The Role Of Platelet Accumulation In Tumor Microenvironment In The Progression Of Hepatocellular Carcinoma

Background & Aims:Hepatocellular carcinoma(HCC)is the sixth most common cancer and the second leading cause of cancer death worldwide.Although great efforts have been made in the diagnosis and therapy in recent years,current treatment for HCC still is a big challenge due to poor understanding of the pathogenesis of HCC.The importance of cancer stem cell in HCC development and therapy has attracted accumulating attention in recent years,but the key factor and regulation of liver CSCs within the HCC microenvironment remain far from clear.Platelets have significant roles during cancer progression due to the expression of a variety of surface molecules and release of a plethora of factors on activation.The interaction between platelets and tumor cells contribute to cancer cell proliferation,angiogenesis,cancer associated-inflammation and chemoresistance.Thus,cancer-associated platelet holds the promise to be a target for cancer therapy.However,the high risk of potentially fatal hemorrhage limits the clinical application of antiplatelet therapy.Therefore,therapeutic approaches that selectively target platelet-cancer cells interaction without affecting the physiological function of platelets are urgently needed for cancer treatment.Liver is one of the major organs that control the production and clearance of platelets.A growing body of evidence highlights a central role for platelets in liver homeostasis and pathobiology,but the precise roles of platelets in the HCC development are not well illustrated.Even though HCC mainly develops in patients with chronic liver diseases which are usually companied by quantitatively and qualitatively abnormal platelets,clinical data demonstrated that high platelet count was associated with a poor prognosis of HCC and a high cumulative incidence of tumor thrombosis.The high prevalence of portal vein thrombosis also suggested the important role of platelet in HCC.Whether and how platelets accumulated within the tumor microenvironment affect HCC development are still not well demonstrated.In our study,we analyzed the clinical relevance of platelet accumulated within HCC microenvironment and studied the mechanisms how platelets affected the HCC development and,specifically,how platelet affected liver cancer stem cell expansion.Our data not only shed new light on how tumor development was regulated by tumor microenvironment but also provide a new potentially effective target for HCC therapy.Methods:Experimental HCC models were established by orthotropic transplantation of Hepa1-6 tumors,or by hydrodynamic injection of oncogenes(c-Met and consitutively activated ?-Catenin),or by subcutaneous injection of Huh7 cells in nude mice.Human HCC samples and associated clinical data as well as prognosis data were collected.The platelet accumulation in experimental HCCs and in human HCCs was determined by immunofluorescence or immunohistochemistry.The association between platelet accumulation in HCC and clinical pathological features was analyzed by Chi-square test.The association between platelet accumulation in human HCC and prognosis were analyzed by Kaplan-Meier survival analysis.The effects of platelet accumulation on HCC progression was determined by using the platelet depletion antibody or antiplatelet agent Clopidogrel.The platelet-HCC cells coculture in vitro was established to determine the effects of platelet on the malignant characteristics of HCC cells,including proliferation,chemoresistance and liver cancer stem cells expansion.Western blot,immunofluorescence,quantitative PCR,RNAi,neutralizing antibody were used to clarify the mechanisms how platelet promoted liver cancer stem cell expansion.Platelet pellet and releasate were isolated to determine whether direct contact between platelet and HCC cells or platelet-released factors promoted liver cancer stem cell expansion.The adhesive molecules that mediated platelet-HCC cells contact were determined by using neutralizing antibodies against platelet adhesive molecules.The effects of anti-P-selectin(CD62P)on HCC growth in vivo was investigated.Results:Platelets were found to accumulate in HCC microenvironment(HME),and platelet accumulation in the HCC microenvironment is associated with poor prognosis.Inhibition of platelets accumulation in HME inhibited HCC development in vivo.Platelets promoted HCC cells proliferation,chemoresistance and liver cancer stem cell expansion.Mechanism study showed that platelet promoted liver stem cell expansion via the JAK2/STAT3 pathway via a direct contact manner.Further studies demonstrated that JAK2/STAT3 pathway activation was induced by the NF-?B/IL-6 pathway,which was evoked in HCC cells by platelets.P-selectin(CD62P)on platelet mediated the direct contact of platelet and HCC cells.P-selectin neutralizing antibody significantly inhibited HCC growth in vivo while the bleeding time was comparable between antibody-treated and control IgG-treated group.Conclusion:Our study proposed a novel paradigm of platelet-tumor cells interaction in which P-selectin on platelet interacted with counterparts on tumor cells,leading to the NF-kB/IL-6/STAT3 activation and subsequent liver CSCs expansion.Neutralization of P-selectin significantly inhibited HCC growth while with minimal interference with the the physiological function of platelets.Therefore,it is tempting to speculate that interruption of platelet-HCC cells interaction by blockage of P-selectin represents an effective and safe method for HCC therapy.Our data thus shed specific light on how tumor microenvironment affects HCC development and provided a new avenue for HCC therapy.