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A Subject seminar on

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 Vivo models In Vitro methods

Chemically
MTT assay for cell proliferation
induced tumors models

Transplantable
Sulphorhodamine B Assay
tumors models

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 (12-
O-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.

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 75-
95% 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 implanted
subcutaneously

B. Orthotopic transplantation
• To the site of origin
• e.g. lung cancer implanted in lung
• Transplantable tumor
models
B. Heterotopic transplantation
• transplantation of tumor cells or tissue at the site
other than its site of origin.
• Subcutaneous transplantation 1. Intraperitoneal transplatation
• A tumor cell suspension is • Tumor cell suspension is
injected into the flank of the injected in peritoneal cavity.
animal.
• Require between a few days to • Grows within few days.
a few months to grow.
• Develop as solid tumor. • Develops as ascites.

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. Tumor size
2. Tumor weight
3. Excision clonogenic assay
4. End point dilution assay
5. 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 Plated on agar

Colony count in lung, spleen colony count on plate


and liver

• 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.
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.
The Rat Aortic Ring Assay
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).
Thank
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