Dichotomous role of the serine/threonine kinase MAP4K4 in pancreatic ductal adenocarcinoma onset and metastasis through control of AKT and ERK pathways

MAP4K4 is a serine/threonine kinase of the STE20 family involved in the regulation of actin cytoskeleton dynamics and cell motility. It has been proposed as a target of angiogenesis and inhibitors show potential in cardioprotection. MAP4K4 also mediates cell invasion in vitro, is overexpressed in various types of cancer, and is associated with poor patient prognosis. Recently, MAP4K4 has been shown to be overexpressed in pancreatic cancer, but its role in tumour initiation, progression, and metastasis is unknown. Here, using the Kras^G12D Trp53^R172H Pdx1-Cre (KPC) mouse model of pancreatic ductal adenocarcinoma (PDAC), we show that deletion of Map4k4 drives tumour initiation and progression. Moreover, we report that the acceleration of tumour onset is also associated with an overactivation of ERK and AKT, two major downstream effectors of KRAS, in vitro and in vivo. In contrast to the accelerated tumour onset caused by loss of MAP4K4, we observed a reduction in metastatic burden with both the KPC model and in an intraperitoneal transplant assay indicating a major role of MAP4K4 in metastatic seeding. In summary, our study sheds light on the dichotomous role of MAP4K4 in the initiation of PDAC onset, progression, and metastatic dissemination. It also identifies MAP4K4 as a possible druggable target against pancreatic cancer spread, but with the caveat that targeting MAP4K4 might accelerate early tumorigenesis. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.     

Imbalance of desmoplastic stromal cell numbers drives aggressive cancer processes

Epithelial tissues have sparse stroma, in contrast to their corresponding tumours. The effect of cancer cells on stromal cells is well recognized. Increasingly, stromal components, such as endothelial and immune cells, are considered indispensable for cancer progression. The role of desmoplastic stroma, in contrast, is poorly understood. Targeting such cellular components within the tumour is attractive. Recent evidence strongly points towards a dynamic stromal cell participation in cancer progression that impacts patient prognosis. The role of specific desmoplastic stromal cells, such as stellate cells and myofibroblasts in pancreatic, oesophageal and skin cancers, was studied in bio‐engineered, physiomimetic organotypic cultures and by regression analysis. For pancreatic cancer, the maximal effect on increasing cancer cell proliferation and invasion, as well as decreasing cancer cell apoptosis, occurs when stromal (pancreatic stellate cells) cells constitute the majority of the cellular population (maximal effect at a stromal cell proportion of 0.66–0.83), accompanied by change in expression of key molecules such as E‐cadherin and β‐catenin. Gene‐expression microarrays, across three tumour types, indicate that stromal cells consistently and significantly alter global cancer cell functions such as cell cycle, cell–cell signalling, cell movement, cell death and inflammatory response. However, these changes are mediated through cancer type‐specific alteration of expression, with very few common targets across tumour types. As highlighted by these in vitro data, the reciprocal relationship of E‐cadherin and polymeric immunoglobulin receptor (PIGR) expression in cancer cells could be shown, in vivo, to be dependent on the stromal content of human pancreatic cancer. These studies demonstrate that context‐specific cancer–stroma crosstalk requires to be precisely defined for effective therapeutic targeting. These data may be relevant to non‐malignant processes where epithelial cells interact with stromal cells, such as chronic inflammatory and fibrotic conditions. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Cancer‐associated fibroblasts predict poor outcome and promote periostin‐dependent invasion in oesophageal adenocarcinoma

Interactions between cancer cells and cancer‐associated fibroblasts (CAFs) play an important role in tumour development and progression. In this study we investigated the functional role of CAFs in oesophageal adenocarcinoma (EAC). We used immunochemistry to analyse a cohort of 183 EAC patients for CAF markers related to disease mortality. We characterized CAFs and normal oesophageal fibroblasts (NOFs) using western blotting, immunofluorescence and gel contraction. Transwell assays, 3D organotypic culture and xenograft models were used to examine the effects on EAC cell function and to dissect molecular mechanisms regulating invasion. Most EACs (93%) contained CAFs with a myofibroblastic (α‐SMA‐positive) phenotype, which correlated significantly with poor survival [p = 0.016; HR 7. 1 (1.7–29.4)]. Primary CAFs isolated from EACs have a contractile, myofibroblastic phenotype and promote EAC cell invasion in vitro (Transwell assays, p ≤ 0.05; organotypic culture, p < 0.001) and in vivo (p ≤ 0.05). In vitro, this pro‐invasive effect is modulated through the matricellular protein periostin. Periostin is secreted by CAFs and acts as a ligand for EAC cell integrins αvβ3 and αvβ5, promoting activation of the PI3kinase–Akt pathway. In patient samples, periostin expression at the tumour cell–stromal interface correlates with poor overall and disease‐free survival. Our study highlights the importance of the tumour stroma in EAC progression. Paracrine interaction between CAF‐secreted periostin and EAC‐expressed integrins results in PI3 kinase–Akt activation and increased tumour cell invasion. Most EACs contain a myofibroblastic CAF‐rich stroma; this may explain the aggressive, highly infiltrative nature of the disease, and suggests that stromal targeting may produce therapeutic benefit in EAC patients. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

胰腺导管腺癌中胰腺星形细胞的研究进展

胰腺星形细胞(pancreatic stellate cells,PSC)是胰腺导管腺癌(pancreatic ductal adenocarcinoma,PDAC)肿瘤微环境中最重要的成分,在PDAC发生发展中具有非常关键的作用.目前大量研究关注于PSC与胰腺癌细胞(pancreatic cancer cells,PCC)之间的相互作用及PSC在PDAC微环境中发挥的作用.PSC在许多情况下发生活化,如乙醇、氧化应激和高血糖等.PDAC早期即可出现PSC的活化,PCC可以诱导刺激PSC发生活化,活化的PSC可以产生大量胶原纤维,形成适宜PCC生长的间质微环境,促进PCC的增殖,减少化疗药物对肿瘤细胞的杀伤作用.另外,PSC还可以与间质中各种细胞成分如内皮细胞和各种免疫细胞相互作用,在血管生成、免疫逃逸和神经侵犯等方面协助肿瘤进展.因此,阐明PSC与肿瘤细胞以及其他间质成分之间复杂的相互作用至关重要.     

Desmoplastic tumour-associated stroma versus neural tissue in central nervous system metastasis: effects of different microenvironments on tumour growth

Chang K‐C, Lu Y‐C, Lin M‐J, Chen H‐Y & Jin Y‐T
(2011) Histopathology 59 , 31–39 Desmoplastic tumour‐associated stroma versus neural tissue in central nervous system metastasis: effects of different microenvironments on tumour growth Aims: Interactions between tumour cells and extracellular matrix (ECM) are critical in the metastatic cascade. We compared effects of desmoplastic stroma versus neural tissue on central nervous system (CNS) metastasis. Methods and results: Using integrins (ECM receptors), ECM (fibronectin, laminin and collagen IV) and CD31 and vascular endothelial growth factor (VEGF) for angiogenesis, this study examined immunohistochemically 69 consecutive cases of CNS metastases. In contrast to low‐level expression in tumour‐embedded neural tissue, ECM [fibronectin (71%), laminin γ‐1 (79%) and collagen IV (92%)] and CD31‐positive microvascular densities (33 versus 4 vessels/field) were significantly richer in desmoplastic tumour stroma, which was present in 90% (53 of 59) of carcinomas, 100% (five of five) of malignant melanomas and 100% (two of two) of sarcomas. Collagen IV expression in tumour stroma was correlated with the expression of fibronectin (P = 0.013) and laminin (P = 0.034) and with infiltrative tumour edges (P = 0.005); fibronectin‐positive tumour stroma was correlated with a higher microvascular density (P = 0.015). In addition, tumour cells expressed integrins (～75%) and laminin (84%) more frequently than VEGF (23%), and tumour expression of laminin was correlated with the presence of desmoplastic stroma (P = 0.006). Interestingly, laminin‐positive tumour stroma was a worse prognosticator (P = 0.072). Conclusions: ECM‐ and vascular‐rich stroma is important in tumour growth, which underlies therapeutic strategies targeting tumour‐associated stroma.     

Podoplanin expression by cancer-associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma

Pula B, Jethon A, Piotrowska A, Gomulkiewicz A, Owczarek T, Calik J, Wojnar A, Witkiewicz W, Rys J, Ugorski M, Dziegiel P & Podhorska‐Okolow M
(2011) Histopathology  59, 1249–1260
Podoplanin expression by cancer‐associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma Aims: It has recently been shown that podoplanin, a mucin‐type glycoprotein, is expressed by cancer cells and cancer‐associated fibroblasts (CAFs), and promotes cancer cell migration and invasiveness. The biological role of podoplanin expression in tumour stroma of invasive ductal carcinoma of the breast (IDC) has not been determined. Methods and results: Podoplanin expression was analysed in 117 cases of IDC and 27 cases of fibrocystic change, as well as in breast cancer cell lines, with the use of immunohistochemistry and real‐time polymerase chain reaction. In 82.1% of analysed tumours, podoplanin was found only in CAFs. Only two of 117 IDC cases (1.7%) were characterized by expression of this glycoprotein in cancer cells. None of the fibrocystic changes or stroma surrounding normal ducts showed podoplanin expression. Podoplanin‐positive CAFs correlated with tumour size (P = 0.0125), grade of malignancy (P = 0.0058), lymph node metastasis (P = 0.0149), lymphovascular invasion (LVI) (P = 0.0486) and Ki67 expression in cancer cells (P = 0.0128). High‐level podoplanin expression (>50% of positive stroma) in the tumour stroma was significantly associated with a negative oestrogen status (P = 0.0201). Univariate, but not multivariate, analysis showed that podoplanin expression by CAFs was associated with poor patient outcome (P = 0.0202). Conclusions: Our results suggest that podoplanin expression by CAFs could be an unfavourable prognostic marker for IDC.     

The epigenetic regulators Bmi1 and Ring1B are differentially regulated in pancreatitis and pancreatic ductal adenocarcinoma

Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are associated with major changes in cell differentiation. These changes may be at the basis of the increased risk for PDAC among patients with chronic pancreatitis. Polycomb proteins are epigenetic silencers expressed in adult stem cells; up‐regulation of Polycomb proteins has been reported to occur in a variety of solid tumours such as colon and breast cancer. We hypothesized that Polycomb might play a role in preneoplastic states in the pancreas and in tumour development/progression. To test these ideas, we determined the expression of PRC1 complex proteins (Bmi1 and Ring1b) during pancreatic development and in pancreatic tissue from mouse models of disease: acute and chronic pancreatic injury, duct ligation, and in K‐Ras^G12V conditional knock‐in and caerulein‐treated K‐Ras^G12V mice. The study was extended to human pancreatic tissue samples. To obtain mechanistic insights, Bmi1 expression in cells undergoing in vitro exocrine cell metaplasia and the effects of Bmi1 depletion in an acinar cancer cell line were studied. We found that Bmi1 and Ring1B are expressed in pancreatic exocrine precursor cells during early development and in ductal and islet cells—but not acinar cells—in the adult pancreas. Bmi1 expression was induced in acinar cells during acute injury, in acinar–ductal metaplastic lesions, as well as in pancreatic intraepithelial neoplasia (PanIN) and PDAC. In contrast, Ring1B expression was only significantly and persistently up‐regulated in high‐grade PanINs and in PDAC. Bmi1 knockdown in cultured acinar tumour cells led to changes in the expression of various digestive enzymes. Our results suggest that Bmi1 and Ring1B are modulated in pancreatic diseases and could contribute differently to tumour development. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Tumour stroma‐derived lipocalin‐2 promotes breast cancer metastasis

Tumour cell‐secreted factors skew infiltrating immune cells towards a tumour‐supporting phenotype, expressing pro‐tumourigenic mediators. However, the influence of lipocalin‐2 (Lcn2) on the metastatic cascade in the tumour micro‐environment is still not clearly defined. Here, we explored the role of stroma‐derived, especially macrophage‐released, Lcn2 in breast cancer progression. Knockdown studies and neutralizing antibody approaches showed that Lcn2 contributes to the early events of metastasis in vitro. The release of Lcn2 from macrophages induced an epithelial–mesenchymal transition programme in MCF‐7 breast cancer cells and enhanced local migration as well as invasion into the extracellular matrix, using a three‐dimensioanl (3D) spheroid model. Moreover, a global Lcn2 deficiency attenuated breast cancer metastasis in both the MMTV–PyMT breast cancer model and a xenograft model inoculating MCF‐7 cells pretreated with supernatants from wild‐type and Lcn2‐knockdown macrophages. To dissect the role of stroma‐derived Lcn2, we employed an orthotopic mammary tumour mouse model. Implantation of wild‐type PyMT tumour cells into Lcn2‐deficient mice left primary mammary tumour formation unaltered, but specifically reduced tumour cell dissemination into the lung. We conclude that stroma‐secreted Lcn2 promotes metastasis in vitro and in vivo, thereby contributing to tumour progression. Our study highlights the tumourigenic potential of stroma‐released Lcn2 and suggests Lcn2 as a putative therapeutic target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

β‐Catenin activation contributes to the pathogenesis of adenomyosis through epithelial–mesenchymal transition

Adenomyosis is defined by the presence of endometrial glands and stroma within the myometrium. Despite its frequent occurrence, the precise aetiology and physiopathology of adenomyosis is still unknown. WNT/β‐catenin signalling molecules are important and should be tightly regulated for uterine function. To investigate the role of β‐catenin signalling in adenomyosis, the expression of β‐catenin was examined. Nuclear and cytoplasmic β‐catenin expression was significantly higher in epithelial cells of human adenomyosis compared to control endometrium. To determine whether constitutive activation of β‐catenin in the murine uterus leads to development of adenomyosis, mice that expressed a dominant stabilized β‐catenin in the uterus were used by crossing PR‐Cre mice with Ctnnb1^f(ex3)/+ mice. Uteri of PR^cre/+ Ctnnb1^f(ex3)/+ mice displayed an abnormal irregular structure and highly active proliferation in the myometrium, and subsequently developed adenomyosis. Interestingly, the expression of E‐cadherin was repressed in epithelial cells of PR^cre/+ Ctnnb1^f(ex3)/+ mice compared to control mice. Repression of E‐cadherin is one of the hallmarks of epithelial–mesenchymal transition (EMT). The expression of SNAIL and ZEB1 was observed in some epithelial cells of the uterus in PR^cre/+ Ctnnb1^f(ex3)/+ mice but not in control mice. Vimentin and COUP‐TFII, mesenchymal cell markers, were expressed in some epithelial cells of PR^cre/+ Ctnnb1^f(ex3)/+ mice. In human adenomyosis, the expression of E‐cadherin was decreased in epithelial cells compared to control endometrium, while CD10, an endometrial stromal marker, was expressed in some epithelial cells of human adenomyosis. These results suggest that abnormal activation of β‐catenin contributes to adenomyosis development through the induction of EMT. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Impact of VEGF‐dependent tumour micro‐environment on EDB fibronectin expression by subcutaneous human tumour xenografts in nude mice

Fibronectin (FN) is an extracellular matrix cell‐adhesive glycoprotein. The alternative spliced isoform EDB‐FN (extra domain B containing FN) is highly expressed in tumour blood vessels and stroma and represents a candidate for tumour targeting. To investigate the impact of different angiogenic micro‐environments on EDB‐FN expression, we used a tumour model in which human endometrial adenocarcinoma Tet‐FGF2 cells overexpressing fibroblast growth factor‐2 (FGF2) driven by the tetracycline‐responsive promoter were further transfected with a VEGF antisense cDNA, generating AS‐VEGF/Tet‐FGF2 cells. In this model, the expression of FGF2 plus VEGF results in fast‐growing, highly vascularized Tet‐FGF2 tumours. Down‐regulation of FGF2 production by tetracycline administration and/or of VEGF production by AS‐VEGF transduction inhibited tumour growth and vascularization, with profound changes in tumour micro‐environment. Quantitative RT‐PCR analysis using human EDB‐FN primers shows that subcutaneous grafting in immunodeficient mice is per se sufficient to cause a dramatic up‐regulation of EDB‐FN expression by these cells, as well as by human oesophageal cancer KYSE 30 cells and renal carcinoma Caki‐1 cells. However, in vivo down‐regulation of VEGF expression, as occurs in AS‐VEGF/Tet‐FGF2 tumours, and to a lesser extent of FGF2 expression, as occurs in tetracycline‐treated Tet‐FGF2 tumour‐bearing animals, causes significant inhibition of EDB‐FN production in tumour grafts, as shown by immunohistochemistry and quantitative RT‐PCR analysis. Accordingly, treatment of Tet‐FGF2 tumour‐bearing animals with the neutralizing anti‐murine VEGF receptor‐2 antibody DC101, or of Caki‐1 tumour‐bearing animals with the anti‐VEGF antibody bevacizumab, inhibited EDB‐FN expression in tumour grafts. EDB‐FN down‐regulation was paralleled by a decrease in vascularity, thus confirming EDB‐FN as a marker of tumour angiogenesis. These data demonstrate that the angiogenic micro‐environment, and in particular the VEGF/VEGFR‐2 system, plays a key role in modulating EDB‐FN expression by tumour cells in vivo. This may have implications for the design of therapeutic strategies targeting EDB‐FN in combination with anti‐angiogenic and/or cytotoxic drugs. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Loss of caveolin‐1 in prostate cancer stroma correlates with reduced relapse‐free survival and is functionally relevant to tumour progression

Levels of caveolin‐1 (Cav‐1) in tumour epithelial cells increase during prostate cancer progression. Conversely, Cav‐1 expression in the stroma can decline in advanced and metastatic prostate cancer. In a large cohort of 724 prostate cancers, we observed significantly decreased levels of stromal Cav‐1 in concordance with increased Gleason score (p = 0.012). Importantly, reduced expression of Cav‐1 in the stroma correlated with reduced relapse‐free survival (p = 0.009), suggesting a role for stromal Cav‐1 in inhibiting advanced disease. Silencing of Cav‐1 by shRNA in WPMY‐1 prostate fibroblasts resulted in up‐regulation of Akt phosphorylation, and significantly altered expression of genes involved in angiogenesis, invasion, and metastasis, including a > 2.5‐fold increase in TGF‐β1 and γ‐synuclein (SNCG) gene expression. Moreover, silencing of Cav‐1 induced migration of prostate cancer cells when stromal cells were used as attractants. Pharmacological inhibition of Akt caused down‐regulation of TGF‐β1 and SNCG, suggesting that loss of Cav‐1 in the stroma can influence Akt‐mediated signalling in the tumour microenvironment. Cav‐1‐depleted stromal cells exhibited increased levels of intracellular cholesterol, a precursor for androgen biosynthesis, steroidogenic enzymes, and testosterone. These findings suggest that loss of Cav‐1 in the tumour microenvironment contributes to the metastatic behaviour of tumour cells by a mechanism that involves up‐regulation of TGF‐β1 and SNCG through Akt activation. They also suggest that intracrine production of androgens, a process relevant to castration resistance, may occur in the stroma. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Syndecan‐1 (CD138) contributes to prostate cancer progression by stabilizing tumour‐initiating cells

Increasing evidence suggests that tumour‐initiating cells (TICs) contribute to the development of prostate cancer. Here, we identified syndecan‐1 as a key molecule maintaining the stability of prostate cancer TICs. Holoclones harbouring the biological properties of stemness were derived from single‐cell cultures of the PC3 human prostate cancer cell line. These holoclones over‐expressed syndecan‐1, but showed reduced expression of NADPH oxidase (NOX) and synthesis of hydrogen peroxide and oxygen radicals. Stable RNA‐mediated silencing of syndecan‐1 gene expression up‐regulated NOX‐dependent generation of reactive oxygen species and reduced the survival of holoclones in vitro. Syndecan‐1 down‐regulation also strongly reduced the number of CD133⁺/CD44⁺ primitive cancer cells and tumour growth in vivo. Interestingly, syndecan‐1 gene knockdown significantly enhanced the tumour‐suppressive effects of docetaxel by inhibiting the docetaxel‐induced increase in CD133⁺/CD44⁺ cells in vivo. In the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model of prostate cancer, early intervention with a syndecan‐1 inhibitor (OGT2115) or syndecan‐1 RNAi reduced the incidence of adenocarcinoma and the number of c‐kit⁺/CD44⁺ cells in cancer foci. Finally, we found that syndecan‐1 immunopositivity in prostate cancer cells was significantly associated with biochemical recurrence after radical prostatectomy. Taken together, our results show that syndecan‐1 contributes to prostatic carcinogenesis by maintaining TICs and may be a target molecule for therapy. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

HR‐MAS MRS of the pancreas reveals reduced lipid and elevated lactate and taurine associated with early pancreatic cancer

The prognosis for patients with pancreatic cancer is extremely poor, as evidenced by the disease's five‐year survival rate of ~5%. New approaches are therefore urgently needed to improve detection, treatment, and monitoring of pancreatic cancer. MRS‐detectable metabolic changes provide useful biomarkers for tumor detection and response‐monitoring in other cancers. The goal of this study was to identify MRS‐detectable biomarkers of pancreatic cancer that could enhance currently available imaging approaches. We used ¹H high‐resolution magic angle spinning MRS to probe metabolite levels in pancreatic tissue samples from mouse models and patients. In mice, the levels of lipids dropped significantly in pancreata with lipopolysaccharide‐induced inflammation, in pancreata with pre‐cancerous metaplasia (4 week old p48‐Cre;LSL‐Kras^G12D mice), and in pancreata with pancreatic intraepithelial neoplasia, which precedes invasive pancreatic cancer (8 week old p48‐Cre LSL‐Kras^G12D mice), to 26 ± 19% (p = 0.03), 19 ± 16% (p = 0.04), and 26 ± 10% (p = 0.05) of controls, respectively. Lactate and taurine remained unchanged in inflammation and in pre‐cancerous metaplasia but increased significantly in pancreatic intraepithelial neoplasia to 266 ± 61% (p = 0.0001) and 999 ± 174% (p < 0.00001) of controls, respectively. Importantly, analysis of patient biopsies was consistent with the mouse findings. Lipids dropped in pancreatitis and in invasive cancer biopsies to 29 ± 15% (p = 0.01) and 26 ± 38% (p = 0.02) of normal tissue. In addition, lactate and taurine levels remained unchanged in inflammation but rose in tumor samples to 244 ± 155% (p = 0.02) and 188 ± 67% (p = 0.02), respectively, compared with normal tissue. Based on these findings, we propose that a drop in lipid levels could serve to inform on pancreatitis and cancer‐associated inflammation, whereas elevated lactate and taurine could serve to identify the presence of pancreatic intraepithelial neoplasia and invasive tumor. Our findings may help enhance current imaging methods to improve early pancreatic cancer detection and monitoring. Copyright © 2014 John Wiley & Sons, Ltd.     

Protease‐activated receptor 2 suppresses lymphangiogenesis and subsequent lymph node metastasis in a murine pancreatic cancer model

Protease‐activated receptor‐2 (PAR‐2) is a G protein‐coupled receptor that functions as a cell‐surface sensor for coagulation factors and other proteases associated with the tumour microenvironment. Pancreatic cancer cells express high levels of PAR‐2 and activation of PAR‐2 may induce their proliferation and migration. Interestingly, however, PAR‐2 expression is increased in stroma‐rich pancreatic cancer regions, suggesting a potential role of PAR‐2 in the tumour microenvironment. Here, we assessed the importance of PAR‐2 in the stromal compartment by utilizing an orthotopic pancreatic cancer model, in which tumour cells are PAR‐2‐positive, whereas stromal cells are PAR‐2‐negative. We assessed tumour weight and volume and analysed proliferation and (lymph)angiogenesis both in vivo and in vitro. We show that genetic ablation of PAR‐2 from the stromal compartment inhibits primary tumour growth, which is accompanied by reduced vascularization in primary tumours and reduced in tube formation of vascular endothelial cells in vitro. In contrast to smaller primary tumours, the number of lymph node metastases was increased in PAR‐2‐deficient animals, which was accompanied by an increased number of lymphatic vessels. In vitro tube‐formation assays show that PAR‐2 does not inhibit the intrinsic tube‐forming capacity of lymphatic endothelial cells, but that PAR‐2 actually inhibits cancer cell‐induced tube formation. Overall, stromal PAR‐2 thus plays a dual role in pancreatic cancer development by potentiating primary tumour growth but limiting lymphangiogenesis and subsequent lymph node metastasis. Our data identify a novel role of PAR‐2 in the tumour microenvironment and pinpoint PAR‐2 as a negative regulator of lymphangiogenesis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Suppression of murine tumour growth through CD8+ cytotoxic T lymphocytes via activated DEC‐205+ dendritic cells by sequential administration of α‐galactosylceramide in vivo

Cancer immunity is mediated through the effective priming and activation of tumour‐specific class I MHC molecule‐restricted CD8⁺ cytotoxic T lymphocytes (CTLs). DEC‐205⁺ dendritic cells (DCs) can cross‐present the epitope(s) of captured tumour antigens associated with class I MHC molecules alongside co‐stimulatory molecules to prime and activate tumour‐specific CD8⁺ CTLs. Immunosuppressive tolerogenic DCs with reduced co‐stimulatory molecules may be a cause of impaired CTL induction. Hepa1‐6‐1 cells were established from the mouse hepatoma cell line Hepa1‐6; these cells grow continuously after subcutaneous implantation into syngeneic C57BL/6 (B6) mice and do not prime CD8⁺ CTLs. In this study, we show that the growth of ongoing tumours was suppressed by activated CD8⁺ CTLs with tumour‐specific cytotoxicity through the administration of the glycolipid α‐galactosylceramide (α‐GalCer), which is a compound known to stimulate invariant natural killer T (iNKT) cells and selectively activate DEC‐205⁺ DCs. Moreover, we demonstrated that sequential repetitive intraperitoneal inoculation with α‐GalCer every 48 hr appeared to convert tolerogenic DEC‐205⁺ DCs into immunogenic DCs with a higher expression of co‐stimulatory molecules and a stronger cross‐presentation capacity, which primed CTL precursors and induced tumour‐specific CD8⁺ CTLs within the tumour environment without activating iNKT cells. These findings provide a new basis for cancer immunotherapy to convert tolerogenic DEC‐205⁺ DCs within tumours into immunogenic DCs through the sequential administration of an immuno‐potent lipid/glycolipid, and then activated immunogenic DCs with sufficient expression of co‐stimulatory molecules prime and activate tumour‐specific CD8⁺ CTLs within the tumour to control tumour growth.