Hepatocyte growth factor activates tumor stromal fibroblasts to promote tumorigenesis in gastric cancer

Cancer-associated fibroblasts (CAFs), as the activated fibroblasts in tumor stroma, are important modifiers of tumor progression. However, the mechanisms underlying stromal fibroblast activation and their promotion of tumor growth remain largely unknown in gastric cancer. Here, we show that normal fibroblasts (NFs) from non-cancerous regions of gastric cancer exhibit the traits of CAFs when grown together with gastric cancer cells in vivo. Activation of NFs can be induced by co-culture with gastric cancer cells, while deprivation of hepatocyte growth factor (HGF) using a neutralizing antibody inhibits the activation of NFs. Moreover, we identify HGF as an important factor from CAFs that acts in a paracrine manner to promote tumorigenesis in vitro and in vivo. Taken together, these results suggest that HGF may play a pivotal role in the regulatory circuit between gastric cancer cells and stromal fibroblasts, and neutralization of HGF inhibits both activation and tumor-promoting properties of CAFs.     

Differential transplantability of tumor-associated stromal cells

At the time of transplantation, tumor fragments contain "passenger" cells: endothelial cells and other stromal cells from the original host. Here, we investigated the fate of genetically labeled endothelial and nonendothelial stromal cells after transplantation in syngeneic mice. We report that angiogenic stroma associated with tumor or adipose tissue persists when transplanted, remains functional, and governs the initial neovascularization of grafted tissue fragments for more than 4 weeks after implantation. Surprisingly, the passenger endothelial cells survive longer than other stromal cells, which are replaced by host-activated fibroblasts after 3 weeks. The transplantability of tumor stroma suggests that the angiogenic potential of a tumor xenograft, which determines its viability, depends on the presence of passenger endothelial cells and other stromal cells within the xenograft. These studies of tumor tissue transplantation provide a platform for exploring the role of epithelial-stromal interactions in studies of tumor heterogeneity and drug resistance.     

Serum levels of iCAF-derived osteoglycin predict favorable outcome in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant stroma, the main cellular constituents of which are cancer-associated fibroblasts (CAFs). Stroma-targeting agents have been proposed to improve the poor outcome of current treatments. However, clinical trials using these agents showed disappointing results. Heterogeneity in the PDAC CAF population was recently delineated demonstrating that both tumor-promoting and tumor-suppressive activities co-exist in the stroma. Here, we aimed to identify biomarkers for the CAF population that contribute to a favorable outcome. RNA-sequencing reads from patient-derived xenografts (PDXs) were mapped to the human and mouse genome to allocate the expression of genes to the tumor or stroma. Survival meta-analysis for stromal genes was performed and applied to human protein atlas data to identify circulating biomarkers. The candidate protein was perturbed in co-cultures and assessed in existing and novel single-cell gene expression analysis from control, pancreatitis, pancreatitis-recovered and PDAC mouse models. Serum levels of the candidate biomarker were measured in two independent cohorts totaling 148 PDAC patients and related them to overall survival. Osteoglycin (OGN) was identified as a candidate serum prognostic marker. Single-cell analysis indicated that Ogn is derived from a subgroup of inflammatory CAFs. Ogn-expressing fibroblasts are distinct from resident healthy pancreatic stellate cells and arise during pancreatitis. Serum OGN levels were prognostic for favorable overall survival in two independent PDAC cohorts (HR = 0.47, P = .042 and HR = 0.53, P = .006). Altogether, we conclude that high circulating OGN levels inform on a previously unrecognized subgroup of CAFs and predict favorable outcomes in resectable PDAC.     

Mesenchymal stem cells regulate epithelial–mesenchymal transition and tumor progression of pancreatic cancer cells

Cancer‐associated fibroblasts contribute to cancer progression that is caused by epithelial–mesenchymal transition (EMT). Recently, mesenchymal stem cells (MSCs) were found to be the major candidate involved in the development of tumor‐promoting cancer stroma. Here we report that α‐smooth muscle actin‐positive myofibroblast‐like cells originating from MSCs contribute to inducing EMT in side population cells of pancreatic cancer. More importantly, MSC‐derived myofibroblasts function to maintain tumor‐initiating stem cell‐like characteristics, including augmenting expression levels of various stemness‐associated genes, enhancing sphere‐ forming activity, promoting tumor formation in a mouse xenograft model, and showing resistance to anticancer drugs. Furthermore, both γ‐secretase inhibitor and siRNA directed against Jagged‐1 attenuated MSC‐associated E‐cadherin suppression and sphere formation in pancreatic cancer side population cells. Thus, our results suggest that MSC‐derived myofibroblasts play important roles in regulating EMT and tumor‐initiating stem cell‐like properties of pancreatic cancer cells through an intermediating Notch signal.     

Stromal responses to carcinomas of the pancreas: juxtatumoral gene expression conforms to the infiltrating pattern and not the biologic subtype

If there is a "science" of tumor-stromal interactions, there must be a set of biologic rules that are organ-site dependent. One way to explore this hypothesis would be to compare the patterns of gene expression of two biologically distinct neoplasms that arise within the same organ site. Using nonradioactive in situ hybridization, we evaluated the gene expression patterns of three genes previously shown to be robust markers of the juxtatumoral stroma within eight infiltrating ductal adenocarcinoma of the pancreas (ApoC1, ApoD and MMP11), and compared these patterns to those associated with seven infiltrating colloid and tubular carcinomas arising in association with intraductal papillary mucinous neoplasms (IPMNs), a histologically distinct form of primary carcinoma of the pancreas, two surgically resected samples of chronic pancreatitis and two surgically resected pancreatic cancer liver metastases. Robust juxtatumoral stromal expression was noted for all three markers within all eight conventional infiltrating ductal adenocarcinoma tissues, but not in samples of chronic pancreatitis. Among the carcinomas arising within an IPMN, expression for all three markers was also noted for five of seven infiltrating carcinomas analyzed. However, when labeling for these three markers was analyzed with respect to infiltrative growth pattern, positive labeling was only seen in areas of tubular (ductal-type) growth and not in areas of colloid carcinoma. This observation was further supported by two infiltrating carcinomas arising in an IPMN that showed both tubular and colloid growth patterns within the same neoplasm indicating the host stromal response observed may relate to infiltrative growth pattern rather than the biology of the primary tumor type. Moreover, these robust patterns within conventional infiltrating ductal adenocarcinomas were not retained within matched metastases to the liver, indicating the importance of the tumor microenvironment in the host stromal response. Juxtatumoral stroma was found to be composed of a least two cell types, tumor-infiltrating macrophages and fibroblasts, highlighting the complexity of tumor-stromal interactions within an infiltrating carcinoma. Since the juxtatumoral gene expression response is the strongest indication of direct communication between stroma and cancer cells, we provide evidence of a stereotypical response to infiltrative growth that might predominate in tumor-stromal interactions independent of cancer type, a finding with clinical implications for therapeutic modalities that target this response in human tumors.     

Stromal expression of connective tissue growth factor promotes angiogenesis and prostate cancer tumorigenesis

Our previous studies have defined reactive stroma in human prostate cancer and have developed the differential reactive stroma (DRS) xenograft model to evaluate mechanisms of how reactive stroma promotes carcinoma tumorigenesis. Analysis of several normal human prostate stromal cell lines in the DRS model showed that some rapidly promoted LNCaP prostate carcinoma cell tumorigenesis and others had no effect. These differential effects were due, in part, to elevated angiogenesis and were transforming growth factor (TGF)-beta1 mediated. The present study was conducted to identify and evaluate candidate genes expressed in prostate stromal cells responsible for this differential tumor-promoting activity. Differential cDNA microarray analyses showed that connective tissue growth factor (CTGF) was expressed at low levels in nontumor-promoting prostate stromal cells and was constitutively expressed in tumor-promoting prostate stromal cells. TGF-beta1 stimulated CTGF message expression in nontumor-promoting prostate stromal cells. To evaluate the role of stromal-expressed CTGF in tumor progression, either engineered mouse prostate stromal fibroblasts expressing retroviral-introduced CTGF or 3T3 fibroblasts engineered with mifepristone-regulated CTGF were combined with LNCaP human prostate cancer cells in the DRS xenograft tumor model under different extracellular matrix conditions. Expression of CTGF in tumor-reactive stroma induced significant increases in microvessel density and xenograft tumor growth under several conditions tested. These data suggest that CTGF is a downstream mediator of TGF-beta1 action in cancer-associated reactive stroma and is likely to be one of the key regulators of angiogenesis in the tumor-reactive stromal microenvironment.     

PDGFRβ expression in tumor stroma of pancreatic adenocarcinoma as a reliable prognostic marker

Pancreatic adenocarcinoma is a lethal disease that often develops a desmoplastic reaction in tumor stroma. In general, desmoplasia is thought to promote tumor growth. However, its molecular pathology and prognostic potential have not been fully investigated. Here, we investigate 26 cases of pancreatic ductal adenocarcinoma and examine the clinicopathological association between survival and expression levels of several molecular markers for stromal cells. These include alpha-smooth muscle actin (SMA) and platelet-derived growth factor (PDGF) receptor β (PDGFRβ). Both are markers of activated fibroblasts or pancreatic stellate cells (PSCs) that play an important role in desmoplasia. The staining patterns of both molecular markers were not uniform, so we categorized them into 3 grades (high, middle, and low) according to intensity. Interestingly, Kaplan-Meier analysis revealed that higher expression of PDGFRβ matched shorter prognosis (p?=?0.0287, log-rank test) as well as lymphatic invasion and lymph node metastasis, whereas SMA did not (p?=?0.6122). Our results suggest the prognostic potential of cancer stroma via PDGF-B signaling. Regulation of PDGF-B-associated signaling crosstalk between cancer cells and stroma cells, therefore, may indicate a possible therapeutic target for desmoplastic malignant tumors such as pancreatic adenocarcinoma.     

Induction of syndecan-1 expression in stromal fibroblasts promotes proliferation of human breast cancer cells

Infiltrating carcinomas characteristically elicit a reactive stromal response, and accumulating evidence indicates that tumor stroma fibroblasts reciprocally promote tumor development and growth. The cell surface heparan sulfate proteoglycan, syndecan-1 (Sdc1), is thought to function as a coreceptor for growth factor and extracellular matrix interactions, and Sdc1 expression is induced in reactive stromal cells in both mice and man. Mice with a targeted mutation in Sdc1 show reduced tumor development in response to oncogene expression and altered responses to other pathological stimuli that are associated with the induction of stromal Sdc1. Here, we test the hypothesis that Sdc1 is required for the growth-promoting activities of reactive stroma. We found that when highly invasive carcinoma cells (MDA-MB-231) were placed in contact with mouse embryonic fibroblasts (MEFs) in a coculture model, Sdc1 expression was induced. Sdc1 was not induced by less invasive or normal cell lines (T47D and NMuMG). Furthermore, the growth of MDA-MB-231 cells was enhanced by 42% when cocultured with Sdc1+/+ MEFs compared with Sdc1-/- MEFs. When T47D cells were cocultured with fibroblasts that expressed transfected Sdc1, these Sdc1-positive fibroblasts stimulated growth of the breast epithelial cells by 85% compared with untransfected controls. The growth-promoting effect was completely abolished when fibroblasts were transfected with mutant Sdc1 lacking heparan sulfate attachment sites. In conclusion, we have demonstrated that a growth-promoting loop exists between breast cancer cells and their stroma that depends on the activity of glycanated Sdc1.     

Assessment of the stromal contribution to Sonic Hedgehog-dependent pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. It is typically detected at an advanced stage, at which the therapeutic options are very limited. One remarkable feature of PDAC that contributes to its resilience to treatment is the extreme stromal activation seen in these tumors. Often, the vast majority of tumor bulk consists of non-tumor cells that together provide a tumor-promoting environment. One of the signals that maintains and activates the stroma is the developmental protein Sonic Hedgehog (SHH). As the disease progresses, tumor cells produce increasing amounts of SHH, which activates the surrounding stroma to aid in tumor progression. To better understand this response and identify targets for inhibition, we aimed to elucidate the proteins that mediate the SHH-driven stromal response in PDAC. For this a novel mixed-species coculture model was set up in which the cancer cells are human, and the stroma is modeled by mouse fibroblasts. In conjunction with next-generation sequencing we were able to use the sequence difference between these species to genetically distinguish between the epithelial and stromal responses to SHH. The stromal SHH-dependent genes from this analysis were validated and their relevance for human disease was subsequently determined in two independent patient cohorts. In non-microdissected tissue from PDAC patients, in which a large amount of stroma is present, the targets were confirmed to associate with tumor stroma versus normal pancreatic tissue. Patient survival analysis and immunohistochemistry identified CDA, EDIL3, ITGB4, PLAUR and SPOCK1 as SHH-dependent stromal factors that are associated with poor prognosis in PDAC patients. Summarizing, the presented data provide insight into the role of the activated stroma in PDAC, and how SHH acts to mediate this response. In addition, the study has yielded several candidates that are interesting therapeutic targets for a disease for which treatment options are still inadequate.     

Stromal cells in tumor microenvironment and breast cancer

Cancer is a systemic disease encompassing multiple components of both tumor cells themselves and host stromal cells. It is now clear that stromal cells in the tumor microenvironment play an important role in cancer development. Molecular events through which reactive stromal cells affect cancer cells can be defined so that biomarkers and therapeutic targets can be identified. Cancer-associated fibroblasts (CAFs) make up the bulk of cancer stroma and affect the tumor microenvironment such that they promote cancer initiation, angiogenesis, invasion, and metastasis. In breast cancer, CAFs not only promote tumor progression but also induce therapeutic resistance. Accordingly, targeting CAFs provides a novel way to control tumors with therapeutic resistance. This review summarizes the current understandings of tumor stroma in breast cancer with a particular emphasis on the role of CAFs and the therapeutic implications of CAFs. In addition, the effects of other stromal components such as endothelial cells, macrophages, and adipocytes in breast cancer are also discussed. Finally, we describe the biologic markers to categorize patients into a specific and confirmed subtype for personalized treatment.     

Notch mediates TGF alpha-induced changes in epithelial differentiation during pancreatic tumorigenesis

Notch signaling regulates cell fate decisions in a wide variety of adult and embryonic tissues. Here we show that Notch pathway components and Notch target genes are upregulated in invasive pancreatic cancer, as well as in pancreatic cancer precursors from both mouse and human. In mouse pancreas, ectopic Notch activation results in accumulation of nestin-positive precursor cells and expansion of metaplastic ductal epithelium, previously identified as a precursor lesion for pancreatic cancer. Notch is also activated as a direct consequence of EGF receptor activation in exocrine pancreas and is required for TGF alpha-induced changes in epithelial differentiation. These findings suggest that Notch mediates the tumor-initiating effects of TG alpha by expanding a population of undifferentiated precursor cells.     

Gene expression profiling of tumor-stromal interactions between pancreatic cancer cells and stromal fibroblasts

The interactions between cancer cells and surrounding stroma play a critical role in tumor progression, but their molecular basis is largely unknown. Global gene expression profiling was performed using oligonucleotide microarrays to determine changes in the gene expression of pancreatic cancer cells (CFPAC1) and stromal fibroblasts induced by coculture. This analysis identified multiple genes as differentially expressed in pancreatic cancer cells and in fibroblasts as a consequence of their mutual interactions, including those that encode for proteins associated with tumor invasion, metastasis, and angiogenesis. Among the genes identified, the cyclooxygenase-2 (COX-2)/PTGS2 gene was of particular interest because COX-2 expression was markedly augmented in both cell types (cancer cells and fibroblasts) in response to coculture. Coculture with fibroblasts also induced COX-2 expression in additional pancreatic cancer cells with an unmethylated COX-2 promoter, but not in those with a methylated COX-2 promoter. Using an in vitro invasion assay, we found an increase in the invasive potential of CFPAC1 cells when they were cocultured with fibroblasts, an effect blocked partially by the addition of a selective COX-2 inhibitor, NS-398, or by COX-2 knockdown with small interfering RNA. Thus, COX-2 inhibitors can decrease the invasive properties of pancreatic cancer cells acquired through tumor-stromal interactions.     

Ectopic Overexpression of Sonic Hedgehog (Shh) Induces Stromal Expansion and Metaplasia in the Adult Murine Pancreas

Ligand-dependent activation of the Hedgehog (Hh) signaling pathway has been implicated in both tumor initiation and metastasis of pancreatic ductal adenocarcinoma (PDAC). Prior studies in genetically engineered mouse models (GEMMs) have assessed the role of Hh signaling by cell autonomous expression of a constitutively active Gli2 within epithelial cells. On the contrary, aberrant pathway reactivation in the human exocrine pancreas occurs principally as a consequence of Sonic Hh ligand (Shh) overexpression from epithelial cells. To recapitulate the cognate pathophysiology of Hh signaling observed in the human pancreas, we examined GEMM where Hh ligand is conditionally overexpressed within the mature exocrine pancreas using a tamoxifen-inducible Elastase-Cre promoter (Ela-CreERT2;LSL-mShh). We also facilitated potential cell autonomous epithelial responsiveness to secreted Hh ligand by generating compound transgenic mice with concomitant expression of the Hh receptor Smoothened (Ela-CreERT2;LSL-mShh;LSL-mSmo). Of interest, none of these mice developed intraductal precursor lesions or PDAC during the follow-up period of up to 12 months after tamoxifen induction. Instead, all animals demonstrated marked expansion of stromal cells, consistent with the previously described epithelial-to-stromal paracrine Hh signaling. Hh responsiveness was mirrored by the expression of primary cilia within the expanded mesenchymal compartment and the absence within mature acinar cells. In the absence of cooperating mutations, Hh ligand overexpression in the mature exocrine pancreas is insufficient to induce neoplasia, even when epithelial cells coexpress the Smo receptor. This autochthonous model serves as a platform for studying epithelial stromal interactions in pancreatic carcinogenesis.     

Isolation of a new cell population in the glioblastoma microenvironment

Glioblastoma (GB) is a highly infiltrative tumor recurring in 90% of cases within a few centimeters of the resection cavity, even in cases of complete tumor resection and adjuvant chemo/radiotherapy. This observation highlights the importance of understanding this special zone of brain tissue surrounding the tumor. It is becoming clear that the nonneoplastic stromal compartment of most solid cancers plays an active role in tumor proliferation, invasion, and metastasis. Very little information, other than that concerning angiogenesis and immune cells, has been collected for stromal cells from GB. As part of a translational research program, we have isolated a new stromal cell population surrounding GB by computer-guided stereotaxic biopsies and primary culture. We named these cells GB-associated stromal cells (GASCs). GASCs are diploid, do not display the genomic alterations typical of GB cells, and have phenotypic and functional properties in common with the cancer-associated fibroblasts (CAFs) described in the stroma of carcinomas. In particular, GASCs express markers associated with CAFs such as fibroblast surface protein, alpha-smooth muscle actin (α-SMA), and platelet-derived growth factor receptor-beta (PDGFRβ). Furthermore, GASCs have a molecular expression profile different from that of control stromal cells derived from non-GB peripheral brain tissues. GASCs were also found to have tumor-promoting effects on glioma cells in vitro and in vivo. The isolation of GASCs in a tumor of neuroepithelial origin was unexpected, and further studies are required to determine their potential as a target for antiglioma treatment.     

Multikinase inhibitor regorafenib inhibits the growth and metastasis of colon cancer with abundant stroma

Interaction between tumor cells and stromal cells plays an important role in the growth and metastasis of colon cancer. We previously found that carcinoma‐associated fibroblasts (CAFs) expressed platelet‐derived growth factor receptor‐β (PDGFR‐β) and that PDGFR targeted therapy using imatinib or nilotinib inhibited stromal reaction. Bone marrow‐derived mesenchymal stem cells (MSCs) migrate to tumor stroma and differentiate into CAFs. A novel oral multikinase inhibitor regorafenib inhibits receptor tyrosine kinases expressed on stromal cells (vascular endothelial growth factor receptor 1–3, TIE2, PDGFR‐β, and fibroblast growth factors) and tumor cells (c‐KIT, RET, and BRAF). These molecules are involved in tumor growth, angiogenesis, lymphangiogenesis, and stromal activation. Therefore, we examined whether regorafenib impaired the tumor‐promoting effect of CAFs/MSCs. KM12SM human colon cancer cells alone or KM12SM cells with MSCs were transplanted into the cecal wall of nude mice. Co‐implantation of KM12SM cells with MSCs into the cecal wall of nude mice produced tumors with abundant stromal component and promoted tumor growth and lymph node metastasis. Single treatment with regorafenib inhibited tumor growth and metastasis by inhibiting both tumor cells and stromal reaction. This tumor‐inhibitory effect of regorafenib was more obvious in tumors developed by co‐implanting KM12SM cells with MSCs. Our data suggested that targeting of the tumor microenvironment with regorafenib affected tumor cell–MSC interaction, which in turn inhibited the growth and metastasis of colon cancer.     

Altered TGF-β signaling in a subpopulation of human stromal cells promotes prostatic carcinogenesis

Carcinoma-associated fibroblasts (CAF) play a critical role in malignant progression. Loss of TGF-β receptor II (TGFβR2) in the prostate stroma is correlated with prostatic tumorigenesis. To determine the mechanisms by which stromal heterogeneity because of loss of TGFβR2 might contribute to cancer progression, we attenuated transforming growth factor beta (TGF-β) signaling in a subpopulation of immortalized human prostate fibroblasts in a model of tumor progression. In a tissue recombination model, loss of TGFβR2 function in 50% of the stromal cell population resulted in malignant transformation of the nontumorigenic human prostate epithelial cell line BPH1. Mixing fibroblasts expressing the empty vector and dominant negative TGFβR2 increased the expression of markers of myofibroblast differentiation [coexpression of vimentin and alpha smooth muscle actin (αSMA)] through elevation of TGF-β1 and activation of the Akt pathway. In combination, these two populations of stromal cells recapitulated the tumor inductive activity of CAFs. TGFβR2 activity in mixed stromal cell populations cultured in vitro caused secretion of factors that are known to promote tumor progression, including TGF-β1, SDF1/CXCL12, and members of the fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) families. In vivo, tissue recombination of fibroblasts overexpressing TGF-β1 and SDF1/CXCL12 not only induced transformation of BPH1 cells, but also promoted a robust growth of highly invasive cells, similar to effects produced by CAFs. While the precise nature and/or origin of the particular stromal cell populations in vivo remain unknown, these findings strongly link heterogeneity in TGF-β signaling to tumor promotion by tumor stromal cells.     

Tumor microenvironment in gastric cancers

The tumor microenvironment favors the growth and expansion of cancer cells. Many cell types are involved in the tumor microenvironment such as inflammatory cells, fibroblasts, nerves, and vascular endothelial cells. These stromal cells contribute to tumor growth by releasing various molecules to either directly activate the growth signaling in cancer cells or remodel surrounding areas. This review introduces recent advances in findings on the interactions within the tumor microenvironment such as in cancer‐associated fibroblasts (CAFs), immune cells, and endothelial cells, in particular those established in mouse gastric cancer models. In mice, myofibroblasts in the gastric stroma secrete R‐spondin and support normal gastric stem cells. Most CAFs promote tumor growth in a paracrine manner, but CAF population appears to be heterogeneous in terms of their function and origin, and include both tumor‐promoting and tumor‐restraining populations. Among immune cell populations, tumor‐associated macrophages, including M1 and M2 macrophages, and myeloid‐derived suppressor cells (MDSCs), are reported to directly or indirectly promote gastric tumorigenesis by secreting soluble factors or modulating immune responses. Endothelial cells or blood vessels not only fuel tumors with nutrients, but also interact with cancer stem cells and immune cells by secreting chemokines or cytokines, and act as a cancer niche. Understanding these interactions within the tumor microenvironment would contribute to unraveling new therapeutic targets.