Establishment Of Carcinogen-induced Common Marmoset Model For Hepatocellular Carcinoma

Objective:HCC(Hepatocellular Carcinoma)is the very common cancer in the world,second only to gastric and lung cancers.As a major disease affecting human beings'health,there is a lack of early diagnostic and therapeutic approaches for HCC in clinic.Also the understanding of its pathogenesis remains limited.To address these issues,one of the key paramerters is an approapriate animal model.Although numerous HCC animal models have been established and extensively utilized for elucidating the pathogenesis of HCC and for screening therapeuticals for HCC,most of them are rodents which are distantly-related to human being genetically,physiologically and anatomically.As a result,a more proper HCC animal model is urgently required for accelerating the development of diagnostic and therapeutic measurements for HCC in clinic research.Common marmoset(Callithrix jacchus),as a new-world nonhuman primate,is a emerging mainstream animal model for translational research due to their unique characteristics i.e.small-sized,high reprodutive rate,short sexual maturation period,low maintenance cost and highly sensitive to pathogens,which confer them superiority as a human disease model to other old-world non-human primates.Particularly,earlier report demonstrated that common marmosets are highly sensitive to tumor by showing tumor-bearing common marmosets died before the tumor reached 2cm in liver.Therefore,in the present study,we aim to establish common marmoset HCC models to recapitulate the pathological process occuring in HCC patients.By utlizing the common marmoset colony in Tianjin Medical University,which have been in captivity for more than 44 years,we examine the suitability of these long-term captive common marmosets for HCC animal model.Various phenotypical,biochemical,behavioral paramertes are examined and compared to wild-type common marmosets,no biological difference is observed.Furthermore,we measure the microorganism flora in the digestive system and other possible infectious pathogens i.e.hepatitis A virus to further confirm the healthy status of long-term captive common marmosets.Through hepatomy,we are able to obtain new-born liver tissues,which are more sensitive to chemical carcinogen.With a low dose,repeated injections of DENA(Diethylnirtosamine),we have successfully established a common marmoset HCC model showing the complexity and typical pathology observed in HCC patients.Meanwhile we use non-invasive imaging systems and biochemical assays to monitor the pathological process during the experiments.In addition,we perform some preliminary studies on common marmoset's reproduction and provide basic data for the establishment of transgenic common marmoset in future.Methods:1.To validate the healthy status of captive common marmosets,we perform a series of assays including body-weight measurement,faecal bacterium assay,blood analysis.Moreover,we conduct mirror reaction test to examine the behavioral changes.2.To optimize the procedure and carcinogens used,we carry out hepatomy with different sizes and test two different carcinogens in rats.One third and two third heaptomy are performed and DENA alone or DENA in combination with AFB-1 are tested in rats via intraperitoneal injection or feeding.3.To optimize the dosing regimen,we perform one thrid hepatomy in two adult common marmosets followed by intraperitoneal injection of escalating dose of DENA from 0.5mg/kg to 8.5mg/kg,increasing lmg/kg every 30 days..4.Based on above studies,we adjust the dosing regimen with a lower dose of 2.5mg/kg,3 times per week.5.During the study,live animal imaging,B-ultrasound and magnetic resonance imaging are used for real-time monitoring.Picrosirius-TNP staining,H&E staining,immunohistochemistry and biochemical analysis are utlized.6.To study the reproduction of common marmosets,immuno radioactive assay is applied to measure progesterone and estradiol.Oscillator is used for stimulating and collecting sperm;immunofluorescence and trypan staining are applied for examing sperms.Result:1.No changes in biological characteristics and health status of captive common marmosets compared to wild-type ones with the exception of behavioral aspects.2.One thrid hepatectomy is superior to two thrid in terms of survival rate.DENA alone shows higher survial rate than DENA in combination with AFB-1.3.Escalating dose of DENA are tested in two adult common marmosets and the results showed higher than 2.5mg/kg leads to high mortality rate.4.The optimal dosing regimen for DENA in common marmosets is 2.5mg/kg,3 times per week.5.During the indcution,typical pathological features are observed including hepatatis,fibrosis,cirrhosis and liver cancer.With B-ultrasound,we find liver hypertrophy,rough liver surface,blood vessels enrichment,increased lymphocytes,larger dark areas and HE staining demonsteates increased inflammatory and apoptotic cells as well as false flocculus and liver fibrosis.Meanwhile higher level of AFP protein in blood from induced group at earlier stage of fibrosis.6.Out study reveal the physiological cycle of common marmosets is 28-33 day with the peak time for progesterone and estradiol is 1-3 days prior to ovulation.We achieve the superovulation of common marmoset with the stimulation of HCG and rsFSH..Conclusion:1.Long-term captivity does not change biological characteristics,health status and intestinal flora situation of common marmosets,with the exception of social behaviors,suggesting captive common marmosets are ideal animal models for biomedical studies.2.The combination of 1/3 hepatectomy and DENA induction is suitable for eliciting HCC in common marmosets.The optimal dosing regimen with 2.5mg/kg(3 times per week)does not cause acute liver injury and drug-related death.3.The common marmoset HCC model recapitulate the complexity of HCC reported in patients involving 4 typical stages hepatitis,fibrosis,cirrhosis and cancer.Notably the early diagnosis marker for HCC in clinic-AFP is highly expressed in induced common marmosets,suggesting common marmosets are ideal model for HCC.4.We have established the platform for collecting oocytes and sperms of common marmosets.