various invivo and invitro methods of screening anticancer drugs

1. SCREENING OF ANTI CANCER AGENTS
1

2. INTRODUCTION
• CANCER: Uncontrolled proliferation of
genetically altered cells.
• Derived from the repeated divisions of a mutant
cell.
• Due to the effects of carcinogens, such as
tobacco smoke, radiation, chemicals or
infectious agents.
• Cancer-promoting mutations may be acquired
through errors in DNA replication.
• Activate the cancer promoting oncogenes and/or
inactivate the tumor suppressor genes.
2

3. •Cancer chemotherapy is a rather young discipline.
•It has been pursued with scientific vigour and
multinational collaborations only since the mid-20th
century.
•Although 92 approved anticancer drugs are available
today for the treatment of more than 200 different tumor
entities, effective therapies for most of these tumors are
lacking.
•Out of the 92 registered drugs, 17 are considered by
oncologists to be more broadly applicable and 12
additional agents are perceived as having certain
advantages in some clinical settings
3

4. •They are mostly cytotoxic in nature and act by a very
limited number of molecular mechanisms.
•Thus, the need for novel drugs to treat malignant disease
requiring systemic therapy is still pressing.
• A preselection, called the screening process, is therefore
required.
•The aim of screening efforts is to identify products that
will produce antitumor effects matching the activity criteria
used to define which compounds can progress to the next
stage in the preclinical development program.
4

5. NEED FOR NOVEL ANTI-CANCER
DRUG
• Development of multidrug resistance in patients.
• Long-term treatment with cancer drugs is also
associated with severe side effects.
• Cytotoxic drugs have the potential to be very
harmful to the body unless they are very specific to
cancer cells.
•New drugs that will be more selective for cancer
cells
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6. ANTI- TUMOUR MODELS
INVITRO METHODS INVIVO METHODS
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7. 7
In-vitro methods

8. Advantages:
•Reduce the usage of animals.
•Less time consuming, cost effective & easy to
manage
•Able to process a larger number of compounds
quickly with minimum quantity.
•Range of concentrations used are comparable to
that expected for in vivo studies.
Disadvantages:
•Difficulty in Maintaining of cultures.
•Show Negative results for the compounds which gets
activated after body metabolism and vice versa.
Impossible to ascertain the Pharmacokinetics. 8

9. Cell Viability
• Functional assay
• DNA labeling
assay
• Morphological
assay
• Reproductive
assay
• Membrane integrity assay
9

10. The major criteria employed in viability assay
Category of
viability assay
Assays Principles
Membrane integrity
assay
-Exclusion dyes
-Fluorescent dyes
-LDH leakage
The determination of membrane integrity via
dye exclusion from live cells
Functional assay
-MTT, XTT assay
-Crystal violet/ Acid
phosphatase(AP) assay
-Alamar Blue oxidation-
reduction assay
- Neutral red assay
-[3H]-thymidin/ BrdU
incorporation
Examining metabolic components that are
necessary for cell growth
DNA labeling
assay
-Fluorescent conjugates
Simultaneous cell selection and viability
assay
Morphological
assay
-Microscopic observation Determination of morphological change
Reproductive
assay
-Colony formation assay Determination of growth rate
10

11. MEMBRANE INTEGRITY ASSAY
Possibly the simplest assay for cell death is measurement
of plasma membrane integrity. This can be assessed in two
ways: The ability of a cell to prevent a fluorescent dye from
entering it and the ability of a cell to retain a fluorescent dye
within it.
As a cell dies it's plasma membrane becomes permeable
allowing fluorescent dyes present outside the cell to enter it
and fluoresce.
The most common dyes used for this purpose are dyes that
label nucleic acids.
11

12. The most commonly used dyes are DAPI, propidium iodide,
7AAD, and ToPro-3.
One of such method includes TRYPHAN BLUE DYE
EXCLUSION ASSAY
The trypan blue dye exclusion assay is the most commonly
used and accepted method for the measurement of cell
viability
 It relies on the alteration in membrane integrity as
determined by the uptake of dye by dead cells, thereby giving
a direct measure of cell viability
 Based on optimal image analysis, the technology allow
precise cell-viability and cell-density determination
 The system performs automatic and reproducible
measurements of human or animal suspension cell densities
as well as standardized differentiation between viable and
dead cells, based on the trypan blue dye exclusion method12

13. 13

14. Other dyes, such as CFDA and Indo-1, are readily taken
up by live cells, and upon internalisation are cleaved into
a fluorescent form that can no longer cross the
membrane.
The result is that live cells become loaded with the dye,
but dead cells, that do not have the enzymes necessary
for cleavage of the non-fluorescent pre-cursor, will not.
As a cell dies, the membrane becomes permeable and
the fluorescent dye leaves the cell resulting in a loss of
fluorescence.
Example includes:
Ethidium bromide (EtBr)/fluorescein diacetate(FDA) and
propidium iodide (PI)
14

15. Intact cell –
PI and FDA is added
Fluorescein in
intact cells
Schematic illustration of the principle of
PI/FDA cell viability assay
● FDA (Fluorescein diacetate)
● PI (Propidium iodide)
Plasma membrane is damaged
; fluorescein leaks out
PI enters and strains
nucleic acids 15

16. •A well-established feature of apoptosis is the
externalisation of the lipid phosphatidyl serine (PS) from
the inner to the outer plasma membrane.
• Annexin-V (five) is a protein that specifically binds PS and
fluorescent labelling of the annexin-V enables the flow
cytometric detection of externalised PS, and hence
apoptotic cells.
•When used in conjunction with a live/dead cell
discriminator that measures membrane integrity (such as
PI, 7AAD, and DAPI), early apoptotic cells (annexin-V
positive only) can be distinguished from late
apoptotic/necrotic cells (annexin-V and PI/7AAD positive)
•The early apoptotic phase can be quite rapid and can
often be missed, making it appear that cells are either live
or late apoptotic/necrotic.
AnnexinV
16

17. 17

18. LDH ASSAY
Test principle
The assay is based on consideration that tumor cells possess high
concentration of intracellular LDH and the cleavage of a tetrazolium salt
when LDH is present in the culture supernatant.
18

19. .
Functional assays
Evaluate viability by examining the metabolic
components that are necessary for cell growth, on the
premise that cellular damage will inevitably result in
the loss of ability to maintain and provide energy for
metabolic function and growth
19

20. Microculture Tetrazolium
Test(MTT Assay)
• MTT assay a quantitative colorimetric assay for
measuring cellular growth, cell survival and cell
Proliferation based on the ability of living cells.
• Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) a tetrazolium salt is
reduced to purple formazan by mitochondrial
dehydrogenase of living cells in which tetrazolium
ring gets cleaved in mitochondria .
20

21. Receipt of Tissues Tissue Shipment Dosing
Rinsing
UVA Exposure and Post-Treatment
Incubation
21

22. Transfer to MTT
Extraction in Isopropanol and Plate
Reading
22

23. Compare with MTT assay and XTT assay
Culture cells in a MTP
for a certain period of time (37℃)
MTT assay XTT assay
Prepare labeling mixture
Incubate cells (0.5-4 h, 37℃)
Add solubilizing solution
(Isopropanol) and incubate
Measure absorbance using an ELISA reader
Add XTT labeling mixtureAdd MTT labeling reagent
Insoluble formazan Soluble formazan
23

24. Example: MTT and XTT
MTT XTT
Jenny G., Mark H., Anna J., Inger K., Douglas Mc., Roland M., 2002.
Evaluation of redox indicators and the use of digital scanners and spectrophotormeter for
quantification of microbial growth in microplates. J. Micro. Methods. 50:63-73
24

25. Other functional assays are:
 Crystal violet dye
elution (CVDE)
 Acid phosphatase (AP)
assay
 Alamar blue oxidation-
reduction assay
 Neutral Red (NR) assay
 [3H]-thymidine and
BrdU incorporation
25

26. DNA labeling assay
Detection of DNA synthesis in proliferating cells relies
on the incorporation of labeled DNA precursors into cellular
DNA during the S phase of the cell cycle.
The labeled DNA precursors, usually pyrimidine
deoxynucleosides, are added to cells during replication,
and their incorporation into genomic DNA is quantified or
visualized after incubation and sample staining.
The same labeled deoxynucleosides can be injected into
experimental animals to assay cellular proliferation in
specific organs and tissues.
26

27. [3H]-thymidine and BrdU incorporation
The most common deoxynucleosides used for assaying
DNA replication are [3H]thymidine and 5-bromo-2-
deoxyuridine (BrdU).
[3H]Thymidine incorporated into DNA is usually detected by
autoradiography, where as detection of BrdU is
accomplished immunologically, through specific anti-BrdU
antibodies.
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29. Morphological assay
Large-scale, morphological changes that occur at the
cell surface, or in the cytoskeleton, can be followed and
related to cell viability.
 Damage can be identified by large decreases in
volume secondary to losses in protein and intracellular
ions of due to altered permeability to sodium or
potassium.
 Necrotic cells: nuclear swelling, chromatin
flocculation, loss of nuclear basophilia
 Apoptotic cells: cell shrinkage, nuclear condansation,
nuclear fragmentation
29

30. Example; Morphological feature
(Human skin keratinocyte)
Fig. Morphological feature of (A) normal human skin keratinocyte, and differentiated
human skin keratinocyte(B).
(A) (B)
30

31. Example; Morphological feature
(Human skin fibroblasts)
Fig. Morphological feature of (A) normal human skin fibroblasts,
and aging human skin fibroblasts(B).
(A) (B)
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32. Reproductive Assay
• Colony-forming Efficiency
Clonogenic Cell:
 Defined as a cell with the capacity for
sustained proliferation
 Have undergone a minimum of 5-6
doublings to give rise to colonies containing
at least 50 cells
32

33. Example; Rat keratinocytes
(A) (B)
(C) (D)
Colony forming Non-colony
forming
48 hr after
subculture
6 days after
subculture
: colony , : Single cells
33

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35. Preclinical Toxicity Studies
• Aimed at predicting
(a) Safe starting dose & dosage regimen for human
clinical trials(P1)
(b) The toxicities of the compound, &
(c) The likely severity and reversibility of drug toxicities.
• Regulatory requirement : Two acute preclinical toxicity
studies
1. Rodent (mice) - single- and multiple-dose lethality
studies.
2. Non rodent (dogs) - single- and multiple-dose
confirmatory toxicity.
• Cytotoxic & non cyotoxic drugs 35

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Tumor model
1. Carcinogen induced models
2. Viral infection models
3. Transplantation Models
4. Genetically Engineered Mouse Models
5. In vivo hollow fibre assay

37. CHEMICAL CARCINOGEN MODEL
DMBA induced mouse skin papillomas
• Two stage experimental carcinogenesis
– Initiator – DMBA (dimethylbenz[a]anthracene),
– Promotor – TPA. (12-O-tetradecanoyl-phorbol-13-
acetate)
• Mice : Single dose – 2.5 µg of DMBA f/b 5 to 10 μg of
TPA in 0.2 ml of acetone twice weekly.
• Papilloma begins to appear after 8 to 10 wks - Tumor
incidence & multiplicity of treatment group is
compared with DMBA control group
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38. RAT MAMMARY GLAND CA
Mouse skin papillomas
38

39. Cancer
site
Cancer
Type
Speci
es
Carcinogen
Colon Adenocarcin
omas
Rat AOM
(azoxymethane)
Prostate Adenocarcin
omas
Rat MNU
(methylnitrosourea)
Esophag
us
Squamous
cell
carcinoma
Rat NMBA(N-nitroso-
methylbenzylamine)
Breast Adenocarcin
oma
Mice NMU (N-Nitroso-N-
methylurea) 39

40. Viral infection models
• Mouse Mammary Tumor Virus (MMTV) was the first
mouse virus, isolated at Jackson labs as the “non-
chromosomal factor” that caused mammary tumors in
the C3H strain of mice.
• Some viruses cause cancer via random integration in
certain cells
• Some viruses carry cellular oncogenes
– Abelson murine leukemia virus – Abl
– Moloneymurine sarcoma virus – Raf
• Engineered viruses now used routinely in the laboratory
to induce cancer.
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41. Transplantation Models
• Tumor cells or tissues (mouse or human)
transplanted into a host mouse.
• Ectopic – Implanted into a different organ
than the original (typically subcutaneous or
kidney capsule)
• Orthotopic – Implanted into the analogous
organ of the original tumor.
• Advantages :
– Typically cheap, fast & easy to use.
– Not covered by patents
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42. 42
On EAC Cells by Liquid Tumour Model
(Ehrlich Ascites Carcinoma) on (Swiss
albino mice)
EXPERIMENTAL PROTOCOL
Induction of ascitic carcinoma - The ascitic tumor
bearing mice (donor) were used for the experiment 12
days after tumor transplantation. The ascitic fluid was
drawn using an 18 gauge needle into a sterile syringe.
A small amount of tumor fluid was tested for microbial
contamination. Tumor viability was determined by
tryphan blue exclusion test and cells were counted
using haemocytometer. The ascitic fluid was suitably
diluted with saline to get a concentration of 10 million
cells/ml of tumor cell suspension. 250 µl of this fluid
was injected in each mouse by i.p. route to obtain
ascitic tumor.

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The mice were weighed on the day of tumor inoculation
and then for each three days. Cisplatin was injected on
two alternative days 1st and 3rd day after tumor
inoculation (intraperitoneally). The drugs were
administered after 24 hours of tumor inoculation and
were administered till 9th day intraperitoneally.
- On 15th day blood was collected from the animal
through the retro orbital plexus to determine the
heamatological parameters and lipid profile.
Normal mouse Tumor bearing mouse

44. Solid tumor model using DLA cell
lines
• The DLA(dalton’s lymphoma ascitic) bearing mouse was
taken 15 days after tumor transplantation. The ascitic
fluid was drawn using a 18 guage needle into a sterile
syringe. A small amount was tested for microbial
contamination
Tumor viability was determined using trypan blue
exclusion method and cells were counted using
haemocytometer.
• The ascitic fluid was suitably diluted in phosphate buffer
saline to get a concentration of 106 cells per ml of
tumor cell suspension.
• Around 0.1ml of this solution was injected
Subcutaneously to the right hind limb of the mice to
produce solid tumor.
• Treatment was started 24 hours after tumor inoculation.
Cisplatin was injected on two alternate days i.e. the 1st
and 3rd day. Extracts were administered till 9th day
intraperitonially.

45. Transplantation Models : Human
Tumor Xenografts
• Athymic “nude”mice developed in 1960’s
• Mutation in nu gene on chromosome 11
• Phenotype: retarded growth, low fertility,
no fur, immunocompromised
– Lack thymus gland, T-cell immunity
• First human tumor xenograft of colon adenocarcinoma
by Rygaard & Poulson, 1969
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46. Xenograft Study Endpoints
• Toxicity Endpoints:
– Drug related death
– Net animal weight loss
• Efficacy Endpoints:
– Tumor weight change
– Treated/control survival ratio
– Tumor growth assay (corrected for tumor
doubling time)
46

47. Human Tumor Xenografts
Advantages
• Many different human
tumor cell lines
transplantable
• Wide representation
of most human solid
tumors
• Good correlation with
drug regimens active
in human lung, colon,
breast, and
melanoma cancers
Disadvantages
• Brain tumors difficult to
model
• Different biological
behavior,metastases rare
– Survival not an ideal
endpoint: death from
bulk of tumor, not
invasion
• Shorter doubling times
than original growth in
human. Difficult to
maintain animals due to
infection risks 47

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49. In Vivo Hollow Fibre Assay
• In vivo screening tool implemented in
1995 by NCI
• 12 human tumor cell lines (lung, breast,
colon, melanoma, ovary, and glioma
• Cells suspended into hollow
polyvinylidene fluoride fibers implanted IP
or SC in lab mice
• After in vivo drug treatment, fibers are
removed and analyzed in vitro
• Antitumor (growth inhibitory) activity
assessed
49

50. 50

51. In Vivo Hollow Fibre
Assay
Subcutaneous
Hollow Fibre
implants
51

52. 52
• Currently, pharmaceutical firms spend a large amount of
money on the compound efficacy and cytotoxicity test.
• There is still a 78% failure rate for all drugs, which may be
devastating to developing companies.
• Effective compounds in vitro may be non-effective in vivo for
many reasons, including differences between in vitro and in
vivo target biology, interrelated biochemical mechanism,
metabolism, poor penetration into solid tissues, etc.

53. 53
• Currently, almost all cell-based assays or biosensors are
developed in 2-D culture systems, although conventional 2-D
cultures usually suffer from contact inhibition and a loss of
native cell morphology and functionality.
• In comparison with 2-D cultures, 3-D cell models create a
more realistic representation of real human tissues, which is
critical to many important cell functions, including
morphogenesis, cell metabolism, gene expression,
differentiation and cell-cell interactions.

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55. 55
• In studying cytotoxicity and drug testing, maintaining cells in
their native functional state in a proper 3-D environment
would improve predictions and have the potential to reduce
clinical trial failures.
• Therefore, although designing 3-D models is much more
complicated than designing the 2-D counterparts, cell- and
tissue-based assays with a 3-D model are superior and are the
assays of choice for HTS (High-throughput screening) of drug
cytotoxicity.

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