Seung Hyun OH; DVM, PhD

가천대학교 약학대학 오승현

Lung cancer mouse models 왜 마우스 모델이 안 만들어질까? 적절한 모델을 계획하고 있는가?윤리적인 암실험은?

세상 끝까지 (1998년)

안녕 내 사랑 (1999년)

가을동화 (2000년)

허준 (2000)

팝콘 (2000년)

아름다운 날들 (2001년)

로즈마리 (2003년)

러브스토리 인 하버드 (2004년)

장미빛 인생 (2005년)

하얀거탑 (2007년)

Cancer in TV drama

2

Who is best model?Humans and pet animals share many environmental risk factors

Humans and pet animals share aspects of tumor biology

• Easily exposed to hazards • Dogs are prone to cancer

Mouse !!!

We know mouse

What kind of mouse models for cancer research?

6

What kind of mouse models for cancer research?

7

1. Spontaneous tumor model

2. Transplanted tumor model

3. Genetically engineered mouse model

• Lung Cancer

• Colon Cancer

Idiopathic

Carcinogen-induced

What kind of mouse models?

Xenograft vs Syngeneic

Ectopic vs Orthotopic

– too slow, low incidence

Carcinogen-induced lung cancer model

Carcinogen-induced lung cancer model

• 1775- Sir Percival Pott, a London surgeon, noticed that chimney sweepers frequently developed a peculiar form of scrotal cancer. He ascribed it to frequent, direct contact with coal tar.

• This launched 244 years of research into the chemical basis of cancer.

• Treatment of mice with carcinogens is the basis of numerous mouse models of cancer.Ø Skin – 7,12-dimethylbenz[α]anthracene (DMBA) + 2-O-tetradecanoylphorbol-13-acetate

(TPA)Ø Lung – NitrosaminesØ Liver – vinyl chlorideØ Breast – N-Nitroso-N-methylurea (NMU)Ø Colon – dimethylhydrazine (DMH) NitrosaminesØ Bladder – Aromatic Amines

Spontaneous tumor model

• Idiopathic

• Carcinogen-induced

• TG/KO

Lung cancer - A/J > SWR > Balb/c > CR > DD... CBA > C3H > DBA > C57BL

Lung cancer - A/J > SWR > Balb/c > CR > DD... CBA > C3H > DBA > C57BL

Carcinogen-induced lung cancer model

American Journal of Pathology, 201012 12

Carcinogen-induced lung cancer model16 weeks

Control NNK+BaP

NNK+BaP

NNK+BaP+Deguelin

JNCI, 2007Bouin’s solution13

Evaluation of mouse lung cancer

14

Control

NNK/BaP

NNK/BaP+

Daurinol50mg/kg

IJC, 2016

Fixation of mouse lung

15

Gross

Fixation

Perfusion

• Spontaneous tumor model

• Transplanted tumor model

*Metastasis model

• Genetically engineered mouse model

• Lung Cancer

• Colon Cancer

Idiopathic

Carcinogen-induced

What kind of laboratory animal?

Xenograft vs Syngeneic

Ectopic vs Orthotopic

Transplanted tumor model model• Transplantation models

– Syngeneic vs xenograft transplantation model

– Transplantation location Ectopic - subcutaneous Orthotopic - Subcutaneous, intraperitoneal, intracranial, intrasplenic, renal subcapsule

§ Syngeneic mouse model Immuno-competent recipient mouse will be used The donor has same genetic background§ Xenograft transplantation models Immuno-incompetent recipient mouse will be used The cell/tissue will be from different species (human)

17

Nude/Athymic mouse

18

• Athymic “nude”mice were first discovered in 1962 by Dr. N. R. Grist at Ruchill Hospital'sBrownlee virology laboratory.

• The mutation arose in a closed but not deliberately inbred albino stock.

• Mutation in nu gene on chromosome 11

• In 2000 the gene responsible for the mutation was identified as a member of the Foxgene family and the nomenclature was updated to Foxn1nu.

• Phenotype: retarded growth, low fertility, no fur, immunocompromised– Lack thymus gland, T-cell immunity

• Because they lack a thymus, nude mice cannot generate mature T lymphocytes. -> Nogeneration of cytotoxic effector cells. AIDS????

• B lymphocyte is normal.

Infectious disease (nude mouse)

19

• Transmission: The bacterium is carried on the skin and in the oral cavity of mice, and is

transmitted by direct contact and by fomite transmission (handling, flake contact, etc.).

• The association of bacterium with disease may be related to lack of hair since the

hairless immunocompetent SKH-1 mouse is also susceptible to bacterial hyperkeratosis

• Pathology: Affected mice may lose weight and are often removed from study because of

reports of poor growth of transplanted tumors or hinderence of immunologic studies.

• Mortality is low and the hyperkeratosis is transient but may recur.

• Diagnosis: The bacterium can be cultured by inoculating blood agar with skin or buccal

swabs and grows in punctiform colonies after 48 hrs of incubation in aerobic conditions.

The bacterium can also be detected in skin and environmental samples by a C. bovis-

specific PCR test.

• Treatment and Control: Antibiotics such as ampicillin or penicillin VK delivered in drinking

water controls the disease but does not eliminate carriage of the bacterium. Control

measures include ordering mice from C. bovis-negative mouse colonies, housing mice in

sterile microisolator cages, and use of oxidant-based disinfectants on forceps or gloves

used for animal handling, and environmental surfaces (including biosafety hoods, animal

restrainers, etc.).

Corynebacterium Bovis

University of Missouri - Comparative Medicine Program and IDEXX-BioResearch

Which immunodefecient mouse?• Significant numbers of T cells that develop with age in an extrathymic pathway (ILAR Journal V34, 1992).

More immonodefecient!20

Nu vs SCID vs NOD scid

Nude – No T cell SCID – No T & B cellfunctional NK & monocyte

NOD – NK & monocyte can’t kill human cell (not functional)

NOD + SCID NOD SCIDNo T & B cellImpaired NK & monocytes

Higher incidence?Bigger tumor?

Metastasis?

“Don’t eat me signal”àPhagocytosis ↓

21

NOD/scid vs NOG, NSG

Taconic Biosciences The Jackson Laboratory

lacks IL2rg#intracytoplasmic domain;cytokine binding but no signaling

IL2rg null mutation; no cytokinebinding or signaling

https://www.jax.org/jax-mice-and-services/in-vivo-pharmacology/humanized-mice/cd34

22

Transplanted tumor model• Tumor transplantation

– Initiated in the 1960’s– Ectopic implantation : implantation of cells or tissue into a different organ than the original : usually subcutaneous- Orthotopic implantation : implantation of cells or tissue into the analogous organ of the original cells or tissue

Brain

Breast

Lung

Colorectal

CancerType

OrthotopicInjection Model

Brain Intracranial(Brain)

Breast Mammary fat pad(breast tissue)

Lung Intrathoracic(thoracic cavity)

Colorectal

Cecal wall(large intestine)

Analysis of data from animal

• Tumor volume : a = tumor length, b = tumor width

V = (a x b2)/2

• Tumor weight change ratio : (g)

• Defined as: treated/control x 100% (g) (V)

• Tumor weight in mg = T/C < 40-50% is considered significant

RNA

Protein

IHCTreatment

Control

24

Cancer Res. 2013 Jan 1;73(1):353-63

Analysis of data from animal

Cancer Res. 2007 Nov 1;67(21):10406-16OncoImmunology 4:7, e1014232; July 2015

Selection of Proper Tumor Model in Drug Screening

J Natl Cancer Inst 2008;100: 1500 – 1510 (Melinda G. Hollingshead)

Analysis of data from animal

Where are you going to inject?

1 2 3

4 5

28

661 662 663 664 665

671 672 673 674 675

661 662 663 664 665

666 667 668 669 670

Where are you going to inject?

29

Tumor size

Day

5,000mm3

P = 0.02

Control

xxx inhibitor

30

What to consider when you start

Why you have 40%tumor incidence in xenograft tumor?

I just chose the cell line from the paper I read!

(Anticancer Drug Development Guide, Humana Press)

31

It’s your fault

Tumor cell culture 1 X 106 cells / 100 ulInject cells into mouse

+-

When did youchange the culturemedium?

How long did it take to inject the cells? Needle size?

Is it tumorigeniccell line? Is it good FBS?

Drug treatment

How do you measurethe size?

32

It’s your fault

Cancer cells in ice

97% 93% 89% 84% 81% 79% 75% 69%

As time goes cell viability

What to consider when you start

• What kind of cell?

• Tumorigenic?

• Which immonodeficient mouse?

• Ramdomization (injection time)

• Metastatic?

• How many cells? ~106 cells/spot

• How many spots? N=mouse no.? or tumor no.?

• When to start to measure?

• When to start drug treatment?

• How to use tissue?

OCT

IHC

Snap-Frozen (RNA)

Cell line authentification

35

36

Cell line contamination

37

Xenograft advantages & disadvantages• Many different type of human/non-human tumor cell lines (breast, lung, prostate,

brain, colon…) are transplantable

• Easy evaluation of therapeutic index (ex, nude mouse, sc injection)

• Very easy to maintain cell lines indefinitely;

• Very fast to generate large numbers of xenograft tumors

38

• Very different biological behavior,

– Checking survival is not an ideal endpoint: death from burden of tumor

– Rare invasion & metastasis

• Too much shorter doubling times than original growth in human (within 2 months)

• Infection risks – difficulty in maintaining facility

• Viral or mycoplasm infection by cell contamination

• Genet ic changes by r epeat ed passages in cell lines

• Host directed therapies (angiogenesis, immune modulation) may not be applicable

– Human vs. murine effects

Syngeneic mouse model

39Oncotargets and Therapy, 2016 Volume 2016:9 P545—555

Orthotopic cancer model

40Con Therapy

3w

11w

Orthotopic cancer model

41

1 d

Imaging 확인 후 군분리XXX-HCl(100mg/kg)처치5회/week, 1회/day

0 4weeks

Cell injection

XXXHCl (100mg/kg)

2012/04/16

0 d

H460-L inj 1일 후 imaging 확인, 투여시작

H460-L, inj,, 5x105 cells/spot

7d 12d

Good ????

Control 신묘한약 10mg/kg

11/051주

A12 Treat start

657 658 659 660 661 662 663 664 676 666 667 668 669 670 671 672 673 674 675665

11/122주

11/203주

11/264주

12/034주

Control 신묘한약 mg/kg

1/1411주

657 658 659 660 661 662 663 664 676 666 667 668 669 670 671 672 673 674 675 665

12/18폐사?

1/12폐사

Orthotopic cancer model

440 7 14

A

B

Control OOO inh.

7d 14d 21d 28d 7d 14d 21d 28d

C

Control

OOO inh.

Phot

onco

nts

(%8d

ay)

Tongue tumor Lymph node metastasis

0

1000

2000

3000

4000

5000

6000

7000

0

500

1000

1500

2000

2500

3000

3500

4000

ControlOOO inh.

(days) 0 7 14 (days)

**

Metastasis

Experimental lung metastasis model

45

Tum

orN

odul

es

02468

10121416

***

EVAd-BP3

Con

Ad-BP3EV

A549 Lateral v.

Lateral a.

Ventral v.Ventral a.

MetastasisInject

cancer cells

Treatment No treatment

Allow time for metastases to appear

Many metastasesFew metastases

Removeinitial tumor

Let initial tumor grow for

several weeks

46

Analysis of metastasis in mouse model

47

0 7 14

A

Phot

onco

nts

(%8d

ay)

Tongue tumor Lymph node metastasis

0

1000

2000

3000

4000

5000

6000

7000

0

500

1000

1500

2000

2500

3000

3500

4000ControlOOO inh.

(days) 0 7 14 (days)

**

Pho

ton

cont

s(%

8day

)

Tongue tumor

0

1000

2000

3000

4000

5000

6000

7000

(days)

*

Lymph node metastasis

0

500

1000

1500

2000

2500

3000

3500

4000ControlOOO inh.

0 7 14 (days)

*

B

0 7 14

or

Metastasis (Syngeneic) model

48JBC, 2014

Cell injection

Cell injection

Cell injection

4T1

4T1-NME1L

4T1-NME1

Genetically engineered mouse model

49

50

Animal Rights Extremism• Many animal rights activists pursue their goals legally,

through protests and information campaigns.

• However, there are animal rights activist groups who believe in violence and extreme measures as acceptable methods to achieve their goal.– Examples:

• Stop Huntington Animal Cruelty (SHAC)• Animal Liberation Front (ALF)

This is the burned remainsof the house of Daniel Vasella,a Swiss pharmaceutical company CEO. This house was allegedly burned by animal rights extremists.

Picture from: http://www.dailymail.co.uk/news/worldnews/article-1204747/Austrian-holiday-home-Swiss-pharmaceutical-boss-torched-British-animal-rights-extremists.html

내분비: stress hormone, ­metabolic rate, ­ heart rate & water retention

면역계:­ Impaired immune functions

순환기계: ­cardiac rate ­systemic vascular resistance peripheral vascular resistance ­coronary vascular resistance ­blood pressure and ­myocardial oxygen consumption소화기계:

Delayed return of gastric and bowel function

근골격계: Decreased muscle function, fatigue and immobility

실험결과 변화실험결과의 재현성 저하

마우스에게 스트레스 => 고통 결과 변화

동물실험에 영향을 미치는 요인

A 4th “R” for Reproducibility

Physical & Environmental factors

Relative humidity

Room temperature

Diet &Water

Contact bedding

Noise

Metabolic state

Light cycle &Quality

Ventilation

Animal care &handling factors

Type of caging

Number of animals per cage

Daily routines

Gentle Handling

Researchmanipulation factors

Time ofmanipulation(s)

Duration ofmanipulation(s)

Experimental stressors

Pain and distress

Animal factors

Age, Sex,Reproductivestate

Disease

Geneticfactors

Metabolic state

Stress

Biological Rhythms

Thank youJu-Hee Kang, PhD Hyunjin Jung, Phd candidate

Minwoo Kim, PhD candidate

Han Le, MS candidate

Hee Namgoong MS candidate

CollaboratorsProf. Je Kyung Seong (SNU)

Prof. Ki-Taek Nam (Yonsei Univ.)

Prof. Jong-Ik Hwang (Korea Univ.)

Dr. Jung Ju Kim (Autophagysciences Co. Ltd)

Dr. Eunju Shin (Univera Co. Ltd)

AutophagySciences

Members

AlumniJong Kyu Woo, PhDYoung-su Jang, PhD

Seung-Ho Choi, PhD

Jung Eun Jang, MS

Myeonga Sung, MS

Ki-Hoon Song, PhD

Don’t worry babyWe don’t catch research miceYou are saving human life

A

B

Tumor

incid

ence

(%)

0

5

10

15

20

100

AD AC

WT(N=10)

AD AC

IGF-1Tg/+(N=37)

T

umor

multip

licity

AD AC0.0

0.1

0.2

0.3

AD AC

WT IGF-1Tg/+(N=10) (N=37)

Tumor

volume(

mm

3 )

AD AC AD AC0.0

5.0

10.0

2.5

7.5

WT IGF-1Tg/+(N=10) (N=37)

IGF-1R

pIGF-1R

pErk1/2

Erk1/2

pAkt (T308)

Akt

IGF-1 Tg/+ WT

1 2 3 1 2 3 : Mouse (#)

pIGF

1R/IG

F1Rb

*

0

0.5

1.0

1.5

pAkt(T308)/Akt

0

0.5

1.0

1.5

pErk1/2

/Erk1/2

IGF-1Tg/+ WT0

0.5

1.0

1.5

Genetically engineered lung cancer model

C

D

Cancer Research, 2009

0 1 2 3 4 9 (Mo)

sacrificeBaP + NNK (once a week)

IGF-1 TgWT

7 8 9 10 11 12 13+ + + + + + +

pIGF-IR

IGF-IR

Mice (#)NNK

pAkt

0

5

10

1520

25

3035

P=0.05

WTAD AC AD AC

Tumor

incid

ence

(%)

IGF-1Tg

Tumors/slide

(n=42) (n=44)WT

AD AC AD AC0

0.1

0.2

0.3

0.4

0.5

P=0.05

IGF-1Tg

0.00.20.40.60.81.01.21.41.61.8

P=0.04

WTAD AC AD AC

P=.79

Tumor

Volume(

mm

3 )

IGF-1Tg(n=42) (n=44)(n=42) (n=44)

IGF-1 TgWT

IGF-1 Accelerates tobacco carcinogen-induced lung tumorigenesis in mice

A B

Effect of IGF-1R Inhibitor on Lung Tumorigenesis Induced by IGF-1 and NNK/BaP

A

B

Control

IGF-6-1 IGF-6-2 IGF-6-3 IGF-6-4 IGF-6-5IGF-1-1 IGF-1-2 IGF-1-3 IGF-1-4 IGF-1-5

IGF1R inhibitor

pERK1/2

pAkt

Akt

ERK1/2

Actin

IGFs Expression during Lung Carcinogenesis in Cancer Cells/Mouse Model

Cancer Research, 2009

A B“Elevated Epithelial Insulin-like Growth Factor Expression Is a Risk Factor for Lung

Cancer Development”0 1 2 3 4 9 (Mo)

sacrificeBaP + NNK (once a week)

IGF-1 TgWT

7 8 9 10 11 12 13+ + + + + + +

pIGF-IR

IGF-IR

Mice (#)NNK

pAkt

0

5

10

1520

25

30

35

P=0.05

WTAD AC AD AC

Tumor

incid

ence

(%)

IGF-1Tg

Tumors/slide

(n=42) (n=44)WT

AD AC AD AC0

0.1

0.2

0.3

0.4

0.5

P=0.05

IGF-1Tg

0.00.20.40.60.81.01.21.41.61.8

P=0.04

WTAD AC AD AC

P=.79

Tumor

Volume(mm

3 )

IGF-1Tg(n=42) (n=44)(n=42) (n=44)

IGF-1 TgWT

58

Do you need mouse?

Thank youJu-Hee Kang, PhD Hyunjin Jung, Phd candidate

Minwoo Kim, PhD candidate

Han Le, MS candidate

Se Yong Park MS candidate

Hee Namgoong MS candidate

CollaboratorsProf. Je Kyung Seong (SNU)

Prof. Ki-Taek Nam (Yonsei Univ.)

Prof. Jong-Ik Hwang (Korea Univ.)

Dr. Jung Ju Kim (Autophagysciences Co. Ltd)

Dr. Eunju Shin (Univera Co. Ltd)

Autophagy Sciences

Members

AlumniJong Kyu Woo, PhDYoung-su Jang, PhD

Seung-Ho Choi, PhD

Jung Eun Jang, MS

Myeonga Sung, MS

Ki-Hoon Song, PhD

Thank youJu-Hee Kang, PhD Hyunjin Jung, Phd candidate

Minwoo Kim, PhD candidate

Han Le, MS candidate

Se Yong Park MS candidate

Hee Namgoong MS candidate

CollaboratorsProf. Je Kyung Seong (SNU)

Prof. Ki-Taek Nam (Yonsei Univ.)

Prof. Jong-Ik Hwang (Korea Univ.)

Dr. Jung Ju Kim (Autophagysciences Co. Ltd)

Dr. Eunju Shin (Univera Co. Ltd)

Autophagy Sciences

Members

AlumniJong Kyu Woo, PhDYoung-su Jang, PhD

Seung-Ho Choi, PhD

Jung Eun Jang, MS

Myeonga Sung, MS

Ki-Hoon Song, PhD