,
and Prostaglandin E2 was assessed by ELISA (DuoSet ELISA De-
velopment kit, R&D systems, Minneapolis, MN, USA). All pro-
cedures were followed according to the manufacturer’s protocol.
3.7. Nitric Oxide Production
NO levels (nmol/ml) were measured in culture supernatants by
the Griess reaction. Briefly, nitrite was measured by adding equal
volumes of Griess reagent (1% sulphanilamide and 0.1% naphth-
ylenediamine in 5% phosphoric acid) to conditioned media sam-
ples. The optical density at 550 nm was measured by using a mi-
croplate reader. Varying concentration solutions of sodium nitrite
prepared in the culture medium were used for standard curve cal-
culations. Fresh medium was used as blank for background absor-
bance of NO production. All chemicals were obtained from Merck
(Darmstadt, Germany).
3.8. Gene Expression Analysis Using qRT-PCR
Gene Expression was assessed using Real-Time PCR. After CAF
isolation, total cellular RNA was extracted using Trizol (Gib-
co-BRL, Life Technologies, MD). Random hexamer-primed
reverse transcription (Metabion) was performed on aliquots (1
µg) of total RNA as a template. The resulting cDNA was used
for Real-Time PCR amplification. Primers for cycloxygenase 2
(COX-2), inducible Nitric Oxide Synthetase (iNOS), Indolamine
Deoxygenase (IDO), Matrix Metalloproteinase 2 (MMP2) and 9
(MMP9) and beta-actin were synthesized based on the reported
sequences as follows:
• COX-2 (145 bp) forward: AGACAGATCATAAGCGAGGAC,
reverse: CCACCAATGACCTGATATTTC;
• INOS (142 bp): forward: TGTGCGAAGTGTCAGTGG, re-
verse: TCCTTTGAGCCCTTTGTG;
• IDO (168 bp): forward: GGATGCGTGACTTTGTGG, reverse:
TGGAAGATGCTGCTCTGG;
• MMP2 (150 bp): forward: AGACAAGTTCTGGAGATA-
CAATG, reverse: GCACCCTTGAAGAAGTAGC;
• MMP9 (136 bp): forward: GGCGTGTCTGGAGATTCG, re-
verse: TGGCAGAAATAGGCTTTGTC
Real-time PCR reaction mixtures (final volume of 30 ul) contained
1 µl cDNA, 30 pmol of each primer, 3 µl of 200 µM dNTP, and
1U Taq-DNA polymerase (MBI Fermentas Inc., Burlington, ON).
Amplification conditions were as follows: 25 cycles of 94°C for 30
s; 55°C for 60 s; and 72°C for 1 min, followed by 72°C incubation
for 10 min (Corbett cycler). The results of primer amplification
were analyzed using Rotor-Gene 6000 and REST software.
4. Statistical Analysis
All data is presented as mean±SEM otherwise stated. Data was
analyzed by SPSS v.19 software and GraphPad Prism v. 8. The rel-
ative expressions and heatmap demonstration were analyzed using
REST software and R studio respectively. A P-value less than 0.05
was considered as statistical significance.
5. Results
5.1. Two Pathological Stages Were Successfully Established in
Tumor Bearing Balb/C Mice
MBL-6 is a cell line isolated from a spontaneous invasive duc-
tal carcinoma of mammary glands of Balb/C mouse. This tumor
was preserved using SC transplantations [15]. This model has the
advantage of long in situ growth prior to metastasis formation,
permitting various evaluations. Subcutaneous growth of cell line
was measured with a digital vernier caliper (Mitutoyo, Japan) and
tumor volume was converted to cell number. When the number of
estimated cells reached 1×109
or 2×109
cells, mice were grouped
as stage II and IV respectively. after tumors became palpable, they
were measured daily. As seen in Figure 1a, after ten days, they
reached stage II. after 1 week they reached stage IV. At this time
point metastasis was evaluated using H&E staining of various tis-
sues. As shown in figure 1b in stage-II none of the resected tissues
showed traces of metastasis where as in stage-IV all the tissues
showed cancer cell growths.
5.2. Stage II CAFs Show Higher Proliferation Capacities
Isolated fibroblasts were evaluated for their surface marker expres-
sion (figure 1c). CD29, CD90, CD105, FAP-1, HLA-DR, CD45,
CD11b and SCA-1 comparison showed that stage-II and -IV CAFs
have similar marker expression. However, the expression of HLA-
DR was higher in stage IV CAFs (P<0.05) (figure 1d). Additional-
ly, immune staining of CAFS in culture showed FAP-1 expression
in stage-II and -IV CAFs (figure 2a). these results confirm that the
isolation technique was able to efficiently isolate carcinoma asso-
ciated fibroblasts from two different stages.
Growth curve of isolated CAFs was evaluated for 13 consecutive
days. Each day, proliferation of 5000 seeded cells were evaluat-
ed using MTT assay. The optical density was corrected using a
MTT-cell standard curve. As shown in figure 2b CAFs growth
curve was similar until day 8 from which stage IV CAFs showed
lower proliferation capacity. Statistical analysis showed that from
day 11 the growth of CAF-II was significantly higher compared
to CAF-IV. Curve equation evaluations calculated the doubling
time of CAF-II as 2.891 and CAF-IV as 1.607 days. The ability
of CAFs to form fibroblastic colonies was evaluated using CFU-F
assay. figure 2c depicts the number of CFU-F cells obtained at
passage 3. CFU-F assay was performed in three concentrations;
clinicsofoncology.com 3
Volume 4 Issue 1 -2021 Research Article
4. 1000, 500,100. Number of colonies was counted in three repeats
for three separate isolated CAF. Statistical analysis showed signif-
icant difference between the numbers of colonies CAF-II was able
to launch (p-value< 0.001). the number of colonies were lower in
CAFs derived from stage IV.
Figure 1. (a) In vivo tumor growth curve of MBL-6 in Balb/C mice showed 100% engraftment. After 10-12 days, tumors reached 109 cells. These mice were grouped as
stage-II. After 4 weeks of inoculation they reached 2×109 cells which were grouped as stage-IV. (b) Metastasis growth in the liver, spleen, lung and lymph node of stage
II and IV of tumor bearing mice compared to healthy tissues. The H&E staining of tissue sections revealed that mice in stage II had minor metastasis to lymph node and
liver (n=1 in 5) and mice in stage IV showed signs of metastasis in all the tissues evaluated (n=5). (c) Flow cytometry was used for comparison of CAFs’ surface markers.
Expression of CD29, CD90, CD105, FAP-1, HLA-DR, CD45, CD11b and SCA-1 was evaluated and data revealed that expression of HLA-DR was higher in stage 4
CAFs (P-value <0.001).
Figure 2: (a) immunocytochemistry of stage IV CAFs stained with rabbit anti-FAP-1 and secondary FITC conjugated goat anti-rabbit IgG. (b) growth curve comparison
of stage II and stage IV CAFs demonstrated higher proliferation capacities of CAF-II cells. (c) CFU-F assay of isolated CAFs revealed significantly higher proliferation
potential in CAF-II cells. Stimulation index of splenocytes in co-culture with (d)CAF-IV (e) MBL-6 and (f) CAF-II cells and their respective conditioned media (CAF2c,
CAF4c and MBL-6c) was performed in four ratios. Coculture with stage-II CAFS (f) significantly augmented splenocyte proliferation in a cell contact dose dependent
manner. The same pattern was observed with stage-IV CAFs however the increase in splenocyte proliferation was significantly higher in stage-II cultures (P value <.000).
Coculture with MBL-6 or its CM suppressed splenocyte proliferation which was greater in the highest ratio (P value >.05 for in group comparisons and CAF-IV, p value
< 0.00 with CAF-II).
clinicsofoncology.com 4
Volume 4 Issue 1 -2021 Research Article
5. 5.3. Stage II and IV CAFs Utilize Different Mechanisms of Im-
mune Suppression
MTT assay was used to measure the proliferation of splenocytes
co-cultured with Triplicates of mitomycin-inactivated CAFs in cell
contact or conditioned media at four ratios. Results showed that the
presence of stage-II CAFs (figure 2f) induced the proliferation of
splenocytes in a cell contact dose dependent manner. On the other
hand, CAF-II secretome had inhibitory properties. The greatest SI
was seen in co-culture of 1:10 ratio of CAFs-II (P value=.000) and
the SI decreased with lowering ratio. What causes the inhibitory
properties of CAF-II secretome? Inhibitory cytokines include IL-
10 and TGF-β. Evaluation of cytokines revealed that CAF-II pro-
duced= copious amounts of TGF-β but negligible levels of IL-10
(figure 3a). However, they were able to induce IL-10 in activated
splenocytes. Therefore, the inhibition of splenocyte proliferation
were due to TGF-β and possibly regulatory T cell differentiation
indicated by IL-10 production. the same result was not observed
in co-cultures with CAF-IV and MBL-6 which suppressed sple-
nocyte proliferation through cellular contact and their secretome.
CAF-IV and MBL-6 did not produce IL-10 or TGF-β but were
able to induce IL-10 in activated splenocytes which efficiently
halts splenocyte proliferation.
Results indicated that IL10 production was limited to splenocytes
and either activated with PHA or not stimulated, the production
of IL10 by splenocytes alone was significantly lower than other
cocultures. CAFs and MBL-6 cell line did not produce IL10 even
under PHA stimulation (figure 3a). Therefore, in cocultures it can
be assumed that the IL10 is of splenocyte origin. Spontaneous pro-
duction of TGF-β1 was seen in activated and not activated sple-
nocytes, however not significantly different. Additionally, Produc-
tion of TGF-β1 in other cocultures was not statistically significant
compared to splenocytes. However, production of TGF-β1 was
highest in stage 2 CAFs. In this case TGF-β1 was produced main-
ly by CAF-II cells. Therefore, its expression in splenocytes needs
further investigation.
5.4. Inflammatory Status of CAFs are Similar in Both Stages
but Different Suppression Mechanisms are Employed
IL-17 is an inflammatory cytokine with both pro- and anti-tumor
functions. Spontaneous production of Splenocytes with/out PHA
was not significant figure 3a and resembled that of media and
CAFs. Therefore, it can be assumed that IL-17 is only produced
from slpenocytes. in the presence of CAFs, splenocytes produced
copious amounts of IL17 (figure 3a). Production of IL17 was in-
duced only in the presence of PHA and in cellular contact with
CAFs (P value < .000). Both stages of CAFs were able to induce
IL17 (P value >.05). The importance of cellular contact for induc-
tion of IL17 indicates involvement of CAFs’ surface molecules
and secretory factors are not able to induce IL17 alone. cell line or
secretome of CAFs did not affect the production of IL17 compara-
ble to spontaneous production of splenocytes.
Figure 3: (a) cytokine pattern of CAFs, MBL-6 and their corresponding cocultures (CC) or with conditioned media (CM), with/out PHA (P) stimulation of splenocytes
(SPL) in vitro were evaluated with ELISA. Similar patterns of IL-10, IL-17 and PGE2 production was observed however the expression of TGF-β1 was significantly
higher in stage 2 CAFs (P<.000). (b) study design included (step 1) initial stage establishment and (step 2) CAF isolation. Afterwards (step 3) CAFs were characterized
using surface marker expression, growth curve and CFU-F assay. in step 4 we evaluated the immunomodulatory properties using splenocyte proliferation and cytokine
profiling of CAFs and in coculture with splenocytes. Step 5, Inflammatory enzymes and mediators were evaluated using real-time qPCR.
Gene expression and ELISA revealed that Both stage 2 and stage
4 CAFs express COX-2 and produce PGE2 and splenocytes, ei-
ther activated or not, and the cell line do not produce any PGE2
(P>0.05). in co cultures of CAFs and splenocytes the production
of PGE2 was significantly higher compared to CM cultures, which
indicates cellular interactions are involved (Figure 4a). The origin
of this PGE2 is not clear however it can be an indication of in-
creased inflammatory milieu in the tumor microenvironment.
clinicsofoncology.com 5
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6. Nonspecific production of NO was seen in splenocytes, with/
out PHA stimulation (figure 4). However, production of NO was
augmented when CAF-IV or cell line or their CM was present.
As shown in figure 4b, MBL-6 had no production of NO, but in
cellular contact with splenocytes, increased amount of NO was
observed. Highest amount of NO was observed from CAF-IV
cultures (P value = .009) and in coculture with splenocytes, the
amount of NO was not significantly altered (P value > .05). Inter-
estingly, significantly low amount of NO was seen in CAF-II and
their cocultures with splenocytes (P value < .001). gene expression
analysis showed that the level of IDO mRNA was higher in stage
2 CAFs by a mean factor of 182.9 (P< .000) and iNOS was signifi-
cantly higher in stage 4 CAFs by a mean factor of 0.490 (P<.000).
The expression of matrix metalloproteinase 2 and 9 were analyzed
by qPCR. The relative mRNA expression, heatmap comparison
and volcano plots are shown in figure 4 c-e. The relative expres-
sions and heatmap demonstration were analyzed using REST soft-
ware and R studio respectively. Real-time PCR analysis revealed
that the expression of MMP2 and MMP9 was similar in both CAFs
(P>.05). however not significant but MMP2 was higher in CAF-II
and MMP9 was higher in CAF-IV.
Figure 4: a production of PGE2 in CAFs, MBL-6 and their corresponding cocultures with splenocytes in vitro was evaluated with ELISA. b Nitric oxide production was
evaluated using Griess reagent. Data revealed that highest amount of NO was produced by stage 4 CAFs (P<.000) and stage 2 CAFs produced very low amounts of NO.
With no production of NO itself, MBL-6 was able to induce NO in splenocytes with higher production in cell-cell contact. relative gene expression analysis (c), heatmap
comparison (d) and volcano plot (d) of MMP2, MMP9, COX-2, IDO, and iNOS expression. mRNAs were extracted from CAFs in passage 2. cDNA was synthesized and
Real-time PCR was used to compare gene expressions. CAF-IV was used as Control and CAF-II as the Target in REST analysis. Significant increase in IDO expression
was observed in CAF-II and iNOS expression was significantly higher in CAF-IV. (***: P<.000)
6. Discussion
Cellular communications can be done through cell to cell or
through molecule to molecule. The immune system employs both
and to fully clarify the interactions influencing the immune re-
sponse in tumors, numerous molecules must be investigated. Ad-
ditionally, the inflammatory milieu of cancer microenvironment is
more divergent, aiding tumor growth and progression. Therefore,
in this study, we investigated the level of cytokines and inflamma-
tory enzymes in cocultures of carcinoma associated fibroblasts and
splenocytes.
The MBL-6 cell line is isolated from a spontaneous tumor in bal-
b/c mice and was able to form stable growing tumors in Balb/C
mice [15]. Using tumor kinetics and metabolic differences, we
were able to correctly predict the time of each stage. This allows
for closer observation of tumor progression and opens the possi-
bility for comparison of different aspects of tumor biology and
treatment in various stages in animal models. In addition, the rela-
tively moderate growth rate in vivo, this line can provide increased
accuracy of experimental settings. Accordingly, a tangible stroma
is provided by this model, which permits tumor microenvironment
assessments. Activated stroma is observed in many cancers [16,
17] and carcinoma associated fibroblasts represent the supporting
stroma.
clinicsofoncology.com 6
Volume 4 Issue 1 -2021 Research Article
7. From both stage II and stage IV tumor tissue, CAFs were isolat-
ed and characterized which showed similar phenotypes. Although
other researches have reported that CAFs do not express HLA-DR
[18, 19], our study showed increased expression of HLA-DR on
stage IV CAFs which can be the result of inflammatory micro-
environment [20, 21]. This variance can be due to evaluations in
different stages in different studies. In the absence of co-stimula-
tion CAFs can induce anergy in activated T cells [22], which was
evident as lower stimulation index in stage IV co-cultures. Similar
study by Peng et. al. showed lower proliferation of T cells in re-
sponse to dendritic cells pre-treated with HCC isolated CAFs [23].
It has been shown that in inflammatory states, fibroblasts are able
to express HLA-DR [24-27] and in some cases antigen presenta-
tion was observed [27, 28]. Therefore, CAFs are not merely mod-
ulators of immune response in the tumor microenvironment, they
can actively alter the response by direct engagement with activated
T cells. In cellular contact, Stage-II CAFs, showed stimulating
properties with significant increase in splenocyte proliferation. As
they express lower levels of HLA-DR, the cytokine milieu may
promote T cell survival [29], albeit, our study revealed that CAF-
II conditioned media suppressed splenocyte proliferation in a dose
dependent manner. studies have shown that interaction of immune
cells and CAFs promote expression of co-inhibitory markers such
as TIM-3, PD-1, CTLA-4 and LAG-3 in proliferating CD4+ and
CD8+ T-cells 18, which indicates Treg differentiation
Cytokines are potent drivers of immune responses and are ex-
pressed not only by immune cells but various cell types are able
to produce cytokines in response to microenvironment stimuli. Al-
though CAFs are not responsible for IL-10 production, they were
able to induce IL-10 production in splenocyte. Looking into the
cause, production of PGE2 in the presence of TGF-β1 increases
the production of IL-10 and inhibits IL-12 [30, 31]. Therefore, the
increased expression of COX-2 in CAFs and TGF-β1 production
could lead to Treg differentiation and IL-10 production in activated
T cells. In addition, nitric oxide also interferes with IL-2 signaling
required for T cell proliferation [32] which is increased in stage-IV
CAFs. Therefore, CAF-IV inhibited immune cell proliferation via
multiple routes and alter their differentiation.
IL-17 is known for its pro-tumorigenic and angiogenic properties
[33-35]. It is shown that PGE2 promotes IL-17 production 36 and
Treg differentiation via PGE2/COX-2 pathway [37, 38]. Our re-
sults showed that IL-17 is produced in both stages, only in cellular
contact and requires PHA stimulation. Studies have shown that
TH17 differentiation and IL-17 production increases with gastric
cancer stage [39] and is involved in promoting metastasis [40].
Other studies have shown the elevated levels of TGF-β1 in stromal
cells of cancerous tissues [41], however the stage in which the
expression was evaluated was not defined. It is noteworthy that
in cocultures with MBL-6 no significant amount of differentiation
cytokine was produced. Although this cannot be extrapolated to
other cell lines, but it can be presumed that the role of CAFs in
immunosuppression is superior to cancer cells.
IDO is a wound healing enzyme [42, 43] and is induced by IFN-γ
[44], IL-12 and IL-18 [45] and PGE2 which requires additional
signaling from other cytokines such as TNF-α [46]. IDO plays an
important role in the immune response by catalyzing the oxidative
degradation of l-tryptophan that contributes to immune-suppres-
sion [47]. In response to TGF-β1, pDCs express IDO and show
tolergenic properties and has a tonic, nonenzymic function that
contributes to TGF-b-driven tolerance [48]. However, IDO is sen-
sitive to NO [49-51], therefore as seen in our results and other
studies, with advancing stage in which iNOS is increasingly ex-
pressed, IDO declines. In a study by Jian-Pei Feng, a cross-regu-
latory pathway was shown between the IDO and NO pathways, in
which the inhibition of IDO stimulated the iNOS pathway and vice
versa [52]. In this study it was shown that only when both path-
ways were inhibited tolerance was abrogated. Thus, both pathways
are involved in tolerance. Hence when considering immune activa-
tion in cancer therapies, numerous pathways must be considered.
In conclusion, the main difference between the CAFs, was the ex-
pression of TGF-β1 and IL-10. TGF-β1 and PGE2 induce IL-10
production [53-55], Additionally, IL-10 inhibits IL-17 and PGE2
production [56-59]. Therefore, increased expression of IL-10 in
stage-IV could be an attempt to control inflammation. There are
differences between TGF-β1 and IL-10 suppression. TGF-β1 sup-
pression is at translational level and consist of 12-16 hours and IL-
10 suppression increases mRNA degradation and take in 3 hours
[60]. Considering these differences, stage-II cancer can be con-
sidered a transforming stage with acute inflammation profile and
stage-IV has an immunosuppressive chronic inflammation profile
(Figure 5). There are many queries regarding the tumor microenvi-
ronment and additional studies are required. In view of our results,
when considering an immunotherapy regiment, it is advisable to
investigate the microenvironment and whether it is compliant to
the type of intended treatment, thereby increasing the probability
of successful therapy.
clinicsofoncology.com 7
Volume 4 Issue 1 -2021 Research Article
8. Figure 5: comparison of different immune suppression mechanisms in stage-II and sage-IV microenvironments. In stage II CAFs produce IDO and COX-2 which in
conjugation with TGF-β induce IL-17 and VEGF and consequently tumor growth and progression. In stage IV, CAFs produce NO and COX-2 leading to Treg cell
differentiation, MMP9 production and EMT.
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