Natural Product Testing: Selecting in vivo Anticancer Assay Model

In vivo anticancer assay model


  • Dr. Norazalina Saad UPM-MAKNA Cancer Research Laboratory (CANRES), Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
  • Suet Lin Chia Department of Microbiology, Faculty of Biotechnology and Biomolecular Science, 43400 UPM Serdang, Selangor, Malaysia
  • Che Azurahanim Che Abdullah Department of Physics, Faculty of Science, 43400 UPM Serdang, Selangor, Malaysia
  • Fadzil Sulaiman Natural Medicine and Product Research Laboratory (NaturMeds), Institute of Bioscience, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia



Animal model, Anticancer model, In-vivo studies, Natural compounds


Phytochemicals and other natural products have been suggested to be effective adjuvants to conventional therapy to reduce potential side effects that arise from cancer treatments. Various natural compounds and synthetic analogues have been studied for their potential anti-cancer properties in the last decade. Prior to administering them to patients in a clinical setting, drug development necessitates a sequence of preclinical testing, beginning with in vitro and progressing to in vivo studies. While numerous drugs and plant extracts were reported to be active in vitro, only several of them had reproducible activity at the tested doses in vivo. Furthermore, breakthroughs in manipulating gene editing have aided scientists in accurately replicating human diseases in animal models.  Therefore, during drug development, in vivo studies are of tremendous help in evaluating a drug candidate's safety, toxicity, and efficacy in complex physiological and biochemical states. Hence, for this purpose, it is crucial to apply and select the animal model that is the most suitable to represent the studied disease or biological process. The current review summarizes various animal models that have been considered for in vivo testing, their advantages and disadvantages, and the experimental guidelines for conducting animal studies.


Amaral RG, Santos SA dos, Andrade LN, Severino P, Carvalho AA (2019) Natural Products as Treatment against Cancer: A Historical and Current Vision. Clinics in Oncology 4(1562):1–5.

Kinghorn AD, Chin YW, Swanson SM (2009) Discovery of natural product anticancer agents from biodiverse organisms. Current Opinion in Drug Discovery & Development 12(2):189–96.

YANG YH, MAO JW, TAN XL (2020) Research progress on the source, production, and anti-cancer mechanisms of paclitaxel. Chin J Nat Med. 18(12): 890-897. doi: 10.1016/S1875-5364(20)60032-2.

Newman DJ, Cragg GM (2016) Natural Products as Sources of New Drugs from 1981 to 2014. Journal of Natural Products79(3):629-661. doi:10.1021/acs.jnatprod.5b01055.

Ranjan A, Ramachandran S, Gupta N, Kaushik I, Wright S, Srivastava S, et al (2019). Role of phytochemicals in cancer prevention. International Journal of Molecular Sciences 2;20(20). doi: 10.3390/ijms20204981.

Quinn B (2014) Preparation and Maintenance of Live Tissues and Primary Cultures for Toxicity Studies [Internet]. Biochemical Ecotoxicology: Principles and Methods. Elsevier Inc; 33–47. doi :10.1016/B978-0-12-411604-7.000039.

McGonigle P, Ruggeri B (2014) Animal models of human disease: Challenges in enabling translation. Biochemical Pharmacology 87(1):162–71. doi: 10.1016/j.bcp.2013.08.006.

Choudhari AS, Mandave PC, Deshpande M, Ranjekar P, Prakash O (2020) Phytochemicals in cancer treatment: From preclinical studies to clinical practice. Frontiers in Pharmacology 10(January):1–17. doi: 10.3389/fphar.2019.01614.

Bianco R, Melisi D, Ciardiello F, Tortora G (2006) Key cancer cell signal transduction pathways as therapeutic targets. European Journal of Cancer 42(3):290–4. doi: 10.1016/j.ejca.2005.07.034.

Shu L, Cheung KL, Khor TO, Chen C, Kong AN (2010) Phytochemicals: Cancer chemoprevention and suppression of tumor onset and metastasis. Cancer Metastasis Rev. 2010;29(3):483–502. doi: 10.1007/s10555-010-9239-y.

Cardot JM, Beyssac E, Alric M (2007) In vitro–in vivo correlation: Importance of dissolution in IVIVC. Dissolution Technologies 14(1):15–9. doi:10.14227/DT140107P15.

Katt ME, Placone AL, Wong AD, Xu ZS, Searson PC (2016) In vitro tumor models: Advantages, disadvantages, variables, and selecting the right platform. Frontiers Bioengineering and Biotechnology 4(12). doi: 10.3389/fbioe.2016.00012.

Ravi M, Paramesh V, Kaviya SR, Anuradha E, Paul Solomon FD (2015) 3D cell culture systems: Advantages and applications. Journal of Cellular Physiology 230(1):16–26. doi: 10.1002/jcp.24683.

Coate L, Cuffe S, Horgan A, Hung RJ, Christiani D, Liu G (2010) Germline genetic variation, cancer outcome, and pharmacogenetics. J Clin Oncol. 2010;28(26):4029–37. doi: 10.1200/JCO.2009.27.2336.

Cirulli ET, Goldstein DB. In vitro assays fail to predict in vivo effects of regulatory polymorphisms. Human Molecular Genetics 15;16(16):1931-9 doi: 10.1093/hmg/ddm140.

Zhu F, Dollé MET, Berton TR, Kuiper R V., Capps C, Espejo A, et al. (2010) Mouse models for the p53 R72P polymorphism mimic human phenotypes. Molecular and Cellular Pathobiology 70(14):5851–9. doi: 10.1158/0008-5472.CAN-09-4646

Kareva I, Wilkie KP, Hahnfeldt P (2014) The Power of the Tumor Microenvironment: A Systemic Approach for a Systemic Disease. Mathematical Oncology 2013: 181–196 2014;181–96. doi: 10.1007/978-1-4939-0458-7__1.

Chen YY, Liang JJ, Wang DL, Chen JB, Cao JP, Wang Y, et al (2022) Nobiletin as a chemopreventive natural product against cancer, a comprehensive review. Critical Reviews in Food Science and Nutrition 63(23):6309-6329. doi: 10.1080/10408398.2022.2030297.

Sun E, Xu F, Qian Q, Cui L, Tan X, Jia X (2014) Ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry analysis of icariside II metabolites in rats. Natural Product Research 28(19):1525–9. doi: 10.1080/14786419.2014.921684.

Wenzel E, Somoza V (2005) Metabolism and bioavailability of trans-resveratrol. Molecular Nutrition and Food Research 49(5):472–81. doi: 10.1002/mnfr.200500010.

Liu Z, Hu M (2007) Natural polyphenol disposition via coupled metabolic pathways. Expert Opinion on Drug Metabolism and Toxicology 3(3):389–406. doi:10.1517/17425255.3.3.389.

Walle T (2011) Bioavailability of resveratrol. Annals of the New York Academy of Sciences 1215(1):9–15. doi:10.1111/j.1749-6632.2010.05842.x.

Mishra SK, Kang JH, Song KH, Park MS, Kim DK, Park YJ, et al. (2013) Inonotus obliquus suppresses proliferation of colorectal cancer cells and tumor growth in mice models by downregulation of β-catenin/NF-κ- signaling pathways. European Journal of Inflammation 11(3):615–29. doi:10.1177/1721727X1301100306.

Wei L, Lin J, Xu W, Cai Q, Shen A, Hong Z, et al (2012) Scutellaria barbata D. Don inhibits tumor angiogenesis via suppression of hedgehog pathway in a mouse model of colorectal cancer. International Journal of Molecular Sciences 13(8):9419–30. doi: 10.3390/ijms13089419.

Yadav VR, Prasad S, Sung B, Gelovani JG, Guha S, Krishnan S, et al. (2012) Boswellic acid inhibits growth and metastasis of human colorectal cancer in orthotopic mouse model by downregulating inflammatory, proliferative, invasive and angiogenic biomarkers. International Journal of Cancer 130(9):2176–84. doi: 10.1002/ijc.26251.

Zou DM, Brewer M, Garcia F, Feugang JM, Wang J, Zang R, et al. (2005) Cactus pear: A natural product in cancer chemoprevention. Nutrition Journal 4:25. doi: 10.1186/1475-2891-4-25.

Richter L, Kropp S, Proksch P, Scheu S. (2029) A mouse model-based screening platform for the identification of immune activating compounds such as natural products for novel cancer immunotherapies. Bioorganic and Medicinal Chemistry 27(23):115145. doi: 10.1016/j.bmc.2019.115145

Phipps HW (2016) Systematic review of traumatic brain injury animal models. Methods in Molecular Biology 1462:61–88. doi: 10.1007/978-1-4939-3816-2_5.

Ruggeri BA, Camp F, Miknyoczki S (2014) Animal models of disease: Pre-clinical animal models of cancer and their applications and utility in drug discovery. Biochemical Pharmacology 87(1):150–61. doi: 10.1016/j.bcp.2013.06.020.

De Jong M, Maina T (2010) Of mice and humans: Are they the same? -- Implications in cancer translational research. Jurnal of Nuclear Medicine 51(4):501–4. doi: 10.2967/jnumed.109.065706.

Schuh JCL (2004) Trials, Tribulations, and Trends in Tumor Modeling in Mice. Toxicologic Pathology 32 (Suppl 1):53–66. doi: 10.1080/01926230490424770.

Li Z, Zheng W, Wang H, Cheng Y, Fang Y, Wu F, et al. (2021) Application of animal models in cancer research: Recent progress and future prospects. Cancer Management and Research 13:2455–75. doi: 10.2147/CMAR.S302565.

Workman P, Aboagye EO, Balkwill F, Balmain A, Bruder G, Chaplin DJ, et al. (2010) Guidelines for the welfare and use of animals in cancer research. British Journal of Cancer 102(11):1555–77. doi:10.1038/sj.bjc.6605642.

Tratar UL, Horvat S, Cemazar M (2018) Transgenic mouse models in cancer research. Frontiers in Oncology 20 (8):268. doi: 10.3389/fonc.2018.00268.

Hanahan D, Wagner EF, Palmiter RD (2007) The origins of oncomice: A history of the first transgenic mice genetically engineered to develop cancer. Genes and Development 21(18):2258–70. doi: 10.1101/gad.1583307

Shah U, Shah R, Acharya S, Acharya N (2013) Novel anticancer agents from plant sources. Chinese Journal of Natural Medicine 11(1):16–23. doi:10.1016/S1875-5364(13)60002-3.

Khan T, Ali M, Khan A, Nisar P, Jan SA, Afridi S, et al. (2019) Anticancer plants: A review of the active phytochemicals, applications in animal models, and regulatory aspects. Biomolecules 10(1). doi: 10.3390/biom10010047.

Pagano JS, Blaser M, Buendia MA, Damania B, Khalili K, Raab-Traub N, et al. (2004) Infectious agents and cancer: Criteria for a causal relation. Seminars in Cancer Biology 14(6):453–71. doi: 10.1016/j.semcancer.2004.06.009.

Tsuda H, Ohshima Y, Nomoto H, Fujita K ichi, Matsuda E, Iigo M, et al (2004) Cancer prevention by natural compounds. Drug Metabolism and Pharmacokinetcs 19(4):245–63. doi: 10.2133/dmpk.19.245.

Liu Z, Brooks RS, Ciappio ED, Kim SJ, Crott JW, Bennett G, et al. (2012) Diet-induced obesity elevates colonic TNF-α in mice and is accompanied by an activation of Wnt signaling: A mechanism for obesity-associated colorectal cancer. Journal of Nutritional Biochemistry 23(10):1207–13. doi: 10.1016/j.jnutbio.2011.07.002

Ooi LL, Zhou H, Kalak R, Zheng Y, Conigrave AD, Seibel MJ, et al. (2010) Vitamin D deficiency promotes human breast cancer growth in a murine model of bone metastasis. Cancer Research 70(5):1835–44. doi:10.1158/0008-5472.CAN-09-3194.

Cheon DJ, Orsulic S (2011) Mouse models of cancer. Annual Review of Pathology 6:95–119. doi: 10.1146/annurev.pathol.3.121806.154244.

Henry NL, Hayes DF (2012) Cancer biomarkers. Molecular Oncology 6(2):140–6. doi: 10.1016/j.molonc.2012.01.010.

Srinivas PR, Kramer BS, Srivastava S (2001) Trends in biomarker research for cancer detection. The Lancet Oncology 2(11):698–704. doi: 10.1016/S1470-2045(01)00560-5.

Slater EP, Strauch K, Rospleszcz S, Ramaswamy A, Esposito I, Klöppel G, et al (2014) MicroRNA-196a and -196b as potential biomarkers for the early detection of familial pancreatic cancer. Translational Oncology 7(4):464–71. doi: 10.1016/j.tranon.2014.05.007.

Brake T, Connor JP, Petereit DG, Lambert PF (2003) Comparative Analysis of Cervical Cancer in Women and in a Human Papillomavirus-Transgenic Mouse Model: Identification of Minichromosome Maintenance Protein 7 as an Informative Biomarker for Human Cervical Cancer. Cancer Research 63(23):8173–80.

Lyons SK (2005) Advances in imaging mouse tumour models in vivo. The Journal of Pathology 205(2):194–205. doi: 10.1002/path.1697.

Li S, Chen L xiang, Peng X hua, Wang C, Qin B yin, Tan D, et al. (2018) Overview of the reporter genes and reporter mouse models. Animal Models and Experimental Medicine 1(1):29–35. doi: 10.1002/ame2.12008.

Lee KW, Bode AM, Dong Z (2011) Molecular targets of phytochemicals for cancer prevention. Nature Reviews Cancer 11(3):211–8. doi: 10.1038/nrc3017.

Navale AM (2013) ANIMAL MODELS OF CANCER: A REVIEW. International Journal of Pharmaceutical Sciences and Research 4(1):19–28.

Chulpanova DS, Kitaeva KV, Rutland CS, Rizvanov AA, Solovyeva VV (2020). Mouse tumor models for advanced cancer immunotherapy. International Journal of Molecular Sciences 21(11):1–15. doi: 10.3390/ijms21114118.

McGrath JC, Drummond GB, McLachlan EM, Kilkenny C, Wainwright CL (2010). Guidelines for reporting experiments involving animals: The ARRIVE guidelines. British Journal of Pharmacology 160(7):1573–6. doi: 10.1111/j.1476-5381.2010.00873.x.

Hollingshead MG (2008) Antitumor efficacy testing in rodents. Journal of the National Cancer Institute 2008;100(21):1500–10. doi: 10.1093/jnci/djn351

Cekanova M, Rathore K (2014) Animal models and therapeutic molecular targets of cancer: utility and limitations Drug Design, Development and Therapy 8:1911–22.doi: 10.2147/DDDT.S49584

Gayatri S, Maheswara Reddy CU, Chitra K, Parthasarathy V (2015) Assessment of in vitro cytotoxicity and in vivo antitumor activity of Sphaeranthus amaranthoides burm.f. Pharmacognosy Research 7(2):198–202. doi: 10.4103/0974-8490.150544

Ayers GD, McKinley ET, Zhao P, Fritz JM, Metry RE, Deal BC, et al (2010) Volume of preclinical xenograft tumors is more accurately assessed by ultrasound imaging than manual caliper measurements. Journal of Ultrasound in Medicine 29(6):891–901. doi: 10.7863/jum.2010.29.6.891

European Medicines Agency. The role of the pathological complete response as an endpoint in neoadjuvant breast cancer studies; 2014. Available at:

Frangioni JV (2008) New technologies for human cancer imaging. Journal of Clinical Oncology 26(24):4012–21. doi: 10.1200/JCO.2007.14.3065

Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, et al. (200) New guidelines to evaluate the response to treatment in solid tumors. Journal of the National Cancer Institute 92(3):205–16. doi: 10.1093/jnci/92.3.205

Narang AS, Desai DS (2009) Anticancer drug development: Unique aspects of pharmaceutical development. Pharmaceutical perspectives of cancer therapeutics. 2009:49-92. doi: 10.1007/978-1-4419-0131-6_2.

Steu S, Baucamp M, Von Dach G, Bawohl M, Dettwiler S, Storz M, et al. (2008) A procedure for tissue freezing and processing applicable to both intra-operative frozen section diagnosis and tissue banking in surgical pathology. Virchows Arch 452(3):305–12. doi: 10.1007/s00428-008-0584-y.

Mayeux R 2004 Biomarkers: Potential Uses and Limitations. NeuroRx 1(2):182–8. doi: 10.1602/neurorx.1.2.182.

McCoy CE 2017 Understanding the intention-to-treat principle in randomized controlled trials. Western Journal of Emergency Medicine 18(6):1075–8. doi: 10.5811/westjem.2017.8.35985.