Establishing murine models of hepatocellular carcinoma using NOD/SCID, nude and Balb/c mice: An initial comparative study

Science & Technology Development Journal, 23(1):454-460 Open Access Full Text Article Original Research 1Stem Cell Institute, University of Science Ho Chi Minh City, Viet Nam 2Cancer Research Laboratory, University of Science Ho Chi Minh City, Viet Nam 3Viet Nam National University Ho Chi Minh City, Viet Nam 4Medical Genetic Institute, Ho Chi Minh City, Viet Nam 5Laboratory of Stem Cell Research and Application, University of Science Ho Chi Minh City, Viet Nam Correspondence Phuc Van Pham, Stem Cell Institute, University of Science Ho Chi Minh City, Viet Nam Cancer Research Laboratory, University of Science Ho Chi Minh City, Viet Nam Viet Nam National University Ho Chi Minh City, Viet Nam Laboratory of Stem Cell Research and Application, University of Science Ho Chi Minh City, Viet Nam Email: pvphuc@hcmuns.edu.vn; phucpham@sci.edu.vn Establishingmurinemodels of hepatocellular carcinoma using NOD/SCID, nude and Balb/cmice: An initial comparative study Nghia Minh Do1,2,3, Sinh Truong Nguyen1,2,3, Phuc Vo Hong1,2,3, Trinh Thi-Phuong Ngo1,3, Kiet Dinh Truong4, Phuc Van Pham1,2,3,5,* Use your smartphone to scan this QR code and download this article ABSTRACT Introduction: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality in the world. Therefore, more and more studies are developing novel therapies to treat this disease. Thepre-clinical trials on animals are a vital step to evaluate the efficacy aswell as side effects of these novel therapies. Hence, this study aimed todevelop themurinemodel of HCCusing 3 kinds ofmice: NOD/SCID, nude and Balb/cmice. Methods: HCCcell lineHepG2was used in this study. Theywere injected into 3 kinds of mice: NOD/SCID, nude and Balb/c mice at three doses: 5 x 106 , 2.5 x 106 , and 1.25 x 106 cells. Tumor size and body weight of themice were recorded daily. To confirm these tumors inmice asmalignant tumors, they were removed and analyzed by histopathology. Results: The results showed that in nude mice, the tumors appeared after 1 day of injection and could be detected by the naked eye; they continuously developed until the end of the study. In NOD/SCID mice, the tumors could be detected by the naked eye after 3 days of injection; their sizes also increased until the end of the experimental study. Meanwhile, in Balb/cmice, although the tumors could be observed by the naked eye on the 3rd day after cell injection, they regressed andmarkedly disappeared after 30 days. The dose of 5 x 106 cells per mouse induced the largest tumors (1.2 cm in diameter) in NOD/SCID mice. The histopathological analysis showed that the tumors collected from nude and NOD/SCID mice also displayed the poorly differentiated malignant carcinoma with muscle tissue invasion. Conclusion: Both nude and NOD/SCID mice are appropriate for future studies to establish HCC murine models using HepG2 injection. Key words: Hepatocellular carcinoma, HepG2, cancer, NOD/SCID mice, NUDE mice, BALB/c mice, HCC model INTRODUCTION Primary liver cancer begins with abnormally prolif- erating liver cells. Some common types of primary liver cancer and variants include: hepatocellular car- cinoma (HCC), fibrolamellar carcinoma, sclerosing hepatic carcinoma, cholangiocarcinoma, epithelioid hemangioendothelioma, and hepatoblastoma. Re- markedly, HCC accounts for 85-90% of all primary malignant tumors. Liver cancer is more common in men than in women. In 2012, there were 782,500 new cases in the world and 745,500 deaths, with China accounting for about 50% of total mortality cases. The highest rates of liver cancer are seen in East and Southeast Asia, North andWest Africa, the lowest re- gion of South-Central Asia, and Europe 1. One of the most frequently used methods of cancer research is xenograft transplantation which allows for observations of growth, invasive process, and metas- tases of human tumors. In xenograft models, the in- hibition of alloimmunization is extremely important and, therefore, immunocompromised mice are essen- tial for use. In 1966, the first human cell line to be successfully transplanted in nude mice was able to be developed and maintained 2. Nude mice carry the mutation in the FoxN1 gene that causes T-cell defi- ciency but still have intact B-cell and innate immune systems3. In 1980, Makino and colleagues created a line of non-obese diabetic mice (NOD) mice with weakened innate immune systems. In 1983, a mu- tation in the PrkdcSCID gene coding for protein ki- nase catalyzing the activation of DNA led to a severely weakened lymphatic system in CB-17 mice 4. Cross- breeding NOD and SCID mice led to the creation of mice with deficiencies in both the innate and adaptive immune systems; this model, called the NOD/SCID mouse model, has been a useful model for human cell transplantation over the past two decades. Nowadays, more than 80% of drug development stud- ies show the potential of new drug candidates for can- cer treatment, with positive results in animal stud- ies. However, there is insufficient or no evidence to show their effectiveness in humans5. This is mainly Cite this article : DoNM, Nguyen S T, Hong P V, Nguyen T T T, Truong KD, PhamP V. Establishingmurine models of hepatocellular carcinomausingNOD/SCID, nude and Balb/cmice: An initial comparative study. Sci. Tech. Dev. J.; 23(1):454-460. 454 History  Received: 15 January 2020  Accepted: 01 March 2020  Published: 19 March 2020 DOI : 10.32508/stdj.v23i1.1767 Copyright © VNU-HCM Press. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Science & Technology Development Journal, 23(1):454-460 due to the use of preclinical models that have not yet simulated human physiological and pathological processes. These models have many characteristics that differ from humans in terms of the physiology of organs, cellular dynamics, and regulatory proteins. Therefore, one of the major challenges of medical de- velopment is to create more humanized models that can easily allow the transplantation of human cell sys- tems into animals. Humanized mouse models can be created by incorporating human genes into mice by transplanting functional cells, tissues or organs 6. In this study, three mouse strains, including nude, NOD/SCID andBalb/cmice, were subcutaneously in- jected with hepatocellular carcinoma. The aims were to investigate the ability of each mouse model to in- duce tumor-carry and tumor formation; as well, tu- mor structure was evaluated by histochemical stain- ing. To our understanding, this study herein is the first to compare the use of these three mouse groups in liver cancer research in vivo. MATERIALS &METHODS Animals Female Balb/c, nude and NOD/SCIDmice, 6-8 weeks old and 20-24 g in weight, were purchased from Jax Laboratory and kept at the Stem Cell Institute (Uni- versity of Science, Ho Chi Minh City, Viet Nam). The mice were maintained in a clean environment with humidity of about 60% and temperature of 25 oC.The light system was automatically controlled at every 12- hour interval. Hepatocellular carcinoma HepG2 cell line Hepatocellular carcinoma HepG2 cells (Manassas, VA, USA) were thawed and expanded. They were cultured in Dulbecco’s Modified Medium/Nutrient Mixture F12 (DMEM/F12; Thermo Fisher Scientific, Waltham, MA), with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO). Injection of HepG2 cells intomice Micewere divided into 4 groups, with each group con- sisting of 3 individuals. Mice were subcutaneously injected with 5 x 106, 2.5 x 106, or 1.25 x 106 cells that were suspended in 100 mL of DMEM/F12 per site; control mice were injected with 100 mL of DMEM/F12 (Figure 1). All manipulations on mice were approved by the Institutional Ethics Committee of the Stem Cell Institute. Evaluation of mouse tumor size After injection of HepG2 cells, all mice were checked for tumor size in a fixed daily time frame (once per day). A straight ruler, with a minimum division of 1 mm, was used to measure the size of the tumor. This experiment was performed for 30 days, or until the mice died, or until tumor size exceeded 2 cm. Histopathological analysis Tumors were collected from mice and fixed in 4% formaldehyde overnight. Then, they were soaked in 30% sucrose solution until they completely sank. Tu- mor blocks were then created in OCT (Optimal Cut- ting Tissue) (Thermo Fisher Scientific,Waltham,MA, USA).The tissue sections were sliced, stained and an- alyzed at Cho Ray Hospital (Ho Chi Minh City, Viet Nam). RESULTS Tumor formation The tumors formed after 5 days (with a probability of 100%) in the three groups injected with HepG2 cells. The control injection site did not show any appear- ance of tumor nor any inflammation, ulcer or necro- sis. In the NOD/SCID and nude mouse groups, at the 10th day after tumor cell injection, there was devel- opment of large and bulging tumors under the skin; around the tumor area, a vein appeared to be directed towards the tumor. The tumor size was seen to in- crease continuously in both the nude andNOD/SCID mice until the 30th day. On the other hand, after the 10th day, the tumor size of the Balb/c group decreased (Figure 2). After 30 days, in the NOD/SCID mice, the largest tu- mor size was recorded at a diameter of 1.2 cmwith the dose concentration of 5 x 106 cells; at the concentra- tions of 2.5 x 106 and 1.25 x 106 cells, tumors arose of similar size (about a 1 cm diameter). Tumors in nude mice were recorded from the first day after injection; their sizes were 0.6, 0.4, and 0.2 cm at the concentra- tions of 5 x 106, 2.5 x 106, and 1.25 x 106 cells, respec- tively. The tumors maintained growth and continu- ously grew to 1.3, 1, and 0.7 cm, respectively. By contrast, after 5 days, tumors in the Balb/c group began to adapt and grow. The largest tumor size was recorded using a dose concentration of 5 x 106 cells; the tumor diameter was about 0.7 cm. For the other two concentrations (2.5 x 106 and 1.25 x 106 cells), the mean tumor size was 0.5 cm and 0.4 cm, respec- tively. After day 10, tumor size began to decline and some tumors appeared to undergo necrosis and the tumors dissolved. In addition, external observations 455 Science & Technology Development Journal, 23(1):454-460 Figure 1: Cell injection sites in NOD/SCID and NUDEmice. Figure 2: Size of tumor of HCC mice model. (A) Size of tumor of HCC NOD/SCID mice model; (B) Size of tumor of HCC NUDE mice model; (C) Size of tumor of HCC Balb/c micemodel; Mice were injected HepG2 cells with three concentrations include 5 x 106 ,2.5 x 106 , 1.25 x 106. also showed that themice did not show signs of ruffled feathers, anorexia or abnormal aggression; indeed, the percentage of mice living until the end of the experi- ment was 100%. In NODmice, themean tumor weight reached 0.4315  0.0999, 0.3067 0.0677, and 0.23 0.0003 g, when injected with hepatocellular carcinoma at a concen- tration of 5 x 106, 2.5 x 106, and 1.25 x 106, respec- tively (Figure 3). In addition, for the injection dose of 2.5 x 106 cells, the injection resulted in 2 small metastatic cancer sites found 1 cmaway from the orig- inal tumor injection site. Similarly, in the nude mice, the tumor weights reached 0.1959, 0.2773, and 0.0948 g at the sites injected with hepatocellular carcinoma cells at a concentrations of 5 x 106, 2.5 x 106, and 1.25 x 106, respectively. In addition, two metastatic sites of cancer were recorded. At the control injection site (DMEM injection), however, no abnormalities were reported. The cell mass was observed to be a large, round solid tumor. In addition, the heterogeneous tumor was di- vided into many parts, with a rough surface separated 456 Science & Technology Development Journal, 23(1):454-460 Figure 3: Tumorfomation weight in HCCmodel mice. Abbreviations: MT: Metatasis Tumors, HCC: Hepatocel- lular carcinomar from the surrounding tissues. Small tumors (believed to have been caused by metastases) appeared about 1 cm away from the main tumor site. The anatomical results also indicated the presence of many blood ves- sels around the tumor (Figure 4). H&E staining Tumor anatomical surgery showed the tissue struc- ture of the cancer contained polymorphic cells ar- ranged in clusters with invasive structure. The cancer cells had an abnormal dividing nucleus. The cells in the tumor center were necrotic or poorly developed, such that nucleus showed little to no staining with hematoxylin dye. This is a characteristic of poorly dif- ferentiated epithelial tumors with invasive muscle tis- sue (Figure 5). DISCUSSION This report aims to, firstly, demonstrate a suitable mouse model for hepatocellular carcinoma research. All injected mice, including immune-deficient and normal mice, formed tumors at the injection site. From this, it can be seen that HepG2 cells have the ability to adapt and develop well in the in vivo envi- ronment of mice. However, there were differences be- tween the mouse strains tested. For experiments in immunodeficient mice, the tumors adapted and de- veloped well, and no elimination nor inflammation took place. The injected HepG2 cells showed the abil- ity to construct tumors between abdominal skin and muscle layer in the HepG2 cell transplantation po- sitions and could adapt to the mouse body through the process of forming new blood vessels. The an- giogenesis involves the proliferation of capillaries and the rebuilding of existing blood vessels. In particular, the NOD/SCIDmice group results provided evidence of continual development/increase in tumor size and weight. On the other hand, the Balb/c group of mice showed a decline in tumor size and, thus, the mouse strain was unsuitable for the HCC model. In experiments by Yu-Huei Liu et al., the authors induced liver cancer tumors in NOD/SCID mice by subcutaneous injection method, using 5 x 106 cells/mouse as the dose of cell injection7. Honghai Xia used a lower cell dose of 106 cells/mouse be- cause the experiments took longer (90 days)8. Al- most the experiments conducted show the adaptive- ness of HepG2 cells in NOD/SCID mice, the tumors formed at the xenograft position, developed, andwere maintained as stable. In the experiments using a dose concentration of 5 x 106 cells, after 15 days of in- jection, the tumors had an average diameter of 0.6 cm7,9. In this experiment, we created a model with subcutaneous injection at a cell dose of 5 x 106; the resulting tumor size at day 15 was 0.65  0.05 cm. It was demonstrated that the procedures used in the study, combined with the specific care conditions in Vietnam, could allow HepG2 cells to adapt well in NOD/SCID mice. Histopathological results of the tumors showed that the development of hepatocellular carcinoma cells in the mouse models were accompanied by characteris- tics such as clumping of polymorphic cells, formation of an invasive strip, and establishment of a necrotic area inside the tumors. In addition, the cells showed an unequally divided nucleus and abnormally small size. In particular, it is clearly observed that cells of 467 Science & Technology Development Journal, 23(1):454-460 Figure4: Imageof surgery tumoronmicemodel. (A) NUDEmicemodel; (B): NOD/SCIDmicemodel. The arrows indicate the blood vessels around the tumor. 458 Science & Technology Development Journal, 23(1):454-460 Figure 5: Result of H&E staining in HepG2 tumor sample of immunodeficient mice. Tumor sample of NUDE mice (A, A1, A2), and NOD/SCID mice (B, B1, B2); green arrows indicate evidence of cancer cell clumps, yellow arrows identify the areas of muscle tissue, gray arrows expose the fibrosis tissues. the tumors had abnormal nuclei, ambiguous bound- aries, invasive surrounding tissues, and proliferating blood vessels. Taken together, these observations show that HepG2 liver cancer cells, indeed, grew well in NOD/SCID mice. The resulting tumors, when im- aged, had morphological and histological structures similar to those observed by Lekshmi R. Nath et al. in their NOD/SCID mouse model10. The results are also consistent with the research of Qiang-Bo Zhang et al., who created a Balb/cmouse model carrying the HepG2 cell tumor to study the activity of Sorafenib11. From these, it is evident to conclude that tumors in the NOD/SCIDmousemodel are established with the typical histological structures of xenografted HepG2 cell tumors in immunodeficient mice. The acquired tumors are poorly differentiated epithelial tumors that have invasive muscle tissues. However, the research herein does have limitations. Our study was conducted with finite funding and, thus, the experiment with the group of immunode- 459 Science & Technology Development Journal, 23(1):454-460 ficient mice was not repeated. In order to more ac- curately assess the potential of immunodeficient mice in an HCC mouse model, the experiments need to be repeated at least twice and evaluated using more in- tensive methods. CONCLUSION HepG2 cells were xenografted into immunodeficient mice (nude and NOD/SCID); notably in NOD/SCID mice, the tumors formed at a rate of 100%, were maintained, and grew in size during the experimental study. Moreover, tumor histology results from these groups of mice also showed that these tumors were malignant tumors. On the other hand, in Balb/cmice, the tumors were present from the 5th to 10th day af- ter injection, then gradually disappeared. Therefore, it can be concluded that immunodeficient mice, espe- cially the NOD/SCIDmodel, is advisable and suitable for the generation amousemodel of human liver can- cer by xenograft transplantation. ABBREVIATIONS HCC: Hepatocellular Carcinoma H&E: Hematoxylin and Eosin OCT: Optimal Cutting Tissue ACKNOWLEDGMENTS This work was supported by the Vietnam National University, Ho Chi Minh City, Vietnam, under grant A2015-18-01. CONFLICT OF INTEREST Theauthors report no conflicts of interest in thiswork. AUTHORS’ CONTRIBUTION All authors equally contributed in this work and ap- proved the final version ofmanuscript for submission. REFERENCES 1. McGlynn K, Meyts ERD, Stang A. Cancer Epidemiology and Prevention: Testicular Cancer. In: Cancer Epidemiology and Prevention. Oxford University Press. 2017;p. 1019–1028. Available from: https://doi.org/10.1093/oso/9780190238667. 003.0054. 2. Flanagan S. ’Nude’, a new hairless gene with pleiotropic effects in the mouse. Genetics Research. 1966;8(3):295– 309. PMID: 5980117. Available from: https://doi.org/10.1017/ S0016672300010168. 3. Ganick DJ, Sarnwick RD, Shahidi NT, Manning DD. Inabil- ity of intravenously injected monocellular suspensions of hu- man bone marrow to establish in the nude mouse . Interna- tional Archives of Allergy and Immunology. 1980;62(3):330– 333. PMID: 6993372. Available from: https://doi.org/10.1159/ 000232530. 4. Bosma GC, Custer RP, Bosma MJ. A severe combined immunodeficiency mutation in the mouse. Nature . 1983;301(5900):527. PMID: 6823332. Available from: https: //doi.org/10.1038/301527a0. 5. Perrin S. Preclinical research: Make mouse studies work. Na- ture . 2014;507(7493):423–425. PMID: 24678540. Available from: https://doi.org/10.1038/507423a. 6. BrehmMA, Shultz LD, Greiner DL. Humanized mouse models to study human diseases. Current opinion in endocrinology, diabetes, and obesity. 2010;17(2):120. PMID: 20150806. Avail- able from: https://doi.org/10.1097/MED.0b013e328337282f. 7. Liu YH, Weng YP, Lin HY, Tang SW, Chen CJ, Liang CJ, et al. Aqueous extract of Polygonumbistortamodulates proteosta- sis by ROS-induced ER stress in human hepatoma cells. Scien- tific reports. 2017;7:41437. PMID: 28134285. Available from: https://doi.org/10.1038/srep41437. 8. Xia H, Cao J, Li Q, Lv Y, Jia W, Ren W, et al. Hepatocellular Carcinoma-propagating Cells are Detectable by Side Popula- tion Analysis and Possess an Expression Profile Reflective of a Primitive Origin. . Scientific reports. 2016;6:34856. PMID: 27725724. Available from: https://doi.org/10.1038/srep34856. 9. Wu YH, Cao JG, Xiang HL, Xia H, Qin Y, Huang AJ, et al. Recombinant vascular basement-membrane-derived multi- functional peptide inhibits angiogenesis and growth of hepa- tocellular carcinoma. World journal of gastroenterology: WJG . 2009;15(14):1744. PMID: 19360918. Available from: https: //doi.org/10.3748/wjg.15.1744. 10. Nath LR, Gorantla JN, Thulasidasan AKT, Vijayakurup V, Shah S, Anwer S, et al. Evaluation of uttroside B, a saponin from Solanum nigrum Linn, as a promising chemotherapeu- tic agent against hepatocellular carcinoma. Scientific Reports. 2016;6:36318. PMID: 27808117. Available from: https://doi. org/10.1038/srep36318. 11. Q-B Z, H-C S, K-Z Z, Q-A J, Y B,MW, et al. Suppression of natural killer cells by sorafenib contributes to prometastatic effects in hepatocellular carcinoma. PLoSOne. 2013;8(2):e55945. PMID: 23409093. Available from: https://doi.org/10.1371/journal. pone.0055945. 460