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In-Vitro, Ex-Vivo and In-Vivo Experimental Models for Evaluation of Probiotics for Cancer Therapy Journal of Critical Reviews ISSN- 2394-5125 Vol 7, Issue 6, 2020 Review Article IN-VITRO, EX-VIVO AND IN-VIVO EXPERIMENTAL MODELS FOR EVALUATION OF PROBIOTICS FOR CANCER THERAPY Rajesh J1, Sai Akilesh M1 And Ashish D Wadhwani1* 1Department of Pharmaceutical Biotechnology, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Ooty – 643001, The Nilgiris, Tamil Nadu, India * Corresponding author: Dr. Ashish D Wadhwani Department of Pharmaceutical Biotechnology ,JSS Academy of Higher Education & Research ,JSS College of Pharmacy, Ooty – 643001 The Nilgiri’s, Tamil Nadu, India E-mail: [email protected] Received: 03.02.2020 Revised: 15.03.2020 Accepted: 17.04.2020 Abstract A disease commonly characterized by the uncontrollable cell proliferation followed by successive metastasis is cancer. Most patients with cancer will have a grossly depleted gut microbiome due to bacterial dysbiosis. Certain probiotic species like Lactobacillus, Bifidobacterium, and Streptococcus are more predominant in healthy tissues than cancer ones of humans. Hence, administration of these bacterial species externally in the form of probiotics may help in alleviating the symptoms of cancer-related dysbiosis as well as in enhancing the immune response of the patients. Recently, several models have emerged in cancer therapy relating to probiotics. The various in-vitro assays and methods are MTT & SRB assay, agar diffusion well plate, Antibiotic susceptibility assay, Cell proliferation assay, Soft agar colony-forming assay, Adherence assay, Cellular uptake study, Cell cycle distribution, and Apoptosis assay. Ex-vivo models include- InTESTine™ system, Engineered probiotics, Ussing chamber, Intestinal enteroids and organoids, Organs over the chip and microfluidic devices, followed by in- vivo models of Immunofluorescence assay, Histological analysis along with protein expression determinations. The ultimate objective for the study is to compile in-vitro, in-vivo and ex-vivo models in cancer research and to bring newer insights to the field of probiotics and its role in oncology. The outcome of this study will allow us to bring a change in current cancer therapy and unlocks the unknown effects on cancer immunology due to probiotic therapy. It also provides potential information on this area for further researches on the same. The impact of this study will serve as a greater purpose in emerging cancer rates worldwide. Keywords: Bacterial dysbiosis; Cancer therapy; Ex-vivo; In-vitro; In-vivo; Immunology; Probiotics © 2019 by Advance Scientific Research. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) DOI: http://dx.doi.org/10.31838/jcr.07.06.138 INTRODUCTION A disease commonly characterized by the uncontrollable cell Gut microbiota proliferation followed by successive metastasis. These cells One of the most critical and complex biodiversity present in the possess the ability to invade the surrounding sites and spread human body is the Gut region which contains host cells, through blood and lymph [3]. As stated by the 2018 statistics, microbiota, nutrients and other such components that cancer is one of the leading causes of mortality worldwide with an communicate with each other. The host and microbiota are estimated 9.6 million deaths [3]. There are five main types and directly dependent on each other for the mutual benefit of several subtypes of cancer worldwide. Carcinoma is a type of survival. This microbiota is generally designated as symbiotes and cancer that begins in the skin or in tissues that line or cover plays a major role in the normal functionalization of the body. internal organs. Their different subtypes include transitional cell They support by neutralizing the metabolites or toxins produced carcinoma, adenocarcinoma, basal cell carcinoma, and squamous in the host cells during the process of digestion or act by cell carcinoma. Sarcoma is a cancer that generally occurs in destroying the system by invasion into tissues leading to other connective or supportive tissues like bone, cartilage, muscle, fat serious consequences [1]. The term associated with this tissues, blood vessels, etc. Leukemia is a cancer that commonly unbalance in the human body is dysbiosis commonly relating to begins in blood-forming tissues such as the bone marrow and the lack of microbiota involvement of infectious microbiota. This causes large numbers of abnormal blood vessels which later may lead to other complications such as cancer, which according enters into the blood stream. Lymphoma and multiple myeloma is to previous other research work suggests that it is mainly due to a cancer that originates in lymphatic immune system which the reduction in active immune response [2]. Though the involves cancer emergence in lymph nodes and bone marrow microbiota plays an essential role in gut physiology, the entire respectively. Central nervous system cancers are cancers that etiology remains a mystery till date. A large number of research is arises from the brain and spinal cord tissues which becomes still in progress to identify the possible role of the microbiota and malignant in a faster rate than other types. Lung cancer, prostate its relation to normal body health. Yet, Studies have still not been cancer, colorectal cancer, stomach cancer and liver cancer are the able to show the possible correlation between the gut microbiota most common types of cancer in males, while breast cancer, and its association with various disease pathophysiologies. Hence colorectal cancer, lung cancer, cervical cancer and thyroid cancer it is imminent to identify the various drug therapies which might are the most common among females. The human metagenome is impact the microbiota. a genetic repository which are almost 100-folds larger than human genome and the host and microbial cells which actively Cancer interact among each of them. It is a complex system which can influence every physiological process in the body while dysbiosis Journal of critical reviews 796 IN-VITRO, EX-VIVO AND IN-VIVO EXPERIMENTAL MODELS FOR EVALUATION OF PROBIOTICS FOR CANCER THERAPY can alter normal physiological processes leading to cancer. Thus, The various probiotic strains can be obtained from recognized the involvement of microbial populations for the homeostasis in collection centers (eg. National Collection of Industrial our system becomes an essential one to consider. Microorganisms (NCIM), India). These cultures are to be maintained by sub-culturing in MRS broth (1% v/v) and growth What are Probiotics? should be monitored with Optical density at a wavelength of 620 Though a majority of bacteria identified have been associated with nm as well as by colony counting on agar plates. The probiotics destructive or unfavorable effects on human health, there have conditioned media (PCM) can be prepared by inoculating been a few identified for its beneficial effects such as digestion, probiotic stains in a 100 ml of Luria- Bertani broth for 18-24 h in destruction of disease-causing cells, production of vitamins or a bacterial incubator.[7][8] essential compounds for the body [4]. Probiotics are ‘live microorganisms which when administered in adequate amounts Preparation of Cell-Free Supernatants (CFS). confer a health benefit on the host’ – definition as per FAO/WHO, CFS are the extracellular substances produced/secreted by the 2002. Most of the microbes in probiotic products are similar to selected probiotic bacteria which can be isolated in RPMI 1640 microbes that naturally live/present in our system. They are medium. Approximately, 105 cfu/ml of probiotics are cultivated in commonly observed in dietary supplements such as yogurt or MRS for 24 h. Further, they should be inoculated in a volume of 14 fermented products. The two commonly observed species of ml of RPMI 1640 and incubated for around 24 h at 37 °C with microorganisms in probiotics are frequent mixing. Incubation must be carried out to optimize the Lactobacillus and Bifidobacterium. In some cases, yeast species of suspensions reach the same concentration of 5 x 108 /ml. After Saccharomyces boulardii has also been used [4]. incubation, samples are to be centrifuged for around 10mins at the rpm of 3000 x g and the pH should result around 6 for lactobacilli Modern treatment approaches for cancer involve chemotherapy, and 7 for S. boulardii. Later, supernatants are sterilized by radiation therapy, and/or surgery, but still, the microbiome plays membrane filtration with 0.22 휇m cellulose filters. CFS then to be a major passive role in the effectiveness of such therapies which stored at 20 °C until use.[7] was unknown in most of the cases. For example, cyclophosphamide – an alkylating drug involved in cancer Cytotoxicity studies models treatment which depends on the health of gut flora for their These studies analysis determines various concentrations of the effectiveness. Some endotoxins such as tumor necrosis factor, product to find IC50, the concentration at which death of exactly have also been linked to metastatic spread. After administering half of the population (50%) of host cells occurred. This obtained these probiotic bacterial groups, they may possess anti- value resembles the average cytotoxicity concentration of carcinogenic properties by decreasing the levels of carcinogenetic compounds treated with healthy living cells. enzymes and causes microflora balance as well as the production of anti-mutagenic organic acids and enhancement of the host's MTT Assay for the determination
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