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

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

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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 of mitochondrial activity immune system. Expression of Hypoxia-inducible factor (HIF), The assay is based on the concept of dead cells or their products Tumor necrosis factor (TNF) & matrix metalloproteinase (MMP- do not reduce tetrazolium while living cells cleave the 3-[4,5- 9) and their pathways that are responsible for the expression of dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) several oncogenes can be suppressed by selected probiotics and into a blue formazan crystals which is a very effective principle for causes high cytotoxic levels in cancer cells. They involve in the cytotoxicity determination. The extent of formazan crystals stimulation of immune cells in-vivo for production of natural killer production by the cells is directly proportional to the number of (NK) cells, IgA-secreting cells, T-lymphocytes (CD4, CD8, and living/alive cells.[9] Cancer cells should be seeded in 96-well CD3), IL-5- and IL-6-producing cells and specific IgG against plates at a density of 2.5 x 104 cells with a volume of 100 µl. The cancer cells and to regulate tumor progression. Commensal cells are incubated to allow adherence to the plate for 24 h. Later, probiotics may influence response to therapy by modulating cells are treated with conditioned culture medium (which is CFS the tumor microenvironment [5]. The following are the list of and PCM suspended in the medium) at concentrations of 50 µg/ml, probiotic organisms which have shown promising results in 100 µg/ml, 250 µg/ml, 500 µg/ml, 1000 µg/ml and 2000 µg/ml cancer therapy: [6] for around 3 d in an incubator (of 37 ºC, 5 % CO2, 95 % air 1. Lactobacillus Rhamnosus strain atmosphere). Later, 50 µl of the solution of MTT (2 mg/ml 2. Lactobacillus acidophilus strain solution) in PBS shall be added to each well and incubated again 3. Lactobacillus casei shirota strain for 4 h at 37 °C to allow the formation of formazan crystals in live 4. Lactobacillus reuteri strain cells. The formazan crystals formed in live cells are solubilized by 5. Lactobacillus crispatus strain adding 50 µl of dimethylsulfoxide (DMSO)/isopropanol and can be 6. Lactobacillus plantarum strain allowed to stay undisturbed for few minutes (Fig. 1). [9] The 7. Lactobacillus thermophiles strain absorbance can be measured at 540 nm using an ELISA microplate 8. Lactobacillus keiri strain reader. The total viability of the test shall be calculated by the 9. Enterococcus lactis strain following formula: 10. Bacillus subtilis CSY191 strain −ퟏ 11. Bacillus polyfermenticus strain 퐀퐬퐚퐦퐩퐥퐞 – 퐀퐛퐥퐚퐧퐤 12. Bifidobacterium adolescentis strain Viability (% of control) = ( ) x 100 퐀퐜퐨퐧퐭퐫퐨퐥 – 퐀퐛퐥퐚퐧퐤 13. Bifidobacterium longum strain This review comprises of the various in-vitro, ex-vivo as well as in- Where, Ablank is the absorbance of culture medium used in this vivo models of probiotics which may aid in cancer research. assay without cells, while Acontrol is the absorbance of untreated (with PCM/CFS) cells. In-vitro models Preparation of probiotic stains

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Fig. 1: Schematic representation of procedure of MTT Assay

Total protein content determination by the assay of the antimicrobial effects of any 5 antibiotics can be measured for Sulphordamine B (SRB) standard [11]. After incubation at 37 °C for 24 h, growth inhibition Under certain minimal acidic conditions, SRB binds to trichloro zones are to be measured with a ruler. From the results of zone of acetic acid fixed cells which specifically binds to protein basic inhibition (ZOI), the report of the activity should be divided into amino acid residues of the fixed cells to provide a relative index of highly potent (diameter ≥20 mm), moderate (20 mm ≤ diameter ≥ cellular protein content which is almost linear around a cell 10 mm), and weakly potent (diameter ≤10 mm).[11] population range of at the minimum of two orders of magnitude. The assay can be carried out on CFS, PCM, and control cell lines. Antibiotic susceptibility test From the cell culture media, PBS is to be removed and successive This method is originally standardized as per ISO 10932/IDF 233 trypsinization must be carried out. After incubation, trypsin is standards with minor modifications. In which, the initial step inactivated and transfer into a falcon tube to determine cell involves swabbing the cultures previously activated on prepared concentration. Cell concentration is adjusted and seeding is agar plates. The antibiotics are in the form of a disc which includes performed to obtain a seeding density of 50 μl in 96 well plates. a wide range of antibiotics like tetracycline (30 μg), ampicillin (30 This is then transferred to a sterile reagent reservoir.[10] The μg), oxacillin (10 μg), cotrimoxazole (25 μg), etc. The observed treatment solutions are then added into the cell suspension (50 μl ZOIs shall be noted after incubation at 37 °C for one day followed of solution added). Set aside three wells for untreated or vehicle by the interpretation of resistance and sensitivity. [12] control. Incubation is carried out followed by cell fixation. This can be performed by the addition of 25 μl of TCA into each well and In-vitro anti-oxidative studies incubated at 4 °C for an hour. They are then washed and air dried This method is carried out by measuring the scavenging activity of to room temperature (RT). Then 50 μl of 0.04 % SRB is added to 2,2-diphenyl-1-picrylhydrazyl radicals. Brieﬂy, PCM & CFS are each well, leave at RT for 1 h and rinse with 1 % Acetic acid (200 added to 1 ml of DPPH solution (of 2 mM/l in methanol). The μl). This is followed by addition of 50 μl to 100 μl of 10 mM Tris mixture should be mixed intensely and incubated for 12 h. The base solution (pH 10.5) to each well and shaken on an orbital absorbance shall be measured at 517 nm until the reaction shaker for 10 min and measurement of absorbance is done at 511 reaches a constant state. Phosphate buffered saline shall be used nm. [10] as sample blank. The free radical scavenging activity is calculated by the following formula: Free radical scaving activity = (1 − Anti-microbial assay As Agar diffusion well ) X 100 Ab This method shall be employed to determine the antimicrobial Where, Ab and As are the absorbance of the blank and sample, activities of the Probiotics against any 10 clinically important respectively. Ascorbic acid can be used as a positive control.(Fig. human pathogens. Suspension of pathogens are cultured and 2)[13 spread on agar plates. 10 μl of each PCM & CSF are added to previously punched wells in the spread plates. For comparison, ]

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Fig. 2: Schematic representation of antioxidant assay

In-vitro anticancer activity prepared and mixed to the cells followed by 4hrs of incubation. Cell proliferation assay The spectrophotometer shall be measured with an absorbance at Cell proliferation is determined by XTT assay and trypan blue 470 nm and a reference wavelength at 640 nm to observe exclusion (TBE) assay. Cell proliferation assay can be carried out colorimetric intensity of wells. For the TBE assay, cells are with Assay Kits and these cells are to be plated in 96-well plates collected and suspended in a suitable culture medium. Both for overnight stabilization PCM & CSF mixed with the absolute trypan blue solution and cell suspension is mixed with an equal medium in one to two ratio are added to the cells and incubated volume ratio and the total number of cells should be determined for around 13 d and regular change of media is carried out. with the help of optical microscopy (Fig. 3)[8] Towards the conclusion of experiments, XTT labelling mixture is

Fig. 3: Schematic representation of cell proliferation assay

Soft agar colony-forming assay contents (like medium and non-adherent bacteria) is All cells are cultured in PCM mixed culture medium in a removed by washing 2-3 times with phosphate-buffered ratio of 1 to 2 and also in absolute culture medium for saline. 200 µl of 1 % Triton X-100 solution is added and control and incubated for 12-14 d and the medium is mixed and then 800 µl of normal saline is added into the changed every 2-3 d. After incubation period, cultured wells. Probiotic bacteria can be counted using a 1 ml cells are suspended in 0.46 % agar solution and plated on sample.[14] 6-well plates which is pre-coated with 0.5 % agar layer. After solidification, medium is added. DOX (1 mg/ml) will Cellular uptake study be added to cancer cells as standard. After incubation at 37 Initially, cells are incubated at 37 °C along with PCM (1 μg/mL) & °C, a photograph of 3 random fields is obtained per well in CFS for 24 h. Later, the cells are microcentrifuged and fixed with a 5 bright field with an inverted microscope with a digital paraformaldehyde of 4 % and mounted with 4’,6-diamino-2- camera. Colonies of larger than 4 mm are accounted for phenylindole. The mounted slide shall be visualized under laser interpretation and considered. [8] scanning microscopy and the cellular uptake of Probiotics shall be determined.[15] Adherence assay Initially, 100 µl of PCM & CFS are mixed with all cell lines in Cell cycle analysis 0.4 ml of incomplete media (without antibiotics) and the This assay of treated and non-treated cells in cell cycle at different mixture is subjected for incubated about 2 h at 37 °C in an phases (Fig. 4) can be analyzed by flow cytometry. [15] incubator (of 5 % CO2). Following the incubation, the

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Fig. 4: Schematic representation of cell cycle – Left: Normal cell and Right: Cancer cell

The cancer cells are seeded at the rate of 1 × 106 cells/well in 6- and incubated at 4 °C for overnight (15). The fixed cells should be well microtitre plates followed by incubation about 24 h. The centrifuged to pelletize, resuspended and treated with a staining wells are then treated with PCM & CFS for 48 h to understand their solution mixture of propidium iodide, RNAse and Triton-X for 40 impact on the cellular level. Followed by the incubation, washing min at 37 °C nucleus staining. Resultant cells after incubation shall of cells with Phosphate buffered saline, followed by harvesting of be analyzed using a flow cytometer. (Fig. 5) [15] cells using trypsin EDTA and to be fixed in 70 % ethanol of ice-cold

Fig. 5: Schematic representation of cell cycle distribution assay

Apoptosis assay requirements for site-specific delivery. Since microbes possess Apoptosis assay can be analyzed by Annexin V and propidium the property of preferential colonization of tumors, the study iodide (PI) dual staining assay which helps to identify the type of carried out by Gurbatri et.al., involved the production process cell death that occurred. This is the most effective method to under control and release of nanobodies inside the tumor which identify apoptotic cells. Initially, cancer cells are seeded in a targets cell death–ligand 1 (PD-L1) and cytotoxic T lymphocyte- microtitre plate at a density of 1 × 105 cells in each well and shall associated protein-4 (CTLA-4) under controlled stable release be allowed for incubation about 24 h for the cells to adhere to the mechanism. The optimized genetic circuit factors to improvise the plate. The cells are then treated with all PCM & CFS samples for 24 therapeutic efficacy are done by computational modeling along h. The non-treated cells can be taken as control. Later, cells should with observational verification of lysis circuit dynamics. Each be rinsed thrice with phosphate-buffer saline and stained by administration in this system will show improved therapeutic adding 2.5 μl of annexin V and PI each at 37 °C for 30 min. To feedback in comparison with other clinically important antibodies identify and count the total amount of apoptotic cells occurred in and hence resulted in regression of the tumor in syngeneic mouse each sample induced wells, the cells are sorted by flow cytometry. models, followed by enhancement of activated T-Cells and [15] memory T-cell from the system of mice. Therapeutic efficacy is expected to get increased in poorly immunogenic syngeneic mice EX-VIVO MODELS OF PROBIOTICS IN CANCER models by combinational therapy with probiotics produced These are the models that contain a whole/part of living tissues cytokine, granulocyte-macrophage colony-stimulating factor with functional environments to the cells found in vivo that will be (GM-CSF).[16] cultured in-vitro and analyzed. InTESTine™ system by TNO Engineered probiotics for local tumor delivery of checkpoint TNO is previously associated with manufacturing of both the types blockade nanobodies of TNO Intestinal Models and Tiny Tim models which are the Checkpoint inhibitors have been vastly used in cancer research models that resemble the digestion and absorption of compounds but work only in a small group of target groups and may reveal a in the small intestine. They have lately come up with the model wide range of toxicities levels, hence recommending the InTESTine™ which makes use of the intestinal tissue from various

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sections of the gastrointestinal tract of healthy and fresh porcine examination of host-microbiome interactions. They now shall be and functions during the absence of microbiota. This model isolated by various methods from different species which are used utilizes a mucus layer and ensures a more successful result in in stem cells cultivation obtained from the biopsy tissues of conjunction with single bacteria or a combinational bacterial humans which are termed as human intestinal enteroids. Since group. Though the data hasn’t been published, it offers a they possess various working cells from the gastrointestinal tract, promising method of studying interactions between the microbes they may also prove to be an effective model in the analysis of the and the host.[17] brain-gut axis.[19]

The Ussing chamber Microfluidics and Organs over the chip devices First developed by Hans Ussing, this model is used to study the The most modern development in bioengineering is the organs on transportation across tissues and becomes a novel tool in the a chip which makes use of the concept of microfluidics. It mimics study of various segments of the intestine. The morphology the complicated multiple organs and the multiple section systems consists of a polarized epithelium separating two proportions and present in-vivo. There are several variations of the same which it is made to separate the apical and basolateral sides. Electrodes currently exist and expand rapidly. The anti-inflammatory are used to measure the short-circuit current in the determination properties along with the analysis of bacterial growth may be of permeability. It is mainly used to analyze the interactions screened using this model. Also, these devices help in subculturing between bacteria and the host through toxins produced by the live microbes and artificially constructed human intestines. bacteria. Studies previously carried out on Ussing Chamber found The recent modifications in the gut in chip technology enabled the the possible activities namely- Intestinal ion transport, Bacterial electrodes to be immersed into chips for understanding the trans- invasion, bacterial and toxin-mediated cellular effects, epithelial electrical resistance. Another human biopsy derived Permeability, prostaglandin E2,Interleukin 8 and lactate organoid intestine on-chip is used to mimic the complex in-vivo dehydrogenase release. Thus, demonstrating this model helps to intestinal epithelium tissue.[20] understand the capability of probiotics to improve/promote the ultimate role of gut barrier with the help of Ussing chamber.[18]. IN-VIVO ANTICANCER MODELS This model is carried out on cancer-induced animals. Animal's The Organoids and intestinal enteroids condition will be monitored continuously during the study and This method makes use of a biopsy tissue as well as a traditional tissue samples shall be collected from those animals for further culture dish system. Dr. Johannes Carolus clever's lab in investigation after the study. The assays after probiotics Netherlands had initially established a technique for segregating administrations (in-vivo) and tissue collection process are; the intestinal sections of mice. Later, they propagated stem cells called leucine rich g-protein coupled receptor 5 cells which are Histological analysis by paraffin inclusion (Sainte Marie’s further sub-cultured and maintained for further studies in-vitro, technique) which is termed as intestinal organoids. Yin et al. worked on Animals should be sacrificed after the sufficient drug treatments directing the differentiation of these stem cells to make it more and the targeted tissue of interest is removed for histological cell-specific which have helped researchers identify cell-type preparations. The isolated tissues are analyzed by paraffin specific responses and would provide a useful tool in the inclusion (Sainte Marie’s technique). (Fig. 6)

Fig. 6: Schematic representation of Histological analysis assay

Immunofluorescence assay for identification of T and the obtained observations shall be stated as the amount of Lymphocytes and IgA positive cells in the sample treated as well as standard and These immunofluorescence assays are used to analyze the control-treated sections. [21] quantity of CD4+, CD8+, and CD3+ T lymphocytes as well as Immunoglobulin alpha (IgA) cells in the histological samples Immunofluorescence assays for identification of Interleukin obtained from the tissues for every time interval during the assay (IL) producing cells along with control groups. IgA cells can be analyzed by the The obtained sections from different samples of different groups sections to be incubated along with the mixture of α-chain are subjected to the measurement of cytokine-positive cells. monospecific antibody and Fluorescein isothiocyanate (FITC) Paraffin sections are immersed with 1 % blocking solution of conjugation. Monoclonal antibodies are linked with FITC which Bovine serum albumin (BSA) and Hanks balanced salt solution can be used to identify CD3+, CD4+ and CD8+ T-lymphocyte (HBSS) and subjected to stand for 30 min at RT as incubation determinants [21]. Using a fluorescent light microscope at a period which is after the process of deparaffinization by magnification of 1000x, the total amount of fluorescent cells immersing in xylene and rehydrating in ethanol. They are rinsed present in the slides shall be counted under suitable magnification for 1-2 times with the solution of saponin-HBSS and goat serum is

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added for further incubation (where goat serum diluted with several greater positive impacts on our system. Commonly 1:50) about 40 min. Again for 75 min at RT, Goat and rabbit anti- observed impacts of probiotics are the stabilization of gut mouse of IL-5 and IL-6 respectively should be applied to the microbiota or its population, improved gastrointestinal barrier sections [21]. Polyclonal anti-IL-1 rat antibody, Monoclonal anti- functions and suppression/neutralization of pathogenic microbes IL-2 rat antibody, anti-IL-10 Monoclonal Antibody and Goat anti- or the existing carcinogenic agents which are resulted from mouse IL-12 antibody should be applied to sections and incubated food/drug metabolites or by pathogenic microbes around the at RT for 75 min. After incubation, sections are subjected to intestinal microenvironment. Probiotics may play an important washing for two times with a saponin-HBSS solution. They are role in minimizing the probability of several serious then treated by a diluted form of rabbit anti-goat antibody coupled diseases/disorders including cancer. Whether by by FITC at RT for 45 min. Again, the sections are washed with the elevating/normalizing the immunity levels of our system or by saponin-HBSS solution. The number of fluorescent cells are directly reducing the proliferation of cancer cells/tumor, counted in a fluorescent light microscope. The results is be probiotics are expected to have a greater impact on the system. expressed as the amount of positive cells in 10 fields of vision. [21] Almost, all models which are used to assess the anticancer activity of selected probiotics represent explanations of exact in-vivo Determination of CD-206 (mannose receptor), CD-68 and conditions. But still, some modifications shall be made to obtain TLR2 positive cells from histological tissues the in-vitro observations to correlate with the in-vivo situation. A The obtained sections from different samples of different groups special amount of care must be undertaken in extrapolating the are subjected for further analysis of CD-206 and TLR-2 positive observations to a larger scale. These models are of great help in cells. Following the process of paraffin removal from the sections selecting specific probiotic candidates and are indispensable in by immersing and rehydrating in xylene and ethanol solutions, the the study of its various mechanisms in the gut. However, factors obtained sample slices are subjected to the addition of blocking such as transient colonization as well as vivo adhesion of solution (which is 1 % BSA) and HBSS and incubated for around probiotics must be verified/confirmed with in-vivo models. This 40 min at RT. Later, the samples are to be rinsed with HBSS for 2- should be executed only in the host in which the probiotics are 3 times (a sufficient number of times). Mouse anti-human CD-206 intended/related due to their mechanism of action in host-specific monoclonal antibodies or polyclonal antibodies of the rabbit can physiology, anatomy and other intestinal microbiotic factors. be added to the segments at RT for 1 h which are in diluted forms Considering all the aspects which are influencing the in-vivo at the ratio of 1:200 and 1:300 respectively [21]. After the models but cannot be simulated in-vitro, there is no proper incubation process, few rinses with HBSS shall be carried out. The resolution in the results of in-vitro and ex-vivo models. Thus, segments are later subjected to the treatment with fluorescein causing in-vivo models as an essential part to analyze whether the isothiocyanate in conjugation with goat anti-rabbit antibody or binding of probiotics to the intestinal mucosa and stimulating the rabbit anti-mouse by equal dilutions at RT for 45 min followed by immune response and its relevance in cancer immunology which washing with HBSS for about 1-2 times. The number of fluorescent is a prerequisite for its physiological functionality or not. Thus cells are counted in a scanning electron microscopy with suitable according to this, further studies are highly suggested, particularly magnifications. In the end, observations can be stated as the abiding clinical studies, which needs to be clarified/analyzed to amount of positive cells present in the sample treated as well as resolve the contention. So, understanding these in-vitro, ex-vivo standard and control-treated sections. [21] and in-vivo models may lead us to understand the exact role/mechanism of probiotics in cancer immunology and Determination of CK7, GAPDH, PGK1, and CK19 protein contributes a novel therapy against cancer. expressions in breast tissue Sample Preparation SOURCE OF FUNDING To attain >70 % tumor cells containing tissue, 50 µm thickness of Nil each section of tumor tissues to be slit and the tissue should be microdissected. The microdissected tissue is distorted by a CONFLICT OF INTEREST mechanical method with the help of small mortar and pestle and The authors declare that they have no conflict of interest all soluble proteins will be extracted with the help of a buffer of volume depending on the amount of tissue (approx. 500 µl) ACKNOWLEDGEMENT containing 2 M thiocarbamide, 4 % CHAPS (3-(3- The authors acknowledge the facilities provided by JSS College of Cholamidopropyl) dimethylammonio-1-propanesulfonate), 7 M Pharmacy, Ooty (JSS Academy of Higher Education & Research) urea and 15 mM Tris (pH - 8.5). The process of lysate follows for caring out original research work on probiotics against various centrifugation for 20 min of about 15,000 rpm to get the cancers. supernatant. The extracted proteins are stored at -80 °C until analysis after measuring the protein concentrations by Bradford REFERENCES assay. Proteins are extracted from 12 mg to 58 mg of tissue whose 1. Thursby E, Juge N. Introduction to the human gut concentrations ranging around 2.6 µg/µl and 19.6 µg/µl microbiota. Biochem J. 2017 Jun 1;474(11):1823–36. respectively. These gathered samples can be used for comparative 2. Carding S, Verbeke K, Vipond DT, Corfe BM, Owen LJ. proteomic studies after this assay. [21] Dysbiosis of the gut microbiota in disease. Microb Ecol Health Dis [Internet]. 2015 Feb 2 [cited 2020 Feb 18];26. Western Blot Available from: Pooled protein groups of individual samples may be identified by https://www.ncbi.nlm.nih.gov/pmc/articles/PMC431577 the Western blot technique. 9/ With the help of staking/resolving gels (SDS-PAGE), 20 µg/sample 3. Blackadar CB. Historical review of the causes of cancer. of protein should be separated and the obtaining resolved World J Clin Oncol. 2016 Feb 10;7(1):54–86. proteins is shifted to 0.2 µm of nitrocellulose membranes at 25 4. Gill HS, Guarner F. Probiotics and human health: a clinical Voltage for about 40 min. The pattern of the proteins obtained perspective. Postgrad Med J. 2004 Sep 1;80(947):516–26. from treated cells is compared with untreated cells[22]. 5. Ding C, Tang W, Fan X, Wu G. Intestinal microbiota: a novel perspective in colorectal cancer biotherapeutics. CONCLUSION OncoTargets Ther. 2018 Aug 13;11:4797–810. Nowadays, wide advantageous impacts of probiotics are found 6. Górska A, Przystupski D, Niemczura MJ, Kulbacka J. due to its developing medical significance on the host health are Probiotic Bacteria: A Promising Tool in Cancer Prevention well discovered lately. Probiotics supplemented orally causes and Therapy. Curr Microbiol. 2019 Aug;76(8):939–49.

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IN-VITRO, EX-VIVO AND IN-VIVO EXPERIMENTAL MODELS FOR EVALUATION OF PROBIOTICS FOR CANCER THERAPY

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