Long pentraxin‐3 as an epithelial–stromal fibroblast growth factor‐targeting inhibitor in prostate cancer

Fibroblast growth factors (FGFs) exert autocrine/paracrine functions in prostate cancer by stimulating angiogenesis and tumour growth. Here dihydrotestosterone (DHT) up‐regulates FGF2 and FGF8b production in murine TRAMP‐C2 prostate cancer cells, activating a FGF‐dependent autocrine loop of stimulation. The soluble pattern recognition receptor long pentraxin‐3 (PTX3) acts as a natural FGF antagonist that binds FGF2 and FGF8b via its N‐terminal domain. We demonstrate that recombinant PTX3 protein and the PTX3‐derived pentapeptide Ac‐ARPCA‐NH₂ abolish the mitogenic response of murine TRAMP‐C2 cells and human LNCaP prostate cancer cells to DHT and FGFs. Also, PTX3 hampers the angiogenic activity of DHT‐activated TRAMP‐C2 cells on the chick embryo chorioallantoic membrane (CAM). Accordingly, human PTX3 overexpression inhibits the mitogenic activity exerted by DHT or FGFs on hPTX3_TRAMP‐C2 cell transfectants and their angiogenic activity. Also, hPTX3_TRAMP‐C2 cells show a dramatic decrease of their angiogenic and tumourigenic potential when grafted in syngeneic or immunodeficient athymic male mice. A similar inhibitory effect is observed when TRAMP‐C2 cells overexpress only the FGF‐binding N‐terminal PTX3 domain. In keeping with the anti‐tumour activity of PTX3 in experimental prostate cancer, immunohistochemical analysis of prostate needle biopsies from primary prostate adenocarcinoma patients shows that parenchymal PTX3 expression, abundant in basal cells of normal glands, is lost in high‐grade prostatic intraepithelial neoplasia and in invasive tumour areas. These results identify PTX3 as a potent FGF antagonist endowed with anti‐angiogenic and anti‐neoplastic activity in prostate cancer. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

MicroRNA‐542‐3p inhibits tumour angiogenesis by targeting Angiopoietin‐2

Angiopoietin‐2 (Angpt2) plays a critical role in angiogenesis and tumour progression. Therapeutic targeting of Angpt2 has been implicated as a promising strategy for cancer treatment. Whereas miRNAs are emerging as important modulators of angiogenesis, regulation of Angpt2 by miRNAs has not been established. Here we firstly report that Ang2 is targeted by a microRNA, miRNA‐542‐3p, which inhibits tumour progression by impairing Ang2's pro‐angiogenic activity. In cultured endothelial cells, miR‐542‐3p inhibited translation of Angpt2 mRNA by binding to its 3′ UTR, and addition of miR‐542‐3p to cultured endothelial cells attenuated angiogenesis. Administration of miR‐542‐3p to tumour‐bearing mice reduced tumour growth, angiogenesis and metastasis. Furthermore, the level of miR‐542‐3p in primary breast carcinomas correlated inversely with clinical progression in primary tumour samples from stage III and IV patients. Together, these findings uncover a novel regulatory pathway whereby an anti‐angiogenic miR‐542‐3p directly targets the key angiogenesis‐promoting protein Angpt2, suggesting that miR‐542‐3p may represent a promising target for anti‐angiogenic therapy and a potential marker for monitoring disease progression. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

Immunomodulatory glc/man‐directed Dolichos lablab lectin (DLL) evokes anti‐tumour response in vivo by counteracting angiogenic gene expressions

Neovascularization and jeopardized immunity has been critically emphasized for the establishment of malignant progression. Lectins are the diverse class of carbohydrate interacting proteins, having great potential as immunopotentiating and anti‐cancer agents. The present investigation sought to demonstrate the anti‐proliferative activity of Dolichos lablab lectin (DLL) encompassing immunomodulatory attributes. DLL specific to glucose and mannose carbohydrate moieties has been purified to homogeneity from the common dietary legume D. lablab. Results elucidated that DLL agglutinated blood cells non‐specifically and displayed striking mitogenicity to human and murine lymphocytes in vitro with interleukin (IL)‐2 production. The DLL‐conditioned medium exerted cytotoxicity towards malignant cells and neoangiogenesis in vitro. Similarly, in‐vivo anti‐tumour investigation of DLL elucidated the regressed proliferation of ascitic and solid tumour cells, which was paralleled with blockade of tumour neovasculature. DLL‐treated mice showed an up‐regulated immunoregulatory cytokine IL‐2 in contrast to severely declined levels in control mice. Mechanistic validation revealed that DLL has abrogated the microvessel formation by weakening the proangiogenic signals, specifically nuclear factor kappa B (NF‐κB), hypoxia inducible factor 1α (HIF‐1 α), matrix metalloproteinase (MMP)‐2 and 9 and vascular endothelial growth factor (VEGF) in malignant cells leading to tumour regression. In summary, it is evident that the dietary lectin DLL potentially dampens the malignant establishment by mitigating neoangiogenesis and immune shutdown. For the first time, to our knowledge, this study illustrates the critical role of DLL as an immunostimulatory and anti‐angiogenic molecule in cancer therapeutics.     

A host deficiency of discoidin domain receptor 2 (DDR2) inhibits both tumour angiogenesis and metastasis

Discoidin domain receptor 2 (DDR2) is a unique receptor tyrosine kinase (RTK) that signals in response to collagen binding and is implicated in tumour malignant phenotypes such as invasion and metastasis. Although it has been reported that DDR2 expression is up‐regulated in activated endothelial cells (ECs), functional studies are lacking. Herein, we found that enforced expression of DDR2 promoted proliferation, migration and tube formation of primary human umbilical vein endothelial cells (HUVECs). The results of immunohistochemical analysis showed a strikingly high level of DDR2 in human tumour ECs. Most significantly, we discovered that a host deficiency of DDR2 inhibits subcutaneous angiogenesis induced by either VEGF or tumour cells. In addition, the remaining tumour vessels in DDR2‐deficient mice exhibit some normalized properties. These vascular phenotypes are accompanied by the up‐regulation of anti‐angiogenic genes and down‐regulation of pro‐angiogenic genes, as well as by alleviated tumour hypoxia. By use of a tail vein metastasis model of melanoma, we uncovered that loss of stromal DDR2 also suppresses tumour metastasis to the lung. Hence, our current data disclose a new mechanism by which DDR2 affects tumour progression, and may strengthen the feasibility of targeting DDR2 as an anticancer strategy. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Endostatin inhibits tumour lymphangiogenesis and lymphatic metastasis via cell surface nucleolin on lymphangiogenic endothelial cells

Endostatin has potent anti‐endothelial and anti‐angiogenic functions. Endostatin was reported to reduce lymphangiogenesis by down‐regulating the level of VEGF‐C in tumour tissues. However, there is little evidence for the direct function of endostatin on lymphangiogenic endothelial cells and lymphangiogenic vessels. Here, we report that cell surface nucleolin, which was reported as an endostatin receptor mediating its anti‐angiogenic and anti‐tumour functions, is also selectively expressed on the cell surface of lymphangiogenic endothelial cells both in vitro and in vivo. Treatment of primary mouse lymphatic endothelial cells (mLECs) by endostatin inhibits mLEC migration, tubule formation, and activation of the Erk pathway in mLECs, while neutralization of cell surface nucleolin or nucleolin knockdown results in loss of the anti‐lymphatic endothelial activities of endostatin. Also, anti‐nucleolin antibody or lentivirus delivered nucleolin siRNA abolishes the anti‐lymphangiogenic function of endostatin in the Matrigel plug assay. Endostatin remarkably inhibits tumour‐associated lymphangiogenesis, leading to reduced lymphatic metastasis. Systemic blockade of nucleolin notably abolishes the anti‐lymphangiogenic and anti‐lymphatic metastatic functions of endostatin. Importantly, endostatin does not affect quiescent lymphatics in normal organs, which is consistent with the lack of expression of cell surface nucleolin in quiescent lymphatics. Taken together, our results demonstrate that endostatin directly acts on lymphangiogenic endothelial cells via cell surface nucleolin, which provides a novel mechanism for the inhibition of tumour lymphangiogenesis and lymphatic metastasis by endostatin. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Local expression of interleukin‐2 by B16 melanoma cells results in decreased tumour growth and long‐term tumour dormancy

The tumour microenvironment is complex containing not only neoplastic cells but also a variety of host cells. The heterogeneous infiltrating immune cells include subsets of cells with opposing functions, whose activities are mediated either directly or through the cytokines they produce. Systemic delivery of cytokines such as interleukin‐2 ( IL‐2) has been used clinically to enhance anti‐tumour responses, but these molecules are generally thought to have evolved to act locally in a paracrine fashion. In this study we examined the effect of local production of IL‐2 on the growth and the immune response to B16 melanoma cells. We found that the local production of IL‐2 enhances the number of interferon‐γ‐expressing CD8 T and natural killer cells in the tumour, as well as inducing expression of vascular cell adhesion molecule 1 on tumour vessels. These responses were largely absent in interferon‐γ knockout mice. The expression of IL‐2 in the tumour microenvironment decreases tumour growth despite also enhancing Foxp3⁺ CD4⁺ regulatory T cells and anti‐inflammatory cytokines such as IL‐10. Higher levels of IL‐2 in the tumour microenvironment eliminated the progressive growth of the B16 cells in vivo, and this inhibition was dependent on the presence of either T cells or, to a lesser extent, natural killer cells. Surprisingly however, the B16 tumours were not completely eliminated but instead were controlled for an extended period of time, suggesting that a form of tumour dormancy was established.     

Accelerated tumour metastasis due to interferon‐γ receptor‐mediated dissociation of perivascular cells from blood vessels

Angiostasis mediated by interferon (IFN)‐γ is a key mechanism of anti‐tumour immunity; however, the effect of IFN‐γ on host vascular endothelial growth factor A (VEGFA)‐expressing cells during tumour progression is still elusive. Here, we developed transgenic mice with IFN‐γ receptor (IFNγR) expression under control of the Vegfa promoter (V‐γR). In these mice, the IFN‐γ responsiveness of VEGFA‐expressing cells led to dramatic growth suppression of transplanted lung carcinoma cells. Surprisingly, increased mortality and tumour metastasis were observed in the tumour‐bearing V‐γR mice, in comparison with the control wild‐type and IFNγR‐deficient mice. Further study showed that perivascular cells were VEGFA‐expressing cells and potential IFN‐γ targets. In vivo, tumour vascular perfusion and pericyte association with blood vessels were massively disrupted in V‐γR mice. In vitro, IFN‐γ inhibited transforming growth factor‐β signalling by upregulating SMAD7, and therefore downregulated N‐cadherin expression in pericytes. Importantly, IFN‐γ neutralization in vivo with a monoclonal antibody reduced tumour metastasis. Together, the results suggest that IFNγR‐mediated dissociation of perivascular cells from blood vessels contributes to the acceleration of tumour metastasis. Thus, the inhibition of tumour growth via IFN‐γ‐induced angiostasis might also accelerate tumour metastasis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

CD169 identifies an anti‐tumour macrophage subpopulation in human hepatocellular carcinoma

Macrophages are a major component of most solid tumours and can exert both anti‐ and pro‐tumourigenic functions. Although the immunosuppressive/pro‐tumour roles of macrophages have been widely examined, significantly less is known about macrophage subpopulations that have potential anti‐tumour properties in humans. In the present study, a population of CD169⁺ macrophages with relatively high expression levels of HLA‐DR and CD86 was identified in human hepatocellular carcinoma tissues. The frequency of CD169‐expressing macrophages within cancer nests was significantly lower than that found in paired non‐tumour areas. In vitro experiments revealed that in the presence of anti‐CD3 stimulation, CD169⁺ macrophages could significantly enhance the proliferation, cytotoxicity, and cytokine production capacity of CD8⁺ T cells in a CD169 molecule‐dependent manner. Autocrine TGF‐β produced by tumour‐stimulated macrophages was involved in the down‐regulation of CD169 expression on these cells. Moreover, the accumulation of CD169⁺ macrophages in tumour tissues was negatively associated with disease progression and predicted favourable survival in hepatocellular carcinoma patients, which was in contrast to the trend observed for total CD68⁺ macrophages. Therefore, CD169 might act as a co‐stimulatory molecule for cytotoxic T‐cell activation, and could define a population of tumour‐infiltrating macrophages with potential anti‐tumour properties in human hepatocellular carcinoma tissues. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Specific chemotherapeutic agents induce metastatic behaviour through stromal‐ and tumour‐derived cytokine and angiogenic factor signalling

Recent studies reveal that chemotherapy can enhance metastasis due to host responses, such as augmented expression of adhesion molecules in endothelial cells and increased populations of myeloid cells. However, it is still unclear how tumour cells contribute to this process. Here, we observed that paclitaxel and carboplatin accelerated lung metastasis in tumour‐bearing mice, while doxorubicin and fluorouracil did not. Mechanistically, paclitaxel and carboplatin induced similar changes in cytokine and angiogenic factors. Increased levels of CXCR2, CXCR4, S1P/S1PR1, PlGF and PDGF‐BB were identified in the serum or primary tumour tissues of tumour‐bearing mice treated by paclitaxel. The serum levels of CXCL1 and PDGF‐BB and the tissue level of CXCR4 were also elevated by carboplatin. On the other hand, doxorubicin and fluorouracil did not induce such changes. The chemotherapy‐induced cytokine and angiogenic factor changes were also confirmed in gene expression datasets from human patients following chemotherapy treatment. These chemotherapy‐enhanced cytokines and angiogenic factors further induced angiogenesis, destabilized vascular integrity, recruited BMDCs to metastatic organs and mediated the proliferation, migration and epithelial‐to‐mesenchymal transition of tumour cells. Interestingly, inhibitors of these factors counteracted chemotherapy‐enhanced metastasis in both tumour‐bearing mice and normal mice injected intravenously with B16F10–GFP cells. In particular, blockade of the SDF‐1α–CXCR4 or S1P–S1PR1 axes not only compromised chemotherapy‐induced metastasis but also prolonged the median survival time by 33.9% and 40.3%, respectively. The current study delineates the mechanism of chemotherapy‐induced metastasis and provides novel therapeutic strategies to counterbalance pro‐metastatic effects of chemo‐drugs via combination treatment with anti‐cytokine/anti‐angiogenic therapy. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Deficiency of bone marrow β3‐integrin enhances non‐functional neovascularization

β3‐Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in β3‐integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on β3‐null endothelial cells. Here we transplanted β3‐null bone marrow (BM) into wild‐type (WT) mice to dissect the role of BM β3‐integrin deficiency in pathological angiogenesis. Mice transplanted with β3‐null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow β3‐integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM β3‐integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with β3‐null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with β3‐null bone marrow a significant proportion of tumour blood vessels are non‐functional when compared with tumour blood vessels in WT‐transplanted controls. Furthermore, β3‐null‐transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT‐transplanted animals. BM β3‐integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF‐induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with β3‐null bone marrow when compared with WT‐transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in β3‐null‐transplanted mice. In conclusion, although BM β3‐integrin is not required for pathological angiogenesis, our studies demonstrate a role for BM β3‐integrin in VEGF‐induced mobilization of bone marrow‐derived cells to the peripheral circulation and for the functionality of those vessels in which BM‐derived cells become incorporated. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Extracellular 2′5′‐oligoadenylate synthetase 2 mediates T‐cell receptor CD3‐ζ chain down‐regulation via caspase‐3 activation in oral cancer

Decreased expression of CD3‐ζ chain, an adaptor protein associated with T‐cell signalling, is well documented in patients with oral cancer, but the mechanistic justifications are fragmentary. Previous studies in patients with oral cancer have shown that decreased expression of CD3‐ζ chain was associated with decreased responsiveness of T cells. Tumours are known to induce localized as well as systemic immune suppression. This study provides evidence that oral tumour‐derived factors promote immune suppression by down‐regulating CD3‐ζ chain expression. 2′5′‐Oligoadenylate synthetase 2 (OAS2) was identified by the proteomic approach and our results established a causative link between CD3‐ζ chain down‐regulation and OAS2 stimulation. The surrogate situation was established by over‐expressing OAS2 in a HEK293 cell line and cell‐free supernatant was collected. These supernatants when incubated with T cells resulted in down‐regulation of CD3‐ζ chain, which shows that the secreted OAS2 is capable of regulating CD3‐ζ chain expression. Incubation of T cells with cell‐free supernatants of oral tumours or recombinant human OAS2 (rh‐OAS2) induced caspase‐3 activation, which resulted in CD3‐ζ chain down‐regulation. Caspase‐3 inhibition/down‐regulation using pharmacological inhibitor or small interfering RNA restored down‐regulated CD3‐ζ chain expression in T cells induced by cell‐free tumour supernatant or rh‐OAS2. Collectively these results show that OAS2 leads to impairment in CD3‐ζ chain expression, so offering an explanation that might be applicable to the CD3‐ζ chain deficiency observed in cancer and diverse disease conditions.     

Simultaneous blockade of programmed death 1 and vascular endothelial growth factor receptor 2 (VEGFR2) induces synergistic anti‐tumour effect in vivo

Recent basic and clinical studies have shown that the programmed death ligand (PD‐L)/PD‐1 pathway has a significant role in tumour immunity, and its blockade has a therapeutic potential against several human cancers. We hypothesized that anti‐angiogeneic treatment might augment the efficacy of PD‐1 blockade. To this end, we evaluated combining the blockade of PD‐1 and vascular endothelial growth factor receptor 2 ( VEGFR2) in a murine cancer model using Colon‐26 adenocarcinoma. Interestingly, simultaneous treatment with anti‐PD‐1 and anti‐VEGFR2 monoclonal antibodies (mAbs) inhibited tumour growth synergistically in vivo without overt toxicity. Blocking VEGFR2 inhibited tumour neovascularization significantly, as demonstrated by the reduced number of microvessels, while PD‐1 blockade had no impact on tumour angiogenesis. PD‐1 blockade might promote T cell infiltration into tumours and significantly enhanced local immune activation, as shown by the up‐regulation of several proinflammatory cytokine expressions. Importantly, VEGFR2 blockade did not interfere with T cell infiltration and immunological activation induced by PD‐1 blockade. In conclusion, simultaneous blockade of PD‐1 and VEGFR2 induced a synergistic in‐vivo anti‐tumour effect, possibly through different mechanisms that might not be mutually exclusive. This unique therapeutic strategy may hold significant promise for future clinical application.     

Altered ratios of pro‐ and anti‐angiogenic VEGF‐A variants and pericyte expression of DLL4 disrupt vascular maturation in infantile haemangioma

Infantile haemangioma (IH), the most common neoplasm in infants, is a slowly resolving vascular tumour. Vascular endothelial growth factor A (VEGF‐A), which consists of both the pro‐ and anti‐angiogenic variants, contributes to the pathogenesis of IH. However, the roles of different VEGF‐A variants in IH progression and its spontaneous involution is unknown. Using patient‐derived cells and surgical specimens, we showed that the relative level of VEGF‐A₁₆₅b was increased in the involuting phase of IH and the relative change in VEGF‐A isoforms may be dependent on endothelial differentiation of IH stem cells. VEGFR signalling regulated IH cell functions and VEGF‐A₁₆₅b inhibited cell proliferation and the angiogenic potential of IH endothelial cells in vitro and in vivo. The inhibition of angiogenesis by VEGF‐A₁₆₅b was associated with the extent of VEGF receptor 2 (VEGFR2) activation and degradation and Delta‐like ligand 4 (DLL4) expression. These results indicate that VEGF‐A variants can be regulated by cell differentiation and are involved in IH progression. We also demonstrated that DLL4 expression was not exclusive to the endothelium in IH but was also present in pericytes, where the expression of VEGFR2 is absent, suggesting that pericyte‐derived DLL4 may prevent sprouting during involution, independently of VEGFR2. Angiogenesis in IH therefore appears to be controlled by DLL4 within the endothelium in a VEGF‐A isoform‐dependent manner, and in perivascular cells in a VEGF‐independent manner. The contribution of VEGF‐A isoforms to disease progression also indicates that IH may be associated with altered splicing. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Immune cells regulate VEGF signalling via release of VEGF and antagonistic soluble VEGF receptor‐1

Vascular endothelial growth factor (VEGF) is an important regulator of physiological and pathological angiogenesis. Besides malignant and stromal cells, local immune cells shape VEGF signalling in the tumour microenvironment. Aminobisphosphonates such as zoledronic acid (Zol) are drugs known to inhibit osteoclast activity and bone resorption, but also have immunomodulatory and anti‐tumour effects. These properties have been linked previously to the down‐regulation of VEGF and interference with tumour neo‐angiogenesis. It was therefore surprising to find that treatment with Zol in combination with low‐dose interleukin (IL)‐2 increased serum VEGF levels in cancer patients. In this study we aimed to characterize the effect of Zol and IL‐2 on VEGF signalling of blood‐derived immune cells in vitro. Upon stimulation with IL‐2, T cells and natural killer (NK) cells increase production of VEGF consecutively to the release of proinflammatory interferon (IFN)‐γ, and Zol accelerates this response specifically in γδ T cells. VEGF can, in turn, be antagonized by soluble VEGF receptor (sVEGFR)‐1, which is released depending on stimulatory conditions and the presence of monocytes. Additionally, malignant cells represented by leukaemia and lymphoma cell lines produce VEGF and some release sVEGFR‐1 simultaneously. Our findings indicate a mechanism by which the VEGF and the sVEGFR‐1 production by immune cells regulates local VEGF signalling. Therefore, immunotherapeutic interventions may enable both pro‐ as well as anti‐tumour effects via immune cell‐mediated alterations of VEGF homeostasis.     

Balance of pro‐ versus anti‐angiogenic splice isoforms of vascular endothelial growth factor as a regulator of neuroblastoma growth

Neuroblastoma (NB) is the second most common extracranial tumour of childhood. Angiogenesis plays a crucial role in the growth and development of NB and vascular endothelial growth factor (VEGF), one of the most potent stimuli of angiogenesis, has been studied extensively in vitro. VEGF₁₆₅ has been shown to be the predominant angiogenic isoform expressed in NB cell lines and tumours. In this study, we investigated the anti‐angiogenic isoform of VEGF‐A, generated from distal splice site selection in the terminal exon of VEGF (VEGF₁₆₅b) and shown to be down‐regulated in epithelial malignancies. The expression of both the pro‐ (VEGF_xxx) and the anti‐angiogenic (VEGF_xxxb) isoforms was compared in a range of NB and ganglioneuroma (GN) tumours. Whereas VEGF_xxxb and VEGF_xxx were both expressed in GN, specific up‐regulation of the VEGF_xxx isoforms was seen in NB at RNA and protein levels. Highly tumourigenic NB cell lines also showed up‐regulation of the angiogenic isoforms relative to VEGF_xxxb compared to less tumourigenic cell lines, and the isoforms were differentially secreted. These results indicate that VEGF₁₆₅ is up‐regulated in NB and that there is a difference in the balance of isoform expression from anti‐angiogenic VEGF₁₆₅b to angiogenic VEGF₁₆₅. Treatment with recombinant human VEGF₁₆₅b significantly reduced the growth rate of established xenografts of SK‐N‐BE(2)‐C cells (4.24 ± 1.01 fold increase in volume) compared with those treated with saline (9.76 ± 3.58, p < 0.01). Microvascular density (MVD) was significantly decreased in rhVEGF₁₆₅b‐treated tumours (19.4 ± 1.9 vessels/mm³) in contrast to the saline‐treated tumours (45.5 ± 8.6 vessels/mm³). VEGF₁₆₅b had no significant effect on the proliferative or apoptotic activity, viability or cytotoxicity of SK‐N‐BE(2)‐C cells after 48 h. In conclusion, VEGF₁₆₅b is an effective inhibitor of NB growth. These findings provide the rationale for further investigation of VEGF₁₆₅b in NB and other paediatric malignancies. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Anti‐interleukin‐10R1 monoclonal antibody in combination with bacillus Calmette–Guérin is protective against bladder cancer metastasis in a murine orthotopic tumour model and demonstrates systemic specific anti‐tumour immunity

Effective treatment of bladder cancer with bacillus Calmette–Guérin (BCG) depends on the induction of a T helper type (Th) 1 immune response. Interleukin (IL)‐10 down‐regulates the Th1 response and is associated with BCG failure. In this study, we investigated whether blocking IL‐10 signalling could enhance the BCG‐induced Th1 response and anti‐tumour immunity in a murine orthotopic tumour model. Treatment with BCG and anti‐IL‐10 receptor 1 monoclonal antibody (anti‐IL‐10R1 mAb) increased the interferon (IFN)‐γ to IL‐10 ratio in both splenocyte cultures and urine. Mice bearing luciferase‐expressing MB49 (MB49‐Luc) tumours were treated and followed for tumour growth by bioluminescent imaging, bladder weight and histology. Mice treated with phosphate‐buffered saline (PBS) (group 1), BCG plus control immunoglobulin (Ig)G1 (group 2) or BCG plus anti‐IL‐10R1 mAb (group 3) showed 0, 6 and 22% tumour regression, respectively. The mean bladder weight of group 3 mice was substantially lower than those of groups 1 and 2 mice. Remarkably, 36% of group 1 and 53% of group 2 mice but no group 3 mice developed lung metastasis (P = 0·02). To investigate the mechanisms underlying the effect of combination therapy, splenocytes were stimulated with S12 peptide (serine mutation at codon 12 of the K‐ras oncogene) known to be expressed in MB49‐Luc cells. Induction of ras mutation‐specific IFN‐γ and cytotoxicity was observed in mice treated with combination therapy. These observations indicate that BCG, in combination with anti‐IL‐10R1 mAb, induces enhanced anti‐tumour immunity that is protective against lung metastasis. Anti‐IL‐10R1 mAb demonstrates systemic effects and may prove useful in clinical practice for treating bladder cancer in high‐risk patients.     

Regulation of autoimmune and anti‐tumour T‐cell responses by PTPN22

A number of polymorphisms in immune‐regulatory genes have been identified as risk factors for the development of autoimmune disease. PTPN22 (that encodes a tyrosine phosphatase) has been associated with the development of several autoimmune diseases, including type 1 diabetes, rheumatoid arthritis and systemic lupus erythematosus. PTPN22 regulates the activity and effector functions of multiple important immune cell types, including lymphocytes, granulocytes and myeloid cells. In this review, we describe the role of PTPN22 in regulating T‐cell activation and effector responses. We discuss progress in our understanding of the impact of PTPN22 in autoimmune disease in humans and mouse models, as well as recent evidence suggesting that genetic manipulation of PTPN22 expression might enhance the efficacy of anti‐tumour T‐cell responses.     

Matrix metalloproteinases inhibition promotes the polyfunctionality of human natural killer cells in therapeutic antibody‐based anti‐tumour immunotherapy

Activation of human natural killer (NK) cells is associated with the cleavage of CD16 from the cell surface, a process mediated by matrix metalloproteinases (MMPs). In this report, we examined whether inhibition of MMPs would lead to improved NK cell antibody‐dependent cell‐mediated cytotoxicity (ADCC) function. Using an in‐vitro ADCC assay, we tested the anti‐tumour function of NK cells with three different therapeutic monoclonal antibodies (mAbs) in the presence of MMPs inhibitor GM6001 or its control. Loss of CD16 was observed when NK cells were co‐cultured with tumour targets in the presence of specific anti‐tumour antibodies, and was found particularly on the majority of degranulating NK responding cells. Treatment with MMPs inhibitors not only prevented CD16 down‐regulation, but improved the quality of the responding cells significantly, as shown by an increase in the percentage of polyfunctional NK cells that are capable of both producing cytokines and degranulation. Furthermore, MMPs inhibition resulted in augmented and sustained CD16‐mediated signalling, as shown by increased tyrosine phosphorylation of CD3ζ and other downstream signalling intermediates, which may account for the improved NK cell function. Collectively, our results provide a foundation for combining MMPs inhibitors and therapeutic mAbs in new clinical trials for cancer treatment.     

Characterization of the cell of origin and propagation potential of the fibroblast growth factor 9‐induced mouse model of lung adenocarcinoma

Fibroblast growth factor 9 (FGF9) is essential for lung development and is highly expressed in a subset of human lung adenocarcinomas. We recently described a mouse model in which FGF9 expression in the lung epithelium caused proliferation of the airway epithelium at the terminal bronchioles and led to rapid development of adenocarcinoma. Here, we used this model to characterize the effects of prolonged FGF9 induction on the proximal and distal lung epithelia, and examined the propagation potential of FGF9‐induced lung tumours. We showed that prolonged FGF9 over‐expression in the lung resulted in the development of adenocarcinomas arising from both alveolar type II and airway secretory cells in the lung parenchyma and airways, respectively. We found that tumour cells harboured tumour‐propagating cells that were able to form secondary tumours in recipient mice, regardless of FGF9 expression. However, the highest degree of tumour propagation was observed when unfractionated tumour cells were co‐administered with autologous, tumour‐associated mesenchymal cells. Although the initiation of lung adenocarcinomas was dependent on activation of the FGF9–FGF receptor 3 (FGFR3) signalling axis, maintenance and propagation of the tumour was independent of this signalling. Activation of an alternative FGF–FGFR axis and the interaction with tumour stromal cells is likely to be responsible for the development of this independence. This study demonstrates the complex role of FGF–FGFR signalling in the initiation, growth and propagation of lung cancer. Our findings suggest that analysing the expressions of FGF–FGFRs in human lung cancer will be a useful tool for guiding customized therapy. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.