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Screening & Evaluation of Anticancer Agents

Cancer
• One type of neoplasm (tumor), which is an abnormal mass of tissue, the growth of cell exceeds & is uncorordinated with that of normal tissue & persists in same excessive manner even after the cessation of the stimuli Two types: – Benign tumor: remain localised, can’t spread to other sites – Malignant tumor: can invade, destroy adjacent structures and spread to distant sites

Pathogenesis
 Mutations in genes, involved in mitosis Oncogenes: stimulate mitosis Eg. SIS gene Tumor suppressor genes: inhibit mitosis Eg. p53 gene  Genes that stimulate angiogenesis

Anticancer Drug Evaluating Systems In Vitro methods

In Vivo models

Chemically induced tumors models Transplantable tumors models

MTT assay for cell proliferation

Sulphorhodamine B Assay

3H-thymidine Uptake Assay

Dye Exclusion Tests

Cell Counting Assay

Short term Cytotoxicity assay

Chemically induced tumors
• Chemicals carcinogens are used to induce cancer in animal models. • Carcinogens require metabolic activation before inducing carcinogenesis. • Experimental carcinogenesis involves following three steps: • Initiation: is due to exposure to carcinogens transforming the normal cell to a cancer cell. • Promotion: is due to the triggering of uncontrolled growth of the transformed cell. • Malignant conversion: is caused due to unlodging of cancer cells from the original site,

DMBA- induced Mouse Skin papillomas
Procedure: • Mice are topically applied a single dose of 2.5 ug DMBA in acetone on the shaved back  followed by 5-10 ug of TPA (12O-tetradecanoyl-phorbol-13-acetate) In 0.2 ml acetone twice weekly on the same site starting one week after DMBA application. Papillomas begin to appear after 6 to 7 weeks of application of TPA. Weekly observations are made to monitor tumor development till the experiment terminates after 18 weeks. Drug under test can be administered either topically or by oral route.

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Evaluation: • • Percent tumor incidence & multiplicity of treatment group is compared with DMBA control group.

MNU-induced Rat Mammary Gland Carcinogenesis Procedure: • Single intravenous injection of 50 mg/kg body weight of MNU (pH 5.0) is given to S D rats, usually at 50 days of age. • In some tests, carcinogen has been administered to 120 days old animal (a better model). • The incidence of tumor produced in this model is 7595% within 130 days post carcinogen. Evaluation: • % reduction in adenoma incidence, • Tumor multiplicity (2-4) or % increase in adenocarcinoma latency(65-80 days) compared with control.

MNU-induced Tracheal Squamous Cell Carcinoma in Hamster
Procedure • 5% solution of MNU in normal saline is administered once a week for 15 weeks using specially designed catheter, which exposes a defined area of the trachea of male Syrian golden hamsters to the carcinogen. • Fifteen weeks MNU administration produces tumors in 40-50% animals within 6 months. Evaluation • percentage reduction of tumor incidence compared with carcinogen control.

N, N-Diethylnitrosamine (DEN)-induced Lung Adenocarcinoma in Hamster
Procedure • 17.8 mg DEN/kg body weight twice weekly by subcutaneous injection for 20 weeks starting at age 7 to 8 weeks usually produces tracheal tumors in 90-100% and lung tumors in 40-50% of male Syrian hamsters. Evaluation: • The percentage reduction in tumor incidence in treatment group is compared control group

DMBA-induced Oral Cancer in Hamster
Procedure: • Oral cancer can be induced in male Syrian hamsters by painting right buccal mucosa, 3 times/ week for 16 weeks with 0.5% solution of DMBA in liquid paraffin (approximately 10 ul containing 100 ug). Evaluation: • Tumor size, • Tumor number and • Tumor burden of drug treated animals can be compared with control animals

Benzopyrene-induced For Stomach Tumors in Mouse
Procedure: • 1 mg of benzopyrene in 0.1 ml peanut oil twice weekly for 4 weeks given to mice by gavage. Evaluation: • Tumor incidence and • Tumor burden in drug treated animals can be observed and compared with carcinogen control animals at the end of the experiment.

• Transplantable tumor

models
• These models are based on the use of cancer cell lines or tissues that can be grown in mice or rats.

• Methods of transplantation
A. Heterotopic transplantation
• To the site other than that of origin • e.g. pancreatic cancer subcutaneously implanted

B. Orthotopic transplantation
• To the site of origin • e.g. lung cancer implanted in lung

• Transplantable tumor
B. Heterotopic transplantation • transplantation of tumor cells or tissue at the site other than its site of origin.
1. Intraperitoneal transplatation • Tumor cell suspension is injected in peritoneal cavity. • Grows within few days. • Develops as ascites.

models

• Subcutaneous transplantation • A tumor cell suspension is injected into the flank of the animal. • Require between a few days to a few months to grow. • Develop as solid tumor.

Simple and less time consuming. Most widely used approach for transplantation.

B. Orthotopic transplantation • It refers to the transplantation of cancer cells to the anatomic location or tissue from which a tumor was derived. • For example, lung tumor is transplanted in lungs. • May be accomplished by 1. Direct injection of tumor cells or 2. Surgical Orthotopic implantation (SOI) - Implantation of the tumor fragments by surgery

A. Syngenic models
Mouse or rat cancer cell line or tissues are transplanted in inbred animals of the same genetic background as the derived cell line

Ehrlich Ascites carcinoma model
Host: Swiss albino mice Procedure: ► I.p. injection of 2x105 tumor cells/animal (day 0) ► After 24 hrs of tumor inoculation drug treatment (i.p.) is started. ► On 5th day, animals are sacrificed and peritoneal fluid is collected. Tumor cells from peritoneal cavity are collected by repeated wash with saline. ► Additional groups of animals can be used for survival time assay.

Evaluation parameters:  Volume of peritoneal fluid  Viability of tumor cells in peritoneal fluid (% viability)  % Increase in survival time as compared to control

A.Xenograft models
• For tumor models that more closely resemble the clinical disease, transplantable tumors of human origin should be used. But transplantation of such human tumors in mice may result in severe immune rejection. For this purpose athymic (nude) mice or severe combined immunodeficiency (scid) mice are used. These animals lack immune response to such foreign transplanted material. Transplantation of tumor cell lines into nude mice can be accomplished via multiple routes: s.c., i.p., i.v., intracranial, intrasplenic, renal subcapsular.

• • • •

1. Intraperitoneal Microencapsulated Tumor Assay
Procedure: • Tumor cells are encapsulated in semipermeable gels that can be formed into microcapsule of from 0.05 to 1 mm  600 MC injected into the peritoneal space of mice. The semipermeability of the capsule protects the tumor cells from host cell–mediated immune cytotoxicity, so that athymic (nude) mice need not be used. At the same time, it allows nutrient and systemic cytotoxic agents to diffuse and reach the tumor cells

Evaluation • Counting viable tumor cells by Heamocytometer in treated versus control animals.

2. Hollow fiber Assay
Procedure: • Polyvinylidene fluoride (PVDF) hollow fibers (500k Da M.wt. exclusion, 1mm i.d.) containing target cells are heat, sealed and cut at 2 cm intervals and implanted into rodents. • 3 or more tumor cell lines can be grown concurrently, in 2 physiologic sites, i.p. and s.c. within each mouse. • The mice are treated with experimental compounds once daily for four days. Fibers are collected 24 hr following the last dose of compound. Evaluation: • After collection the viable cell mass is determined using an MTT dye conversion assay. • The cytostatic/cytocidal effect of a compound is determined from differences in the viable cell mass in fibers from compound treated Vs diluent treated mice.

• Methods for evaluation of drug

effect
1. 2. 3. 4. 5. Tumor size Tumor weight Excision clonogenic assay End point dilution assay Increase in Survival time

Excision clonogenic assay
• Determines the fraction of cells in a tumor population retaining proliferative capability after drug exposure. • Tumor bearing animals are treated with drug. • At 24 hours, the tumors are excised from treated and untreated animals. Tumor Cell suspension Inject i.v. in animal Colony count in lung, spleen and liver • • Plated on agar colony count on plate

Colony-forming efficiency (CE) = no. of tumor colonies counted
no. of tumor cells plated Surviving Fraction (SF) = CE treated CE control

TD50 (End point dilution assay)
• • • • • It is the tumor cell inoculum that produces tumor growth in 50% of inoculated animals It is a measurement of the number of cells required to produce tumors from inocula in vivo A cell suspension is prepared from both treated and untreated animals. Various dilutions for each tumor are prepared depending on the expected value of TD50. The suspension is inoculated into groups of test animals subcutaneously, intramuscularly, or intradermally for solid tumors and intraperitoneally or intravenously for leukemias. The percentage of tumor take versus cell number inoculated for each treatment is determined and compared to control animals to determine TD50.

Survival time assay
 Survival of animal on drug treatment is compared with that of control.  Sum total of interactions between tumor, drug, and host  Since drug toxicity and tumor growth both have independent effects on survival, a judgment can be made about therapeutic index.

In vitro methods
1. Short term Cytotoxicity assay
• • Specific number of cells is incubated with drug for 3 hrs at 37°C. After 3 hrs, number of viable cells is counted using trypan blue dye exclusion method using haemocytometer. % reduction in cell viability as compared to control is calculated. IC50 for the drug is calculated using this data.

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2. Microculture Tetrazolium Test
Procedure: • 100μL of cell suspension (containing specific amount of cells) is dispensed in respective wells of 96 well plate. • Different concentration of drug is added to each well. Each concentration in triplicate. • The plate is incubated at 37°C, 5% CO2 and 95% humidity. • After 18 to 22 hr, 20μL of 0.5% MTT is added to each well. • Incubate for 4 hrs. • Then 100µL of 25% SDS is added to each well to solubilize the formed formazan crystals. • The plate is kept at room temperature for next 17 to 20 hrs. • The optical density of wells is recorded at 570nm using a multiwell plate reader. • The cell number correlating with the optical density values can be read from a calibration curve previously built using known number of cells. • % inhibition of cell proliferation by drug as compared to control is calculated. • IC50 for drug is obtained from this data.

3H-thymidine Uptake Assay
• Tumor cell suspensions are exposed to the drug continuously for 5 days, • After which a radiolabeled precursor (3H-thymidine) is added during the final 48 hours of the assay. • The replicating cells will incorporate [3H]-thymidine into their DNA, which can then be determined either by autoradiography or by liquid scintillation counting. • Autoradiographic determination of the [3H]-thymidine provides information on tumor growth kinetics. This can generate DNA histograms, which can provide information on the ploidy status of the cells. • This assay looks at cells, which have actively replicating DNA and hence are viable. • Nonreplicating or dead cells will not be counted in this case.

Fluorescence
• Fluorescent dyes may be used in conjunction with microscopic evaluation methods as an in vitro chemosensitivity assay. • Cells are exposed to fluorescent-labeled precursors after drug-exposure. • The replicating cells will incorporate labeled precursor into their DNA and the resulting florescence is then measured by flow cytometry. • This assay also looks at actively replicating cells and hence dead or nonreplicating cells are not counted.

Differential staining cytotoxicity assay
• The DISC assay is drug sensitive assay, which relies on structural integrity of the cells. • In this assay, cells are incubated with drugs for 4 days. • Dead cells are stained in suspension with fast green dye with or without nigrosin. • The specimen is centrifuged and discs of cells are collected in the microscopic slides. • Live cells are then stained with hemotoxylin-eosin. • As control duck erythrocytes are used. • The end point of the study is the morphologic identification of tumor-cell cytotoxicity compared with the internal control standard of duck erythrocytes. • The DiSC assay measures cell kill in both dividing and nondividing tumor cell population.

Cell Counting Assay
• Cells are cultured in the presence of drug for 2-5 culture-doubling times, • After which the cell number is estimated using a hemocytometer or a cell counter. • The assay is easy to perform, rapid and can be used for both adherent and suspension cell lines. • However, dead and nonreplicating cells can be counted in this assay by the cell counter. • The IC50 values can be calculated in all the above assays.

Clonogenic Assays
• • • • • • It is the most direct method of measuring cytotoxic activity of a drug. In cologenic assays single-cell suspension are prepared from tumor biopsies and exposed to anticancer agents to be tested. Cells are then rinsed and plated in a semisolid medium (agar or methyl cellulose), a medium that precludes proliferation of nonmalignant cells in the specimen. After 14 to 28 days, some cells will have undergone several divisions and will have formed tumor colonies, which can be quantified in a visual or semi automated fashion. Nonreplicating and dead cells are not counted in this case. The number of colonies from the treated cells is compared with the number of colonies from the untreated control cells and the fraction of control growth provides an index of drug activity.

Inhibition of angiogenesis
Endothelial cell proliferation
Rationale:
• Proliferation of endothelial cells is an important process of angiogenesis. Human umbilical vein endothelial cells (HUVEC) are used to study endothelial cell proliferation

Procedure:
• The HUV-ECs are cultured at 37 °C and 5% CO2 in 90% Ham’s F12K, 10% fetal bovine serum, 30 μg/ml endothelial cell growth factor, 100 μg/ml heparin, and 4 mM L-glutamine.at a density of 3 × 103 cells/well in 24-well plates. After 24 hrs, the test compound in various concentrations of the vehicle are added, and plates are incubated for 72 h. Cells are then harvested with trypsin/EDTA and counted by a hemocytometer.

• •

Evaluation:
• Inhibition of cell proliferation is compared in vehicle treated and compound treated cultures.

Chorioallantoic membrane assay
Rationale:
• Angiogenesis on chorioallantoic membrane of chicken eggs is used as a model system.

Procedure:
• • • • Fertilized White Leghorn chicken eggs  incubated at 37 °C On day 3, a square window is opened in the shell and 2 to 3 ml of albumen is removed to allow detachment of the developing CAM. The window is sealed with a glass and the eggs are returned to the incubator. On day 8, 1 mm 3 gelatin sponges loaded with 3 μl phosphate buffered saline (negative control) or containing 3 μg of the basic fibroblast growth factor (positive control), or with various doses of test compound, are implanted on top of the CAM. CAM are examined daily until day 12, when the angiogenic response peaks.

Evaluation:
• On day 12, blood vessels entering the sponge within the focal plane of the CAM are recognized, counted and photographed. Only transversely sectioned microvessels i.e. capillaries and venules with or without a 3 to 10 μm lumen are counted

Mesenteric window angiogenesis model
Rationale: • The mesenteric window assay in rats for quantitative measurement of induction and inhibition of angiogenesis Procedure: • i.p. injection of the mast cell secretagogue compound 48/80 twice daily for 4-5 days to male S D rats ( 225 g.) • Test compounds or saline are injected s.c. 1 h before each injection of compound 48/80. • Specimens are fixed on slides and stained with toluidine blue to measure the relative vascularized area. • Three randomly selected vascular view fields per mesenteric window spread are analyzed for microvascular length per unit area of vascularized tissue. • The total microvascular length is computed from the vascularized area of each animal multiplied by the mean microvascular length for the corresponding treatment group. Evaluation: • The total microvascular length in control and drug treated groups are compared by statistical analysis of the observations.

Procedure: It involves three steps: Preparation of the aorta • Thoracic and abdominal aorta is isolated  transferred to a culture dish containing MCDB 131 culture medium remove the surrounding fibro-adipose tissues  cut into 1mm ring sections and rinsed in culture medium to remove blood residues. Making agarose culture wells • Concentric rings from solidified agarose are prepared in a Petri dish and 4 wells are transferred in each culture dish. Culturing of the aortic rings • Bottom of each well is coated with 150μl of clotting fibrinogen solution. • Aortic rings are embedded into the well by filling up all the agarose wells completely with fibrinogen solution. • Then 10ml of MCDB 131 culture medium is added into each Petri dish and kept at 37 °C with 5% CO2. Evaluation: • Quantification of microvessel growth is done manually or by computer assisted image analysis (i.e. pixel integrated density is calculated from the image).

The Rat Aortic Ring Assay

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