Carcinoma-associated Fibroblasts Research Articles

25 Different subsets of carcinoma-associated fibroblasts promote oral carcinoma cell invasion by distinct mechanisms

Bidirectional tumor–stroma interactions have tumor-promoting effects, and heterogeneity of carcinoma-associated fibroblasts (CAFs) has been showed, but its functional relevance for tumor development and invasion is still poorly understood. This study identified two CAF subtypes in oral squamous cell carcinoma (OSCC) based on transcriptome analysis. Approximately 40% of significantly up-regulated genes when CAFs were compared to normal fibroblasts (NFs) were transforming growth factor-β (TGF-β) targets, highlighting the crucial role of this factor in CAF activation. One subgroup had a transcriptome closer to NFs and thus was termed CAF-N; the other showed a more divergent gene expression pattern and was termed CAF-D. Both CAF subgroups supported significantly higher tumor take in NOD/SCID Ilγ2 deficient mice and deeper invasion of malignant keratinocytes than NFs or dysplasia-associated fibroblasts (DAF), but CAF-Ns supported a significantly deeper invasion and increased tumor formation than CAF-Ds. CAF-N were characterized by a secretome closer to NFs, included a significantly higher proportion of motile fibroblasts, and showed enhanced migration in response to TGF-β1. Migratory enhanced NFs (by TGF-β1-pretreated) induced invasive properties in transformed oral keratinocytes, while inhibition of CAF-N migration impaired invasion of malignant oral keratinocytes, showing the essential role for fibroblast motility in oral carcinoma invasion supported by CAF-Ns. In contrast, CAF-Ds synthesized significantly higher levels of TGF-β1, but contained only a low percentage of motile fibroblasts and did not respond to TGF-β1. CAF-Ds and TGF-β1 induced enhanced migration of malignant keratinocytes and increased expression of EMT markers. Taken together, these data shows that different subsets of CAFs promote tumor formation and carcinoma cell invasion by different mechanisms.

P10 Interactions of fibroblasts derived from irradiated subcutaneous connective tissue with HNSCC

Cancer cells show a variety of interactions with cells of the surrounding connective tissue, mainly consisting of fibroblasts. Human skin-fibroblasts which are co-cultivated with tumor cells have the ability to convert into carcinoma-associated fibroblasts (CAF’s) which are known to support tumor growth and invasiveness. The aim of the current study was to determine the impact of irradiation on fibroblast-tumor interaction. Primary human fibroblasts of subcutaneous connective tissue of pre-irradiated or non-irradiated skin were co-cultivated with the HNSCC cell line FaDu for 3 days. MTT assay, annexin V-propidium iodide test and ELISA were performed to analyze tumor viability, apoptosis and secretion of interleukin (IL)-6 and IL-8. Markers for epithelial-mesenchymal transition (EMT) were assessed by western blot. Co-culture with pre-irradiated fibroblasts showed a significant decrease in tumor viability and an increase in apoptosis. Levels of IL-8 significantly decreased in irradiated compared to non-irradiated fibroblasts. After irradiation, the cells did not promote EMT anymore. Previous radiation influences fundamental properties of the complex tumor and stroma orchestration. These interactions have to be evaluated in detail in order to further illuminate possible options of intervention.

28 Expression of integrin [formula omitted]-11 by carcinoma associated fibroblasts modulates oral squamous cell carcinoma behavior

Background and Aim We have previously shown that integrin α -11 is up-regulated in cancer associated fibroblasts (CAF) in oral squamous cell carcinoma (OSCC). The aim of this study was to investigate its role on behavior of OSCC cells. Materials and methods Primary fibroblasts from OSCC patients with different levels of integrin α -11 expression were isolated and propagated in culture after informed consent. Cultured CAFs were transfected with retroviral vector constructs for down regulation of integrin α -11 or with empty vectors (as control), and then co-injected (100,000 CAFs) with luciferase transfected dysplastic oral keratinocytes (DOK cell line, 10,000 DOKluc per injection) in NOD/SCID-IL γ 2 deficient mice (n = 7 for each type of CAFs). Results The vectors targeted towards ITGA11 efficiently (more than 50%) suppressed gene expression in CAFs (CAF α 11−) at both protein and mRNA levels, when compared with empty vector transfected CAFs (CAF α 11+). CAF α 11− had similar morphology and growth potential as CAF α 11+. No tumors were formed when either of CAFs were injected in NOD/SCID-IL γ 2 deficient mice. Bigger tumors were formed when DOKluc were co-injected with empty vector CAFs (CAF α 11+) than with CAF α 11− (28.46 ± 6.46 mm3 versus 9.56 ± 3.31 mm3). The manual measurements of tumor volume were confirmed by the bioluminescence readings that showed an average reduction of 26% in the bioluminescence of tumors formed by DOKluc when co-injected with CAF α 11−. Conclusions The results of this study suggest that the expression of integrin α -11 by CAFs is able to modulate the behavior of OSCC cells.

9 Matrix remodeling in head and neck squamous cell carcinomais

Extracellular matrix (ECM) remodeling is an inherent characteristic of invasive head and neck squamous cell carcinoma (HNSCC). We investigated the regulation of gene expression, synthesis and activation of ECM components and matrix remodeling enzymes in a co-culture model of head and neck squamous cell carcinoma (HNSCC) and in HNSCC tissue. Periodontal ligament fibroblasts (PDL) and SCC-25 HNSCC cells were co-cultured for seven days, followed by mRNA and protein expression analysis of ECM components, ECM receptors, matrix metalloproteinases (MMPs) and proteinase inhibitors. The activity of MMPs was analyzed using gelatinase zymography. MMP-9, fibronectin and “antiadhesion proteins” were investigated by immunohistochemistry in HNSCC tissue. In co-cultured fibroblasts, in addition to fibronectin (FN), the “antiadhesion proteins” tenascin C (TSC), thrombospondin 1 (TSP1) and latent TGF-beta binding protein 2 (LTBP2) were upregulated. In lower levels: FN, TSC and TSP1 were also produced by the tumor cells. In co-cultured fibroblasts, alpha 1 and 5 and beta 1 integrins, in co-cultured tumor cells alpha 3 and 5 integrins were upregulated. MMP-1 was upregulated in both co-cultured tumor cells and fibroblasts, MMP-2 in co-cultured fibroblasts, and MMP-9 in co-cultured tumor cells. MMP inhibitors were mainly produced in fibroblasts. The activation of MMP-2 required the co-culture with tumor cells. MMP-9 was exclusively produced in tumor cells. In sections of formalin-fixed paraffin embedded HNSCC tissue, FN and LTBP2 were detected in fibroblastic stroma surrounding tumor cell nests; TSP1 was detected both in tumor cell nests and in the surrounding fibroblastic stroma. Extended MMP-9 immunohistochemical staining was found in 27 of 280 HNSCC tissue samples, in a subpopulation of cells in tumor cell nests. Fibroblast – tumor cell communication is a major pathway of matrix remodeling in HNSCC tumor tissue. Carcinoma-associated fibroblasts produce new matrix elements as TSP1, TSC and LTBP2, both tumor cells and fibroblasts upregulate integrin receptors, and they also share tasks in synthesis and activation of matrix remodeling enzymes. Intensive matrix re-organization takes place at the borders of tumor cell nests.

Analysis of the effects of T3, T4 and tetrac on mesenchymal stem cell biology

Mesenchymal stem cells (MSCs) are actively recruited into growing tumour stroma, where they differentiate to carcinoma-associated fibroblasts (CAFs) and form the tumour's fibrovascular network. Thyroid hormones T3 and T4 play a critical role in tumour stroma formation mediated through non-genomic mechanisms via integrin αvβ3. To improve our understanding of thyroid hormone action in tumour stroma formation, we examined the effects of T3, T4 and the integrin-specific inhibitor tetrac on MSC biology.

Int6 reduction activates stromal fibroblasts to enhance transforming activity in breast epithelial cells.

BackgroundThe INT6 gene was first discovered as a site of integration in mouse mammary tumors by the mouse mammary tumor virus; however, INT6’s role in the development of human breast cancer remains largely unknown. By gene silencing, we have previously shown that repressing INT6 promotes transforming activity in untransformed human mammary epithelial cells. In the present study, guided by microarray data of human tumors, we have discovered a role of Int6 in stromal fibroblasts.ResultsWe searched microarray databases of human tumors to assess Int6’s role in breast cancer. While INT6 expression levels, as expected, were lower in breast tumors than in adjacent normal breast tissue samples, INT6 expression levels were also substantially lower in tumor stroma. By immunohistochemistry, we determined that the low levels of INT6 mRNA observed in the microarray databases most likely occurs in stromal fibroblasts, because far fewer fibroblasts in the tumor tissue showed detectable levels of the Int6 protein. To directly investigate the effects of Int6 repression on fibroblasts, we silenced INT6 expression in immortalized human mammary fibroblasts (HMFs). When these INT6-repressed HMFs were co-cultured with breast cancer cells, the abilities of the latter to form colonies in soft agar and to invade were enhanced. We analyzed INT6-repressed HMFs and found an increase in the levels of a key carcinoma-associated fibroblast (CAF) marker, smooth muscle actin. Furthermore, like CAFs, these INT6-repressed HMFs secreted more stromal cell–derived factor 1 (SDF-1), and the addition of an SDF-1 antagonist attenuated the INT6-repressed HMFs’ ability to enhance soft agar colony formation when co-cultured with cancer cells. These INT6-repressed HMFs also expressed high levels of mesenchymal markers such as vimentin and N-cadherin. Intriguingly, when mesenchymal stem cells (MSCs) were induced to form CAFs, Int6 levels were reduced.ConclusionThese data suggest that besides enhancing transforming activity in epithelial cells, INT6 repression can also induce fibroblasts, and possibly MSCs as well, via mesenchymal-mesenchymal transitions to promote the formation of CAFs, leading to a proinvasive microenvironment for tumorigenesis.

Supportive and rejective functions of tumor stroma on tumor cell growth, survival, and invasivity: the cancer evolution.

Tumors are complex organs composed of reciprocally interacting cell types including cancer-initiating cells, more or less differentiated cancer cells, extracellular matrix (ECM), and a variety of stromal cells such as endothelial cells, immune cells, pericytes, adipocytes, and fibroblasts (1). The tumor microenvironment (TME) comprises a variety of cell types lying among a network of various ECM fibers merged within the interstitial fluid and gradients of several chemical compounds, which constantly interplay with malignant cells (2). The TME is also energetically linked to cancer cells, which orchestrates functional changes in mitochondria of tumor cells that accompanies resistance to intrinsic apoptotic stimuli and contributes to hypoxia (3). The interaction of tumor resident cells impacts on the composition and texture of the matrix, including its stiffness. Tumor cells interacting with fibroblastic microenviroment might undergo a peculiar phenotypic change, known as epithelial–mesenchymal transition (2, 4, 5), and gain a pseudomesenchymal phenotype. These transdifferentiated cancer cells promote stiffening of their environment, which in turn feeds back in form of loss of tissue architecture or induction of cell invasion (2). Additionally, the ECM components shape the immune properties of dendritic cells (DCs) in the tumor (6), and finally contribute to an anergic state in these cells, favoring tumor immune-escape. Benencia et al. in this Research Topic discuss that this “immune paralysis” of cancer-associated DCs can be overcome in an experimental setting by blocking interleukin (IL)-10R while simultaneously activating specific pattern recognition receptors (6). In agreement, also Accolla et al. describes that the establishment of a pro-tumor environment is not the cause but simply the consequence of the tumor strategy to primarily counteract components of the adaptive cellular immunity, particularly T helper lymphocytes (7). Stroma cells within the TME might show diverse polarization states, which is well-known in case of immune cells (8), but is a novel assumption in case of carcinoma-associated fibroblasts (CAFs) (1). Within the TME immune cells might destroy cancer cells, or they may promote tumor growth and dissemination, which is largely due to different polarization of them (8). Also, the CAFs have more populations, including the one, which is assigned with pro-tumorigenic effects as stimulating tumor growth and progression, and also the one, which might have tumor-inhibitory effects (1). The novel point of view is the acknowledgment of the plasticity and polarized phenotype both in the tumor cell populations and also in the microenvironment (1, 8). The polarized phenotypes and plasticity in both tumor cells and stroma enables a dynamic array of inhibitory and stimulatory signals in the tumor organ, which represents a new challenge for investigation of interactive processes instead of assuming cells with predefined roles. This is the basis of a continuous evolution of the tumor tissue. According to contributions to this research topic (6, 7) the dynamism of TME might be influenced. The optimal initiation of the adaptive immune response dictated by adequate antigen availability, as evidenced by several research groups and reviewed by Accolla et al. is sufficient to reorient the TME from a pro-tumor to an anti-tumor one (7). Similarly to immune cells, also fibroblasts might contribute to pro and to anti-TME. In an original article of Kaler et al. (9), authors report that normal intestinal fibroblasts activate STAT1 signaling in colon cancer cells and, in contrast to CAFs, inhibit growth of tumor cells (9). Interestingly, the proinflammatory cytokine TNFα could release this inhibition. Detailed analysis of not only the potent mediators but also the signaling components is required in the stromal component of the tumor tissue in addition to tumor cells. Investigation of the signaling in stroma independently from tumor cells is a novel challenge, which is elegantly addressed by Wallace et al. They utilized two model systems: one where protein kinase C β (PKCβ) was deleted in both the epithelial and stromal compartments, and second, where PKCβ was deleted only in the stromal compartment (10). Mice globally lacking PKCβ were living longer and developed smaller tumors, and when PKCβ was exclusively lacking in the stroma, breast cancer tumor cells formed smaller tumors as well, with diminished collagen deposition. Interestingly, the stroma PKCβ-loss had no effect on tumor cell proliferation, vascularization, or macrophage infiltration (10). In summary, this research topic highlights the importance of the investigation and targeting of TME dynamism, and of understanding the stroma cell polarization.

Molecular mechanism underlying the tumor-promoting functions of carcinoma-associated fibroblasts.

Tumor microenvironment is composed of all the untransformed elements in the vicinity of tumor, mainly including a large number of stromal cells and extracellular matrix proteins, which play an active role in most solid tumor initiation and progression. Carcinoma-associated fibroblasts (CAFs), one of the most common stromal cell types in the tumor microenvironment, have been demonstrated to be involved in tumor growth, invasion, and metastasis. Therefore, they are becoming a promising target for anti-cancer therapies. In this review, we firstly summarize the current understandings of CAFs' molecular biology, including the heterogeneous cellular origins and molecular markers, and then, we focus on reviewing their various tumor-promoting phenotypes involved in complex mechanisms, which can be summarized to the CAF-conveyed paracrine signals in tumor cells, cancer stem cells, and metastasis-initiating cancer cells, as well as the CAF-enhanced extrinsic tumor-promoting processes including angiogenesis, extracellular matrix remodeling, and tumor-related inflammation; finally, we describe the available directions of CAF-based target therapy and suggest research areas which need to be further explored so as to deepen the understanding of tumor evolution and provide new therapeutic targets for cancer treatment.

Mesenchymal stem cells promote epithelial to mesenchymal transition and metastasis in gastric cancer though paracrine cues and close physical contact.

Mesenchymal stem cells (MSCs) have been shown to integrate into the tumor stroma; however, the precise mechanisms of this process are still elusive. In this study, the EMT phenotype and the enhanced metastatic ability of tumor cells were observed using transwell and trans-endothelial migration assays, respectively, as well as by using electron and laser confocal microscopy. Critical genes were screened and validated using gene arrays and clinical samples, and the changes at the protein level were examined both in vitro and in vivo. Cancer cells acquired an "activated" carcinoma-associated fibroblasts (CAFs) phenotype after being in close contact with MSCs and enhancing tumor metastasis and growth in vivo. Paracrine signals also induced EMT and promoted transwell and trans-endothelial migration, the changes were dependent on β-catenin, MMP-16, snail and twist. Notably, the higher expression levels of β-catenin and MMP-16 were correlated with tumor invasion and distant organ and lymph node metastases in intestinal type gastric cancer. MSCs within the tumor niche significantly facilitated tumor growth and metastasis by paracrine cues and close physical connection. This occurred partly through snail, twist and its downstream targets, specifically β-catenin/MMP-16.

Abstract B39: Stromal miRNAs as mediators of mitogen activated protein kinase-induced estrogen repression in breast cancer

Abstract Over the past decade an accumulating body of evidence has demonstrated the pivotal role of stromal cells in promoting breast cancer progression. In breast cancer, the stroma is mainly comprised of carcinoma-associated fibroblasts (CAFs). CAFs secrete various growth factors and cytokines that are known to activate MAPK pathway. We have previously established that hyperactivation of MAPK in breast cancer cells leads to repression of estrogen receptor (ER) expression. Aberrant mitogenic signaling also affects expression of microRNAs (miRNAs). We have recently identified a miRNA signature associated with hyperactivation of MAPK signaling (hMAPK-miRNAs). Most ER- breast cancers and a population of ER+ breast cancers are identified by this signature, and these hMAPK-miRNA tumors exhibit significantly decreased disease-free survival and overall survival compared to tumors that are not hMAPK-miRNA. Some of these hMAPK-miRNAs have been established as targeting ER. To further investigate the contribution of CAFs in activation of MAPK signaling in cancer cells, we isolated CAF cell cultures from three different primary breast tumors: CAF19 (from a luminal A tumor), CAF21 (from a Her-2 like tumor) and CAF23 (from a basal like tumor) and tumor cell cultures (DTs) from 5 different primary breast tumors. There is differential expression of hMAPK miRNAs between these CAFs and DTs, including those that target ER. Thus, we propose that interactions between cancer and stromal cells lead to activation of MAPK signaling in the cancer cell and aberrant miRNA expression within the TME that contribute to the biology of ER-negative tumors. To further investigate the connection between hMAPK-miRNAs and stroma, we analysed TCGA breast cancer datasets and found that hMAPK-miRNAs are significantly associated with tumors that possess high stromal scores. Co-culture of CAFs with ER+ breast cancer cells, as well as treatment of ER+ breast cancer cells with conditioned media (CM) from CAFs, induces both MAPK activation and altered expression of miRNAs, specifically MAPK miRNAs within the breast cancer cells. Treatment of ER+ breast cancer cells with CM from CAFs represses both ER protein and mRNA expression, and importantly, this ER repression is specific to CAFs isolated from ER-negative breast tumors (CAF21 and CAF23) compared to the CAFs obtained from ER-positive tumors (CAF19). Additionally, CM from CAF21 and CAF23 caused repression of an ER 3'UTR reporter construct transfected into ER+ breast cancer cells, indicating a role for miRNAs either secreted into the CM or induced in the cancer cell in repressing ER. To investigate a role for hMAPK-miRNAs secreted by CAFs in mediating ER repression, nanostring analysis of miRNAs isolated from ER+ breast cancer cells treated with CAF CM was performed and differentially expressed miRNAs between CM from CAFs obtained from ER-positive (DT19) vs ER-negative tumors (DT21 and DT23) were identified. qPCR analysis of conditioned media also confirmed the presence of hMAPK-miRNAs in CAF CM. Collectively, these data indicate a role for CAFs in regulating hMAPK-miRNAs, and thus in establishing an ER-negative breast cancer phenotype, and importantly, differences between CAFs from ER-negative as compared to ER-positive tumors in this role. They further suggest that hMAPK-miRNAs secreted from CAFs participate in the induction of the ER-negative phenotype induced by MAPK. Citation Format: Sanket H. Shah, Phil Miller, Emilio Issa, Katherine Drews-Elger, Dorraya El-Ashry. Stromal miRNAs as mediators of mitogen activated protein kinase-induced estrogen repression in breast cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B39. doi:10.1158/1538-7445.CHTME14-B39

Abstract B14: Activin A regulates cell interactions in the microenvironment of oral squamous cell carcinomas

Abstract Although the molecular mechanisms by which carcinoma-associated fibroblasts (CAFs) influence oral squamous cell carcinomas (OSCC) remain largely unknown, previous studies demonstrated that presence of CAFs in the stroma of OSCCs is an important risk factor of patient's shortened survival. Here we showed that activin A is produced in higher levels by CAFs compared to normal oral fibroblasts, and that activin A released by CAFs significantly increases OSCC cell proliferation and xenograft tumor volume, whereas down-regulation of activin A decreases OSCC proliferation. Furthermore, activin A reduced OSCC cell death even when challenged by staurosporine, and promoted migration, invasion and the expression of epithelial-mesenchymal transition (EMT) markers (decreases E-cadherin and increases vimentin and N-cadherin) of OSCC cells. Immunohistochemical analysis of 115 OSCC samples revealed that increased activin A expression is significantly correlated with presence of regional metastasis (N stage, p=0.034), poorly differentiated tumors (p=0.013) and shown to be predictive of a shortened disease-free survival (HR: 1.74, 95% CI: 1.39-2.97, p=0.016). Importantly, immunohistochemical expression of activin A was significantly associated with presence of occult lymph node metastasis (p=0.006), which has an important impact on clinical management and prognostication of patients with OSCCs. In conclusion, our data demonstrate that activin A is an important molecule for OSCCs, and its pleiotropic effects on proliferation and invasion are important on control of the aggressiveness of OSCCs. Citation Format: Ricardo D. Coletta, Andreia Bufalino, Lays M. Sobral, Priscila C. Rodrigues, Edgard Graner, Luis P. Kowalski, Tuula Salo. Activin A regulates cell interactions in the microenvironment of oral squamous cell carcinomas. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B14. doi:10.1158/1538-7445.CHTME14-B14

Abstract A46: The role of circulating fibroblast precursors in promoting metastatic sarcoma

Abstract Soft tissue sarcomas are malignant tumors that develop in connective tissue. Treatment with standard chemotherapeutic agents is often minimally effective and approximately half of all patients with soft tissue sarcoma experience disease recurrence and die of metastatic cancer. Therefore, the purpose of this study is to gain a better understanding of the soft tissue sarcoma metastatic environment in order to lead to the development of more effective therapies. Studies have shown that the tumor microenvironment plays a pivotal role in promoting tumor metastasis and one of the most prominent cell types of the tumor microenvironment is the carcinoma associated fibroblast (CAF). Our studies, based upon a murine single hematopoietic stem cell transplantation model have demonstrated that hematopoietic stem cells (HSCs) are a novel source for CAFs and their circulating fibroblast precursors (CFPs). Our research has also shown that murine CFPs are present in the peripheral blood, increase with tumor burden, and contribute to tumor growth. In vitro studies have shown that HSC-derived CFPs enhance tumor cell proliferation, migration, and invasion, processes critical to tumor metastasis in vivo. The role of human CFPs in metastasis of soft tissue sarcoma, however, has yet to be determined. It is our hypothesis that HSC derived CFPs promote sarcoma cell proliferation, migration, and invasion. In order to determine the role of CFPs in sarcoma metastasis, we established a method for culturing fibroblasts from peripheral blood obtained from patients diagnosed with metastatic sarcoma. These cultured cells were then characterized by immunohistochemistry to profile fibroblast and hematopoietic markers. In order to address the functional role of CFPs during sarcoma metastasis, we examined the ability of conditioned media from cultured CFPs from patients with metastatic sarcoma to influence the proliferation, migration, and invasion of HT-1080 cells, a human sarcoma line. Our immunohistochemical findings demonstrate that fibroblasts can be isolated from the circulation of patients with metastatic sarcoma and results from our functional assays indicate that cultured CFPs from patients with metastatic sarcoma promote the proliferation, migration, and invasion of HT-1080 cells. Given that proliferation, migration, and invasion of tumor cells are essential steps in the metastatic cascade, these findings suggest that CFPs promote tumor progression. Ongoing studies involve analysis of CFPs at various time points based upon chemotherapeutic treatment in order to evaluate differences in numbers of CFPs, marker expression, growth factor production, and ability to promote metastasis. Long term, these studies may identify novel biomarkers for disease progression as well as treatment efficacy, potentially leading to the development of more effective therapies for soft tissue sarcomas. Citation Format: Dayvia A. Laws, Andrew S. Kraft, Lee R. Leddy, Amanda C. LaRue. The role of circulating fibroblast precursors in promoting metastatic sarcoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A46. doi:10.1158/1538-7445.CHTME14-A46

Stromal Carcinoma Associated Fibroblasts Promote Drug Resistance of Human Pancreatic Cancer Cells by Modulation of ROS via CXCR4/CXCL12 Signaling

Pancreatic cancer is one of the most aggressive malignancies, with a 5-year overall survival of less than 5%. Tumor drug resistance to conventional chemotherapy, such as Gemcitabine, is often a significant contributor to poor overall survival. One of the common mechanisms of Gemcitabine resistance is activation of cell signaling via increased phosphorylation of mitogen-Activated kinase (MAP) kinases, leading to increased tumor survival and reduced sensitivity to chemotherapeutic agents. A growing body of evidence suggests that the CXCL12/CXCR4 signal transduction axis in the tumor microenvironment is an important mediator of tumor migration, growth, and drug resistance. We hypothesized that stromal cells such as carcinoma-associated fibroblasts (CAFs), an important cellular component of the tumor microenvironment (TME), play a contributory role in the growth, invasiveness, and drug response of pancreatic cancer cells (PCCs)

Cancer-associated fibroblasts-induced proliferation of breast cancer cell depends on WNT10B up-regulation

Objective To study how fibroblasts can influence breast cancer cells MCF-7 proliferation and the expression of WNT10B.Methods Transwell co-culture of 1.0 × 105 fibroblasts and 1.0 × 105MCF-7 was done for 2 days.Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of WNT10B in MCF-7 cells.Three-dimensional co-culture 5.0 × 103 fibroblasts and 2.0 ×103 MCF-7 cells were subjected to three-dimensional co-culture for 12 days for assaying the clone formation rate (CFE) of MCF-7 cells.Results Cancer-associated fibroblasts (CAFs) increased significantly the mRNA expression of WNT10B as compared with normal mammary fibroblasts (NBFs) (3.864 2 ± 0.105 2 vs.2.170 6 ±0.064 6,P ＜0.05).CAFs increased significantly the CFE of MCF-7 cells as compared with the controls [(37.83 ± 2.02) ％ vs.(6.67 ± 1.89) ％,P ＜ 0.05],but NBFs did not [(8.83 ± 2.52) ％ vs.(6.67 ± 1.89) ％,P ＞ 0.05],the neutralizing WNT antibody decreased the CFE of MCF-7 cells in CAFs three-dimensional culture [(11.67 ± 2.56) ％ vs.(37.83 ± 2.02) ％,P ＜ 0.05].Conclusion Our results suggest that WNT10B signaling is critical for CAFs-induced growth of human breast cancer cell lines. Key words: WNT10B; Carcinoma associated fibroblasts; Breast cancer

Reciprocity in breast cancer progression.

Reciprocity in breast cancer progression.

Colorectal Cancer-Associated Fibroblasts are Genotypically Distinct.

Cells in the stromal microenvironment facilitate colorectal cancer (CRC) progression and "co-evolve" with the epithelial cancer cells. Genetic and epigenetic differences between normal colorectal mucosa fibroblasts (NF) and carcinoma-associated fibroblasts (CAF) are not known. The aim of this study is to identify differentially expressed genes and promoter methylation between NF and CAF in human CRC. RNA and DNA were extracted from cultured NF and CAF from CRC resections. Genome-wide gene expression and methylation analyses were performed using the Illumina Human HT-12 v4.0 Expression and Illumina Human Methylation 27 BeadChips. Gene expression values between NF and CAF were compared and correlated with methylation patterns. Data was analyzed using Partek Genomics Suite using one-way ANOVA and p<0.05 as significant. Ingenuity iReport™ was performed to identify potential differences in biological functions and pathways between the NF and CAF. Paired methylation and gene expression analyses from 11 NF and 10 CAF colorectal samples are reported. Unsupervised analysis of differentially expressed genes using iReport™ identified "Top Diseases" as "Cancer" and "Colorectal Cancer". Previous genome wide studies have focused on the cancer cells. We have identified differentially expressed genes and differentially methylated promoter regions that are CAF-specific in CRC.

Reciprocal Interaction between Carcinoma-Associated Fibroblasts and Squamous Carcinoma Cells through Interleukin-1α Induces Cancer Progression

Crosstalk between cancer cells and carcinoma-associated fibroblasts (CAFs) has earned recognition as an interaction that plays a pivotal role in carcinogenesis. Thus, we attempted to clarify whether increase in the level of CAFs promotes cancer progression by proportionally enhancing the interaction between cancer cells and CAFs. We first analyzed clinical correlation between the levels of fibroblasts and cancer progression and found that the level of CAFs made a noticeable difference on the prognosis of patients with oral squamous cell carcinoma (OSCC). In vivo animal study also demonstrated that tumor volume depended on the dose of CAFs that was co-injected with OSCC cells. The same tendency was observed in an in vitro study. We also found that interleukin-1α (IL-1α) secreted from OSCC cells had dual effects on CAFs: IL-1α not only promoted the proliferation of CAFs but also upregulated the secretion of cytokines in CAFs such as CCL7, CXCL1, and IL-8. The induction activity of cytokine secretion by IL-1α surpassed that of proliferation in OSCC cells. In summary, we unraveled an important interactive mechanism of carcinogenesis: IL-1α released from carcinoma stimulates the proliferation of CAFs and the simultaneous increase in cytokine secretion from CAFs promotes cancer progression in human OSCC. On the basis of these findings, we propose that the level of CAFs is eligible for being selected as a prognostic factor that will be useful in routine diagnosis. We also propose that blockage of reciprocal interaction between cancer cells and CAFs will provide an insight for developing novel chemotherapeutic strategy.

Elevated CXCL1 expression in breast cancer stroma predicts poor prognosis and is inversely associated with expression of TGF-β signaling proteins.

BackgroundCXCL1 is a chemotactic cytokine shown to regulate breast cancer progression and chemo-resistance. However, the prognostic significance of CXCL1 expression in breast cancer has not been fully characterized. Fibroblasts are important cellular components of the breast tumor microenvironment, and recent studies indicate that this cell type is a potential source of CXCL1 expression in breast tumors. The goal of this study was to further characterize the expression patterns of CXCL1 in breast cancer stroma, determine the prognostic significance of stromal CXCL1 expression, and identify factors affecting stromal CXCL1 expression.MethodsStromal CXCL1 protein expression was analyzed in 54 normal and 83 breast carcinomas by immunohistochemistry staining. RNA expression of CXCL1 in breast cancer stroma was analyzed through data mining in http://www.Oncomine.org. The relationships between CXCL1 expression and prognostic factors were analyzed by univariate analysis. Co-immunofluorescence staining for CXCL1, α-Smooth Muscle Actin (α-SMA) and Fibroblast Specific Protein 1 (FSP1) expression was performed to analyze expression of CXCL1 in fibroblasts. By candidate profiling, the TGF-β signaling pathway was identified as a regulator of CXCL1 expression in fibroblasts. Expression of TGF-β and SMAD gene products were analyzed by immunohistochemistry and data mining analysis. The relationships between stromal CXCL1 and TGF-β signaling components were analyzed by univariate analysis. Carcinoma associated fibroblasts isolated from MMTV-PyVmT mammary tumors were treated with recombinant TGF-β and analyzed for CXCL1 promoter activity by luciferase assay, and protein secretion by ELISA.ResultsElevated CXCL1 expression in breast cancer stroma correlated with tumor grade, disease recurrence and decreased patient survival. By co-immunofluorescence staining, CXCL1 expression overlapped with expression of α-SMA and FSP1 proteins. Expression of stromal CXCL1 protein expression inversely correlated with expression of TGF-β signaling components. Treatment of fibroblasts with TGF-β suppressed CXCL1 secretion and promoter activity.ConclusionsIncreased CXCL1 expression in breast cancer stroma correlates with poor patient prognosis. Furthermore, CXCL1 expression is localized to α-SMA and FSP1 positive fibroblasts, and is negatively regulated by TGF-β signaling. These studies indicate that decreased TGF-β signaling in carcinoma associated fibroblasts enhances CXCL1 expression in fibroblasts, which could contribute to breast cancer progression.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-781) contains supplementary material, which is available to authorized users.

Expression of galectin-1 in carcinoma-associated fibroblasts promotes gastric cancer cell invasion through upregulation of integrin β1.

Increased expression of galectin-1 (Gal-1) in carcinoma-associated fibroblasts (CAFs) has been reported to correlate with progression and prognosis in many cancers. However, rarely have reports sought to determine whether high Gal-1 expression in CAFs in gastric cancer is involved in the tumor process, and the specific mechanism by which it promotes the evolution of gastric cancer is still unknown. In this study, we cultured gastric cancer CAFs, which showed strong expression of Gal-1, and established a co-culture system of CAFs with gastric cancer cells. Specific siRNA and in vitro migration and invasion assays were used to explore the effects of the interaction between Gal-1 expression of CAFs and gastric cancer cells on cell migration and invasion. We found that the overexpression of Gal-1 in CAFs enhanced gastric cancer cell migration and invasion, and these stimulatory effects could be blocked by specific siRNA which reduced the Gal-1 expression level. A set of cancer invasion-associated genes were then chosen to identify the possible mechanism of Gal-1-induced cell invasion. Among these genes, integrin β1 expression in cancer cells was considered to be associated with Gal-1 expression. Pre-blocking of the integrin β1 expression in gastric cancer cells with siRNA could interrupt the invasion-promoting effect of CAFs with high Gal-1 expression. Furthermore, immunohistochemical assay confirmed a positive correlation between Gal-1 and integrin β1 expression. Our results showed that high expression of Gal-1 in CAFs might facilitate gastric cancer cell migration and invasion by upregulating integrin β1 expression in gastric cancer.

Differential regulation of extracellular matrix protein expression in carcinoma-associated fibroblasts by TGF-β1 regulates cancer cell spreading but not adhesion.

Cancer progression is characterized by a complex reciprocity between neoplastic epithelium and adjacent stromal cells. In ductal carcinoma in situ (DCIS) of the breast, both reduced stromal decorin expression and myxoid stroma are correlated with increased recurrence risk. In this study, we aimed to investigate paracrine regulation of expression of decorin and related extracellular matrix (ECM) proteins in cancer-associated fibroblasts (CAFs). Transforming growth factor-β1 (TGF-β1) was identified as a competent ECM modulator, as it reduced decorin and strongly enhanced versican, biglycan and type I collagen expression. Similar but less pronounced effects were observed when fibroblasts were treated with basic fibroblast growth factor (bFGF). Despite this concerted ECM modulation, TGF-β1 and bFGF differentially regulated alpha-smooth muscle actin (α-SMA) expression, which is often proposed as a CAF-marker. Cancer cell-derived secretomes induced versican and biglycan expression in fibroblasts. Immunohistochemistry on twenty DCIS specimens showed a trend toward periductal versican overexpression in DCIS with myxoid stroma. Cancer cell adhesion was inhibited by decorin, but not by CAF-derived matrices. Cancer cells presented significantly enhanced spreading when seeded on matrices derived from TGF-β1-treated CAF. Altogether these data indicate that preinvasive cancerous lesions might modulate the composition of surrounding stroma through TGF-β1 release to obtain an invasion-permissive microenvironment.