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Supplementary Figure Legend from Angiotensinogen Delays Angiogenesis and Tumor Growth of Hepatocarcinoma in Transgenic Mice

Angiotensinogen, a member of the serpin family, is involved in the suppression of tumor growth and metastasis. To investigate whether human angiotensinogen protects against tumor progression in vivo, we established an original bitransgenic model in which transgenic mice expressing human angiotensinogen (Hu-AGT-TG mice) were crossed with a transgenic mouse model of hepatocellular carcinoma (HCC-TG mice). Bitransgenic mice overexpressing human angiotensinogen (HCC/Hu-AGT-TG) had a significantly longer survival time than the HCC-TG mice and a reduction of both tumor growth and blood flow velocities in the liver. This antitumor effect of angiotensinogen is related to a reduced angiogenesis, impaired expression of endothelial arterial markers (active Notch4, Delta-like 4 ligand, and ephrin B2) with a decrease of arterial vessel density in HCC/Hu-AGT-TG mice liver. Overexpression of human angiotensinogen decreases angiogenesis, and prevents tumor sinusoids from remodeling and arterialization, thus delaying tumor progression in vivo. [Cancer Res 2009;69(7):2853–60]

Supplementary Figure 1 from Angiotensinogen Delays Angiogenesis and Tumor Growth of Hepatocarcinoma in Transgenic Mice

Background: Non-alcoholic fatty liver disease (NAFLD), especially non-alcoholic steatohepatitis (NASH) is a chronic liver disease commonly associated with hepatic fibrosis. NASH patients have an increased risk for hepatocellular carcinoma (HCC). Due to western way of life, NASH incidence is rising and is predicted to become the leading cause of HCC in the next decades. Therefore, there is an urgent need for robust animal models fully recapitulating the NASH-related HCC carcinogenesis. In this study, we develop and characterize specific diet-induced variants from our transgenic HCC mouse model, focusing on immune landscape. Methods: To mimic NASH, ASV-B, a transgenic mouse model (C57BL/6J) that spontaneously develops a reproducible stage-defined HCC (hyperplasia at week(W)8, nodular stage at W12, and diffuse carcinoma at W16-20) was exposed to 5 different diets. Ten ASV-B and 5 control mice were fed as follows: classic diet as control (yellow), or a high-fat diet (blue), a diet enriched with saturated fatty acids + 1.25% cholesterol (green), a diet containing 22% of vegetal oil + 0.2% cholesterol (orange), and a 1.25% cholesterol diet containing 21% of milkfat (red). All mice fed with special diets also received 30% fructose in the drink water. RNA was extracted from frozen livers at W20 for 40 immune markers analysis using qRT-PCR (LightCycler, Roche). Immune populations were assessed using automated immunohistochemistry (IHC) (Bond Max, Leica). Results: ASV-B model shows an increase in liver volume and angiogenesis, ASV-B livers harboring marked arterialization and capillarization as compared to control. Assessing immune markers on 7 evaluable tumor specimens, we observed an increase in CD8, Foxp3, INOS, CD11b, PD-1, PD-L1, IL1β, IFN-γ, TNF-α, IL17A and IL17F mRNA expression, as frequently observed in human inflammatory HCC. In addition, IHC staining showed intratumoral infiltration of lymphocytes (CD8+) and macrophages (F4/80+, a well-characterized and extensively referenced mouse macrophage marker). ASV-B mice receiving yellow, blue, and green regimens showed similar liver volumes and weight. By macroscopic analysis, we observed increased liver steatosis, and fibrosis in the red and orange regimen compared to others. Moreover, we observed a 40% mortality rate in the orange regimen, and a 20% mortality rate in the blue and the green regimens. At the conference, we will show the morphologic changes of the livers using HPS staining and the immune landscape in the livers of the diet-variants. Conclusion: ASV-B transgenic mouse model mimics several characteristics of human HCC developing on healthy liver including inflammatory reaction and immune cell infiltration. In the ASV-B model, we have been able to develop specific-diets variants aiming at mimicking NASH that could be used for drug testing. Citation Format: Annemilaï Tijeras-Raballand, Christian Hobeika, Benoit Rousseau, Patricia Hainaud, Philippe Bonnin, Aurélie Rodrigues, Fouad Ladfil, Marc Pocard, Valérie Paradis, Armand de Gramont, Eric Raymond, Evelyne Dupuy, Clarisse Eveno, Sandrine Faivre. Diet-variants and immune characterization of a stage-defined, transgenic immunocompetent mouse model of HCC (ASV-B) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 91.

Tumor angiogenesis is closely associated with clinical staging and has been proposed to correlate with clinical response in terms of subsequent metastases following primary resection. Netrin-4 (NT-4) regulates angiogenic responses. Therefore, we sought to examine the effects of NT-4 on the primary tumor growth of colon cancer cells, liver and lung metastases of colon cancer cells, and responses following primary tumor resection. We used 3 different mouse models of orthotopic primary tumor and liver and lung metastases, comparing 2 human colon cancer cells lines: wild-type (low expression of NT-4) and NT-4 (overexpression of NT-4) LS174 cells. NT-4 overexpression inhibited the primary tumor growth of colorectal LS174 xenografts in nude mice (144.3±12.9 vs. 62.4±4.5 mm3; p

We hypothesized that in vitro treatment of peripheral blood mononuclear cells (PB-MNCs) from diabetic patients with ephrin-B2/Fc (EFNB2) improves their proangiogenic therapeutic potential in diabetic ischemic experimental models. Diabetes was induced in nude athymic mice by streptozotocin injections. At 9 weeks after hyperglycemia, 105 PB-MNCs from diabetic patients, pretreated by EFNB2, were intravenously injected in diabetic mice with hindlimb ischemia. Two weeks later, the postischemic neovascularization was evaluated. The mechanisms involved were investigated by flow cytometry analysis and in vitro cell biological assays. Paw skin blood flow, angiographic score, and capillary density were significantly increased in ischemic leg of diabetic mice receiving EFNB2-activated diabetic PB-MNCs versus those receiving nontreated diabetic PB-MNCs. EFNB2 bound to PB-MNCs and increased the adhesion and transmigration of PB-MNCs. Finally, EFNB2-activated PB-MNCs raised the number of circulating vascular progenitor cells in diabetic nude mice and increased the ability of endogenous bone marrow MNCs to differentiate into cells with endothelial phenotype and enhanced their proangiogenic potential. Therefore, EFNB2 treatment of PB-MNCs abrogates the diabetes-induced stem/progenitor cell dysfunction and opens a new avenue for the clinical development of an innovative and accessible strategy in diabetic patients with critical ischemic diseases.

Background: Hepatocellular carcinoma (HCC) is a complex multistep malignancy in need of new therapeutic options often arising on underlying chronic liver disease. HCC animal models relevant to clinical situations are crucial to investigate new anticancer drugs, alone or in combination. Mice bearing xenografts and transgenic mice are the two main models used for preclinical drug development, but most of them fail to mimic the different step of HCC observed in patients. In this study, we aim to describe a stage-defined, transgenic immunocompetent HCC mouse model. Methods: ASV-B is a transgenic mouse model that spontaneously develops, upon SV40 T-Ag oncogene expression in hepatocytes, a reproducible stage-defined HCC. Hyperplasia at week(W)8 is followed by nodular stage at W12 (multiple well-delimited tumor nodules of about 1 mm, growing progressively up to 1 cm), then diffuse carcinoma stage at W16-20. HCC is restricted to male, backcrossed with C57BL/6J mice, the female littermates being used as controls. Results: Liver volume assessed by ultrasound at each step of carcinogenesis showed a 2.7-, 2.7-, 3.9-, and 5.8-fold increase (p Conclusion: ASV-B transgenic mouse model mimics several characteristics of human HCC developing on healthy liver including multinodular disease, vessel abnormalities, and dedifferentiation toward mesenchymal phenotype. Further steps will consist of characterizing the adjacent nontumor liver, and developing nonalcoolic steatohepatitis (NASH) on ASV-B model using specific diets. Citation Format: Annemilaï Tijeras-Raballand, Patricia Hainaud, Christian Hobeika, Clarisse Eveno, Marc Pocard, Philippe Bonnin, Valérie Paradis, Mohamed Bouattour, Eric Raymond, Armand de Gramont, Evelyne Dupuy, Sandrine Faivre. Stage-defined, transgenic immunocompetent mouse model (ASV-B) to investigate new drugs for hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4096.

Background: Hepatocellular carcinoma (HCC) is characterized by strong abnormalities of liver vasculature linked to proangiogenic pathways in the tumor and its microenvironment. Upregulation of Notch4 and its ligand Dll4 in endothelial cells (ECs), as well as PlGF is involved in liver tumor growth, arterialization and sinusoid remodeling. However, the specific role of PlGF and its receptor VEGFR-1 toward Dll4/Notch4 in ECs and tumor vascular remodeling remains to be characterized. Material and Methods: In vitro, we used primary culture of HUVECs, isolated in our laboratory from human umbilical cords. Active forms of Notch4 and Dll4 in HUVECs were assessed by Western blot. In vivo, differential expression of VEGF-A, PlGF and their receptors were evaluated by qRT-PCR and immunostaining in ASV-B transgenic mice developing stage-defined HCC. Finally, we assessed in vivo PlGF and Notch inhibition, using siRNA PlGF, DAPT and a Notch inhibitor, LY3039478, alone or in combination with sorafenib. Results: In vitro, we first investigated the relative contribution of VEGF-A, VEGF-E and PlGF on Notch4 pathway, showing that all 3 growth factors increased Dll4 and Notch4 expression in HUVECs, the effect being more pronounced with PlGF. Downregulation of VEGFR-1 by si RNA abrogated VEGF-A and PlGF stimulation on Dll4 expression and Notch4 activation, whereas VEGFR-2 siRNA had no effect. PlGF silencing significantly reduced Dll4 dependent expression and Notch4 activation in response to VEGF-E and VEGF-A. To confirm in vivo the prevalence of PlGF/VEGFR-1 pathway on vessel abnormalities, we evaluated the differential expression of PlGF, VEGF-A, VEGFR-1 and VEGFR-2 in our ASV-B HCC model. VEGFR-1 expression increased with HCC progression and was restricted to macrophages and sinusoidal endothelial cells. PlGF levels were maximal at the stage that coincides with the beginning of liver vessels abnormalities in HCC. Silencing in vivo PlGF or blocking Notch4 using DAPT delayed tumor growth, reduced arterial vessel length, and sinusoids abnormalities while decreasing Dll4 and active Notch4 expression. Using a clinically relevant Notch inhibitor, LY3039478, alone or in combination with sorafenib, liver size and tumor macronodules number were significantly lower in all treatment arms vs placebo, Y3039478/sorafenib combination showing increased tumor control. Angiogenesis measured by mean blood flow in the coeliac trunk (TCm) decreased in all treatment arms vs placebo, confirmed by CD31 staining. Conclusions: These data suggest that PlGF/VEGFR-1-Dll4/Notch4 pathways are involved in vessel remodeling and tumor growth. In vivo, silencing PlGF or inhibiting Notch4 prevent tumor vessels remodeling/arterialization and delay tumor growth, further warranting new antiangiogenic strategies for HCC treatment. Citation Format: Annemilaï Tijeras-Raballand, Christian Hobeika, Jean-Olivier Contreres, Patricia Hainaud, Matthieu Martinet, Philippe Bonnin, Clarisse Eveno, Evelyne Dupuy, Marc Pocard, Valerie Paradis, Eric Raymond, Sandrine Faivre, Armand de Gramont. PlGF/VEGFR-1-dependent activation of Dll4/Notch4 pathway contributes to liver vessel abnormalities and tumor growth in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2043.

Angiotensinogen, a member of the serpin family, is involved in the suppression of tumor growth and metastasis. To investigate whether human angiotensinogen protects against tumor progression in vivo, we established an original bitransgenic model in which transgenic mice expressing human angiotensinogen (Hu-AGT-TG mice) were crossed with a transgenic mouse model of hepatocellular carcinoma (HCC-TG mice). Bitransgenic mice overexpressing human angiotensinogen (HCC/Hu-AGT-TG) had a significantly longer survival time than the HCC-TG mice and a reduction of both tumor growth and blood flow velocities in the liver. This antitumor effect of angiotensinogen is related to a reduced angiogenesis, impaired expression of endothelial arterial markers (active Notch4, Delta-like 4 ligand, and ephrin B2) with a decrease of arterial vessel density in HCC/Hu-AGT-TG mice liver. Overexpression of human angiotensinogen decreases angiogenesis, and prevents tumor sinusoids from remodeling and arterialization, thus delaying tumor progression in vivo. [Cancer Res 2009;69(7):2853–60]

To explore the possible role of a residual or variant alphaIIbbeta3 integrin (alphaIIbbeta3) in thrombogenesis, we used a new ex vivo perfusion chamber model to examine blood from patients with different subtypes of Glanzmann's thrombasthenia (GT). Non-anticoagulated blood was perfused through capillaries coated with type III collagen for 4.5 min (shear rate: 1600/s). Platelet deposition was quantified as platelet adhesion and mean thrombus size volume; fibrin and von Willebrand Factor (VWF) were specifically revealed by immunohistochemistry. In two patients with variant and in one patient with type II GT, platelet adhesion was maximal and we observed an unexpected formation of thrombi that were smaller than normal in size. These thrombi were surrounded by a thick meshwork that displayed a strong staining for fibrin and VWF. In two patients with heterozygous GT, platelet adhesion and thrombogenesis were normal. In two patients with type I GT, there was no thrombus formation, although platelet adhesion was also maximal. These data suggest the existence of a substitute pathway for thrombogenesis mediated by fibrin and possibly alphaIIbbeta3 (alphaIIbbeta3 at a reduced level, as in type II, and/or abnormal) as this fibrin network was not observed in type I GT with no alphaIIbbeta3. These interactions might facilitate haemostasis and even lead to thrombosis under certain favourable conditions. Furthermore, these data might have pharmacological relevance to the development of anti-alphaIIbbeta3 antithrombotic agents.

Background An interaction between tumor cells and the microenvironment, as well as the development of angiogenesis, are required to form liver metastases (LMs). Material and methods Immunofluorescence detection of α-smooth muscle actin, desmin, Ki67, laminin, and CD31 was used to analyze the kinetics of tumor angiogenesis determinants, especially the contribution of hepatic stellate cells (HSCs) to angiogenesis in hepatic metastasis produced by intrasplenically injected LS174 colorectal cancer cells. Immunostaining was performed at various times (days 9, 14, 28, and 39). Results At the earliest stage, micrometastases consisted of proliferating cancer cells, a well-organized network of activated HSCs and laminin deposits. No vascular network was observed. As the LMs grew in size, an organized vascular network appeared; the laminin network colocalized with CD31 immunostaining. At the later stages, all the immunostained markers became peripheral as a central necrosis developed. Purified activated HSCs isolated from transgenic mice livers developing hepatocellular carcinoma secreted laminin and showed enhanced human umbilical vein EC network formation in a Matrigel assay. In a coinjection LM experiment, activated HSCs enhanced the metastatic process. Moreover, colorectal LMs from six patients were analyzed, and a pattern of marker distribution similar to the coinjection experiment was found in human LMs. Conclusions For the first time, our results show that HSCs play a crucial role in organizing and accelerating the progression of metastasis in modulating the prometastatic niche, interacting with colorectal cancer cell recruitment, and the organization of angiogenesis during colorectal LM development. Therefore, HSCs may be an early therapeutic target in colorectal cancer therapies.

We studied the effect of SR33805, a calcium channel blocker, in vitro on the proliferation of vascular smooth muscle cells (SMC) stimulated by foetal calf serum, basic fibroblast growth factor and platelet derived growth factor, and in vivo with regard to SMC migration and proliferation which occurred following injury of the porcine carotid artery. The intimal lesion was induced by a silasten collar surgically positioned around the carotid artery and by a stenosis reducing blood flow by 50% for 30 days. Animals received SR33805 (5 mg/kg/day) 8 days before the induction of the lesion and up to 30 days after. In vitro, SR33805 inhibited in a dose-dependent manner growth factor-induced proliferation of SMC (0.20IC(50)0.46 microM). In vivo, SR33805 reduced the intima/media ratio of the cross sectional surface area (decrease of 60%, P0.05) without affecting neointimal SMC density. The medial SMC density was 40% lower in treated than in control animals (upstream, P0.05 and downstream to the stenosis, P0.01). Thus, it appears that SR33805 significantly reduced intimal hyperplasia, which occurred after perivascular manipulation of the artery, an effect consistent with its in vitro proliferation inhibitory activity, suggesting that long-term treatment with SR33805 may reduce or delay SMC proliferation.

Antithrombotic activity of two recombinant GPIb-binding fragments of vWF, RG12986 (residues 445-733), and VCL (residues 504-728), were assessed in an ex vivo capillary perfusion chamber exposing human type III collagen to native nonanticoagulated guinea pig blood. Platelet adhesion and thrombus formation were evaluated by computer assisted morphometry for two shear rates (650 and 1800 s-1) and for two perfusion times (1.5 and 4 min). At 1800 s-1 and 4 min of perfusion, platelet adhesion decreased from 63 +/- 7% for control, to 46 +/- 4% for 20 mg/kg RG12986, and to 29 +/- 5% for 4 mg/kg VCL, and the mean thrombus height dropped from 40 +/- 8 microns to 24 +/- 3 microns and 7.5 +/- 1 microns, respectively. The two doses did not change bleeding time values. Our results suggest that guinea pig blood and the circular perfusion chamber represent a good model for the evaluation of limited amount of GPIb/IX-vWF axis inhibitors.

In their study, they evaluated transplantation of theKM12SM human colon cancer cell line mixed with mesen-chymal stem cells (MSCs) into the spleen. They reported asignificantly greater number of liver metastases at 4 weekswith the cell mixture than with the transplantation ofKM12SM cells alone. Additionally, MSCs surrounding thetumour were functionally incorporated into the stroma of theorthotopic colon tumour and expressed carcinoma-associatedfibroblast markers.This manuscript prompted us to analyze the relevance ofthe murine model.To induce liver metastasis, a human colon cancer line(LS174) was injected into the spleen of nude mice. First, wecharacterized the bone marrow-derived cell (BMDC) popula-tion by fluorescence-activated cell sorting isolated on greenmice C57 BL/6-Tg obtained after a density gradient. Thepopulation contained 30% of BM-derived endothelial precur-sor cells and derived haematopoietic precursor cells thatexpressed CD 133. At day 0, we injected BMDCs or PBS intothe tail veins of tumor-bearing mice.At day 38, the injection of BMDCs enhanced the metastaticprocess in our model of liver metastasis with a number ofmicro or macrometastases in 56% of the BMDC group ( n ¼16) versus 20% in the PBS group (n ¼ 20), p < 0.05. Immuno-fluorescent staining of cut liver specimens has demonstratedthat BMDCs labelled with Green fluorescent protein (GFP)always localized to the tumour invasion front and colocalizedwith endothelial cells (Fig. 1).There is increasing evidence that BMDCs participate in thetumour microenvironment and metastatic progression

Background and Aims. An arterial blood supply and phenotypic changes of the sinusoids characterise the liver vasculature in human hepatocellular carcinoma (HCC). We investigated the effects of rosuvastatin on liver vessel anomalies, tumour growth and survival in HCC.Methods. We treated transgenic mice developing HCC, characterized by vessel anomalies similar to those of human HCC, with rosuvastatin.Results. In the rosuvastatin group, the survival time was longer(P<.001), and liver weight(P<.01)and nodule surface(P<.01)were reduced. Rosuvastatin decreased the number of smooth muscle actin-positive arteries(P<.05)and prevented the sinusoid anomalies, with decreased laminin expression(P<.001), activated hepatic stellate cells(P<.001), and active Notch4 expression. Furthermore, rosuvastatin inhibited endothelial cell but not tumour hepatocyte functions.Conclusions. Rosuvastatin reduced the vessel anomalies and tumour growth and prolonged survival in HCC. These results represent new mechanisms of the effects of statin on tumour angiogenesis and a potential target therapy in HCC.

SummaryOur findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.

Background: Hepatocellular carcinoma (HCC), is characterized by severe vessel anomalies with an intense arterial blood supply, and acquisition of a basal membrane rich in laminin by sinusoids. A role of the VEGF-A/Dll4-Notch4/ephrin B2 cascade in tumor vessel anomalies has been demonstrated, raising the question of the nature of the VEGF receptor pathway implicated in triggering this cascade. This study was undertaken to determinate the role of PlGF and its receptor VEGFR-1 in the development of vascular anomalies in HCC and to evaluate the role of the downstream Notch4 pathway. Material and Methods: In vitro, we used primary culture of HUVECs, isolated in our laboratory from human umbilical cords. HUVECs were cultured in EBM2 medium, with 20% of FBS and 2ng/ml of FGF-2. The expression of the active form of Notch4 and Dll4 in HUVECs were assessed by Western Blot. In vivo, the differential expression of VEGF-A, PlGF and their receptors were evaluated by qRT-PCR and immunostaining in transgenic mice developing stage-defined HCC. Results: In vitro, to determine which growth factor/receptor combination was predominant in activating the Notch4 pathway, we compared the effects of VEGF-A, VEGF-E and PlGF on Dll4 expression and Notch4 activation in HUVECs. Treatment with all 3 growth factors increased Dll4 and active Notch4 expressions whose levels were significantly greater in PlGF than VEGF-A and VEGF-E treated HUVECs. Moreover, downregulation of VEGFR-1 by si RNA, but not VEGFR-2, abrogated the effects of VEGF-A and PlGF stimulation on Dll4 expression and Notch4 activation. PlGF silencing significantly reduced Dll4 dependent expression and Notch4 activation in response to VEGF-E and VEGF-A. To confirm in vivo the prevalence of the PlGF/VEGFR-1 pathway in induction of vessel anomalies, we evaluated the differential expression of PlGF, VEGF-A, VEGFR-1 and VEGFR-2 in our transgenic HCC model at different stages. VEGFR-1 expression is increased with HCC progression and restricted in macrophages and sinusoidal endothelial cells. Moreover, PlGF levels were maximal at the stage that coincides with the beginning of liver vessels anomalies in HCC. Silencing PlGF or blocking Notch4 activation, using a γ-secretase inhibitor (DAPT), in vivo delayed tumor growth, reduced arterial vessel length, and sinusoids anomalies with a decrease in Dll4 and active Notch4 expression levels without affecting microvascular density or normal liver. Conclusions: Together, these data suggest that the activation of PlGF-VEGFR-1/ Dll4- Notch4/ephrin B2 cascades played a role in vessel remodeling and tumor growth. Inhibitor of γ-secretase or silencing PlGF in vivo reduced activation of Notch4, prevent tumor vessels remodelling and arterialization, leading to a delay in tumor growth. These results would argue that targeting PlGF or active Notch4 may be valuable new anti-angiogenic strategies in the treatment of HCC. Citation Format: Annemilai Tijeras-Raballand, Jean-Olivier Contreres, Patricia Hainaud-Hakim, Carole Le Hénaff, Marc Pocard, Evelyne Dupuy, Armand de Gramont. PlGF/VEGFR-1-dependent activation of the Dll4-Notch4/Ephrin B2 cascade contributes to liver vessel anomalies in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3564. doi:10.1158/1538-7445.AM2015-3564

SummaryThe occurrence of a thrombocytopathy concomitantly to the development of a malignant haemopathy has been reported for some time, but little is known about the mechanism(s) involved in the platelet dysfunction. Platelet glycoprotein VI (GPVI) has now been identified as a principal platelet receptor for collagen. In this paper, we report the cases of two patients with a myelodysplasia and a B lymphopathy, respectively, who presented with thrombocytopathy in relation to a defective GPVI-mediated platelet reactivity to collagen. Thus, with regard to the different steps of adhesion, activation secretion or aggregation, patients’ platelet responses to collagen and to the GPVI specific agonists, collagen related peptide (CRP) or convulxin were null or dramatically impaired. Platelet responses to other agonists ADP, TRAP,Arachidonic acid were normal or showed only a moderate decrease. GPVI content was repeatedly normal, and binding of specific ligands, such as convulxin, satisfactory. Nevertheless, specific activating monoclonal antibodies and convulxin failed to induce platelet secretion; collagen, CRP or convulxin were unable to provoke calcium mobilisation. Furthermore, using a perfusion chamber model, we showed that ex vivo collagen-induced thrombi formation was very impaired.Taken together,these data provide evidence, for the first time, of an acquired defect in GPVI-mediated platelet reactivity to collagen, which reflects data observed in constitutional GPVI deficiencies, in two patients with malignant haemopathies.

Background/Aims : The hypervascularity described in hepatocellular carcinoma varies according to the progression and the differentiation of the tumor, suggesting an angiogenic switch during tumor development. Methods : We used a transgenic mouse model of hepatocellular carcinoma induced by the expression of SV40-T antigen, in which male mice developed hepatic tumors at various temporal and histological stages, whereas female mice remained tumor-free. We analyzed, by immunostaining and reverse transcription–polymerase chain reaction, factors involved in tumoral angiogenesis. Results : We demonstrated that tumoral angiogenesis occurred before the development of diffuse hepatocarcinoma. We showed that some SV40-T-positive cells with an endothelial phenotype are involved in angiogenic processes, suggesting a partial vasculogenic mimicry. This tumoral angiogenesis is associated with platelet activation due to tissue factor expression in endothelial cells and invading macrophages. Normal and transgenic livers exhibited different pattern of expression of hypoxia-inducible factor 1 α (HIF-1α) and vascular endothelial growth factor (VEGF) mRNA. Conclusions : This model of hepatocellular carcinoma displays marked tumoral angiogenesis, with proliferation, remodeling and arterialization of hepatic sinusoids, probably associated with a partial vasculogenic mimicry. Abnormal angiogenesis observed in hepatocarcinoma was associated with platelet activation by tissue factor (TF) produced by endothelial cells and invading macrophages. In this transgenic model, HIF-1α, VEGF, and TF play a crucial role in tumoral angiogenesis.

To explore the possible role of a residual or variant alphaIIbbeta3 integrin (alphaIIbbeta3) in thrombogenesis, we used a new ex vivo perfusion chamber model to examine blood from patients with different subtypes of Glanzmann's thrombasthenia (GT). Non-anticoagulated blood was perfused through capillaries coated with type III collagen for 4.5 min (shear rate: 1600/s). Platelet deposition was quantified as platelet adhesion and mean thrombus size volume; fibrin and von Willebrand Factor (VWF) were specifically revealed by immunohistochemistry. In two patients with variant and in one patient with type II GT, platelet adhesion was maximal and we observed an unexpected formation of thrombi that were smaller than normal in size. These thrombi were surrounded by a thick meshwork that displayed a strong staining for fibrin and VWF. In two patients with heterozygous GT, platelet adhesion and thrombogenesis were normal. In two patients with type I GT, there was no thrombus formation, although platelet adhesion was also maximal. These data suggest the existence of a substitute pathway for thrombogenesis mediated by fibrin and possibly alphaIIbbeta3 (alphaIIbbeta3 at a reduced level, as in type II, and/or abnormal) as this fibrin network was not observed in type I GT with no alphaIIbbeta3. These interactions might facilitate haemostasis and even lead to thrombosis under certain favourable conditions. Furthermore, these data might have pharmacological relevance to the development of anti-alphaIIbbeta3 antithrombotic agents.

Introduction: Hepatocellular carcinoma (HCC), is characterized by severe vessel anomalies with an intense arterial blood supply (arterialisation), and acquisition ofa basal membrane rich in laminin by sinusoids(sinusoid capillarisation). A role of the VEGF-A/Dll4-Notch4/ephrin B2 cascade in tumor vessel anomalies has been demonstrated, raising the question of the nature of theVEGF receptor pathway implicated in triggering this cascade. This study was aimed at determining whether PlGF/VEGFR-1 or VEGF-A/VEGFR-2 pathways were implicated in the vessel anomalies observed during HCC development. Material and Methods:In vitro, we used primary culture of HUVECs, isolated in our laboratory from human umbilical cords. HUVECs were cultures in EBM2 medium, complemented with 20% of FBS and 2ng/ml of FGF-2. To determine which pathway was predominant in the activation of the Dll4/Notch4 cascade, the effects of PlGF (a specific ligand of VEGFR-1), VEGF-E (a specific ligand of VEGFR-2) and VEGF-A (a ligand of both receptors) on the expression of the active form of Notch4 and Dll4 in HUVECs were compared by Western Blot. In vivo, () the differential expression of VEGF-A, PlGF and their receptors were evaluated by qRT-PCR and immunostainings in transgenic mice developing stage-defined HCC. Results: In vitro, HUVECs stimulation by either VEGF-A, VEGF-E or PlGF increased Dll4 expression and Notch 4 activation. However, PlGF had the most important effect with a 5-fold increase of Dll4 expression level and a 4-fold increase in Notch 4 activation, compared to control HUVECs. Moreover, downregulation of VEGFR-1 by small interfering RNA, but not VEGFR-2, abrogated the effects of VEGF-A and PlGF stimulation on Dll4 expression and Notch4 activation. To confirm in vivothe prevalence of the PlGF/VEGFR-1 pathway in induction of vessel anomalies, we first evaluated the differential expression of PlGF, VEGF-A, VEGFR-1 and VEGFR-2 in our transgenic HCC model at different stages of the disease. We showed that VEGFR-1 increased with HCC progression and was expressed by macrophages and sinusoidal endothelial cells. Moreover, PlGF levels were maximal at the stage that coincides with the beginning of liver vessels anomalies in HCC. Silencing PlGF in vivo delayed tumor growth (p Conclusions: We demonstrated that PlGF and VEGFR-1 played a key role in the development of vessel anomalies in HCC and that silencing PlGF prevents liver vessel anomalies and delays tumor growth. Our results bring a new insight in the mechanism responsible for vessel anomalies observed in HCC, giving a strong rationale for the development of new antiangiogenic strategies based on PlGF inhibition in HCC. Citation Format: Annemilai Tijeras-Raballand, Armand de Gramont, Patricia Hainaud, Jean-Olivier Contreres, Carole Le Hénaff, Marc Pocard, Evelyne Dupuy. PlGF/VEGFR-1 dependent activation of the Dll4-Notch4/Ephrin B2 cascade contributes to liver vessel anomalies in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 19. doi:10.1158/1538-7445.AM2014-19

Abstract The relative involvement of the glycoprotein (GP) Ib/IX–von Willebrand factor (vWF) axis and GPIIb/IIIa in thrombus growth at high shear rates was assessed and compared by testing the pharmacological effects of VCL, a recombinant GPIb-binding fragment of vWF (residues 504-728), aurintricarboxylic acid (ATA), which binds to the 509-695 disulfide loop of vWF, and lamifiban, a specific synthetic GPIIb/IIIa antagonist. In vivo, their effects were evaluated in guinea pig mesenteric arteries, in a model of a laser-induced cyclic thrombotic process, and ex vivo, at a shear rate of 1800 s −1 , in a capillary perfusion chamber model, in which collagen-adherent platelets are exposed to nonanticoagulated guinea pig blood. In vivo, VCL, ATA, and lamifiban administered 2 minutes after intimal injuries stopped thrombus growth, prevented the cyclic thrombotic process, and induced gradual thrombus dissolution. Ex vivo, at 1800 s −1 , collagen exposure to untreated blood for 2 minutes, 4 minutes, or two consecutive periods of 2 minutes each resulted in similar platelet adhesion, 56%, 59%, and 61%, respectively, with an average thrombus volume of 6, 19, and 17.5 μm 3 /μm 2 , respectively, without any fibrin formation. This indicated that the two consecutive perfusions did not affect the dynamic process of thrombus growth. When collagen-adherent platelets deposited after the first 2-minute perfusion were perfused for 2 minutes with VCL-, ATA-, or lamifiban-treated blood, thrombus growth was prevented and platelet adhesion remained unchanged, but fibrin formation increased on and around the predeposited platelets. These results suggest that both the GPIb/IX-vWF axis and GPIIb/IIIa are involved in in vivo platelet-to-platelet interactions at high shear rates in the guinea pig.

The use of experimental models of arterial thrombosis both in vivo and ex vivo in animals and ex vivo in humans is an obligatory step for the understanding of mechanisms involved in thrombogenesis as well as in the evaluation of anti-thrombotic therapeutics. Arterial thrombogenesis is a complex phenomenon which involves multiple systems, mechanisms and parameters. Therefore studies of thrombogenesis from a pathological as well as a therapeutic point are necessary for understanding this problem in its entirety. For these reasons, it is necessary to use models as representative as possible of the human pathological condition. Besides these theoretical requirements, practical needs have also to be fulfilled (accessibility of the models, adaptation to the type of the technique to different animal model and/or of the size of the animal to the amount of molecule available, cost...) which necessary lead to some promises. In this review we have tried to underline the criteria for the choice, characteristics, advantages and disadvantages of the major models commonly accepted and used, in such a form that the reader who may not be an expert in the field would be led either to a choice of a particular model for a specific purpose or to appreciate a paper or a report based on an experimental model of arterial thrombosis. In vitro models of arterial thrombosis are so far removed from reality and due to their nature can generate so much artifacts that we have omitted their discussion from this paper.

member of the Dkk gene family, Dkk3, has recently been knocked out and is presently undergoing functional analysis. Regulatory effects of Dkk2 are of high biological and developmental evidence as it was previously shown that that Dkk2-mediated repression of the Wnt/bcatenin pathway is an essential prerequisite for the differentiation of corneal epithelial progenitor cells into a nonkeratinizing stratified epithelium. As the Wnt signaling pathway has been demonstrated to be essential to HCC development, we analyzed the tumor development and growth in these livers after DEN/Phenobarbital induction. Methods: To induce liver carcinogenesis a single diethylnitrosamine (DEN) i.p. injection was performed at day 7 post partum. Promotion of carcinogenesis was achived by adding continouisly phenobarbital to drinking water. Results: Dkk2 deleted mice demonstrated an enhanced growth of liver cancer by means of tumor size and number compared to wildtype mice. In addition, immunohistochemistry demonstrated a significantly enhanced proliferation already in untreated Dkk2 deleted mice. By means of RT_PCR we were able to demonstrate an increased b-catenin level in Dkk2 KO mice. Detailed molecular analyses are currently underway. Conclusion: We established a significant role of Dkk2 deletion in liver cancer development, as we were able to demonstrate that Dkk2 deleted mice develop significantly more HCC after DEN/Phenobarbital induction.

To overcome the resistance to VEGF-inhibitor treatment, additional anti-angiogenic agents need to be valorized. Placental growth factor (PlGF), a VEGF homologue, stimulates endothelial, tumor and inflammatory cells. Studies in independently generated PlGF−/− mice by us and Regeneron identified a role for PlGF in inflammatory, ischemic and malignant disease, while the anti-PlGF mAb 5D11D4 blocks growth of implanted tumors. Several clinical studies also show that PlGF levels correlate with poor prognosis in diverse types of cancer, while the humanized anti-human PlGF mAb TB403 has been succesfully tested in a phase I trial in out-treated cancer patients. However, genetic evidence for a disease-candidate role of PlGF or an effect of the anti-PlGF mAb 5D11D4 in spontaneous cancer models has been lacking so far. Here, we show that loss of PlGF inhibited a carcinogen-induced skin tumor model papilloma formation and angiogenesis. Furthermore, silencing of PlGF in a transgenic hepatocellular carcinoma (HCC) model reduced tumor size and incidence. In a carcinogen-induced HCC model, most WT but only a minority of PlGF−/− mice died, while fewer tumor nodules developed in PlGF−/− mice. When WT mice with established HCC were treated with 5D11D4, more control IgG than 5D11D4 treated mice died. PlGF blockage inhibited the characteristic arterialization response in these tumors and blocked vessel abnormalization in HCC leading to reduced levels of hypoxia, known to stimulate HCC growth, however, without affecting microvascular density. These findings indicate that the anti-cancer activity of PlGF-blockage is not reflected by a change in vessel density alone. In the Rip1Tag2 model, no differences between WT and PlGF−/− mice or 5D11D4-treated mice in tumor burden or incidence were found, indicating that 5D11D4 does not have off-target effects. Resistance of this model is likely due to the lack of infiltration by Gr1+ neutrophils by PlGF blockage. These findings and previously published results of a Phase I trial with TB403 warrant furter analysis of anti-PlGF strategies for (non)-oncology indications. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-362.