유방암, 대장암동물모델의제작과적용 2020 한국실험동물학회실험동물기술원교육강연동계심포지엄 2 ( ) 강원도평창알펜시아컨벤션센터 ( 금 ) 마우스종양모델과적용 박준석, PhD, DVM 실험지원팀장, 책임연구원 실험동물센터융합연구부대구경북

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1 2020 한국실험동물학회실험동물기술원교육강연동계심포지엄 2 ( ) 강원도평창알펜시아컨벤션센터 ( 금 ) 마우스종양모델과적용 박준석, PhD, DVM 실험지원팀장, 책임연구원 실험동물센터융합연구부대구경북첨단의료산업진흥재단 (DGMIF)

2 I 마우스종양동물모델 Ⅱ 유방암동물모델 Ⅲ Ⅲ Ⅳ 생체영상기법을대장암동물모델활용한유효성평가생체영상기법을유효성평가활용한유효성평가

3 I 마우스종양동물모델 Ⅱ 유방암동물모델 Ⅲ Ⅲ Ⅳ 생체영상기법을대장암동물모델활용한유효성평가생체영상기법을유효성평가활용한유효성평가

4 Preclinical models to evaluate anticancer drugs Ocana, A. et al. (2010) Preclinical development of molecular-targeted agents for cancer Nat. Rev. Clin. Oncol.

5 Translation of preclinical findings for the benefit of patients but mice are not humans! 5

6 Preclinical Tumor models 1. Transplantation Models Murine Human 2. Chemo (Carcinogen)-Induced Tumor Models 3. Viral Infection Models 4. Genetically Engineering Mouse (GEM) Models

7

8 Human Tumor Xenograft 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 Human cancers grown in immune-deficient animals. First human tumor xenograft of colon adenocarcinoma by Rygaard & Poulson, 1969 Subcutaneous Xenograft model

9 Transplantation Models Tumor cells or tissues (mouse or human) transplanted into a host mouse. Ectopic (Heterotopic) Implanted into a different organ than the original 이소이식 (typically subcutaneous or kidney capsule) Orthotopic Implanted into the analogous organ of the original tumor. 동소이식 ( 정소이식 )

10 Transplantable tumor models Nature reviews

11 Xenograft Model human derived cancer cell line Breast cancer cell line (MDA-MB-435, MDA-MB-231, MCF-7.) Colon Cancer (Lovo, SW480, Colo205, HT29, HCT116..) Brain tumor (U87-MG, U373-MG, U251- MG.) Lung Cancer (PC14-PE6, A549, H23, H460.) Lymphoma (Raji, Ramos, Daudi, BJAB, Toledo, SKW 6.3.) Other Cancer Cell lines..

12 Subcutaneous Xenograft Tumor Weight Change Tumor weight change ratio (used by the NCI in xenograft evaluation) Defined as: treated/control x 100% Tumor mass volume= (a x b 2 )/2 a = tumor length b = tumor width T/C < 40-50% is considered significant

13 Experimental Design: Xenograft model (S.C.)

14 Are subcutaneous models adequate?

15 Advantages and Disadvantages of Orthotopic Model Advantages Resembles the original tumors morphologically, biologically and biochemically Important for the research of cancer metastasis Short-term screening of variable cancer therapy strategy Disadvantages Necessary to have skillful technique Wide variation

16 Schematic of Orthotopic Tumor Model

17 A long journey to develop metastatic tumors Nat Rev Immunol Feb; 15(2):

18 Preclinical models of metastasis Nature Reviews Cancer 11, (Feb 2012) Nature Reviews Drug Discovery 11, (June 2012) 18

19 Tumor cell lines cannot predict clinical efficacy

20 Tumor Heterogeneity Nature 501, 2013

21 Tumour heterogeneity in metastatic spread Nature Review CANCER 12, 2012

22 How can we improve the efficiency of drug development in oncology? A way to increase the predictivity of preclinical studies for Success in the clinic would be: Ø To use preclinical models which more closely recapitulate the heterogeneity of human cancers Ø Perform studies for genotype/response correlation on a population scale, to achieve scientific solidity

23 Patient-Derived Tumor Xenograft Model Compound efficacy testing in mice engrafted with patient-derived tumor xenografts Established PDX models for many tumor types (lung, brain, pancreas, etc.) Readouts: Survival, tumor volume and growth rate, histopathology, Non-invasive bioluminescence imaging Preclinical Development Avatar Human DNA RNA Histopathology Biomarker

24 Establishment and testing of PDTX models

25 항암제개발을위한전임상플랫폼구축 항암제개발을위한수요자맞춤형전임상유효성평가지원

26 Key molecular imaging modalities used for preclinical and/or clinical applications

27 유효성 / 안정성평가 - 생체영상활용 Current Methodology = 15 animals over three treatment points 0h 2h 4h Imaging Methodology = 5 animals over four treatment points - 각 time point 별동물희생 - 개체들의평균값 - 낮은효율성 0h 2h 4h - 동일한개체사용 - 신뢰도증가 - 높은효율성 Same group of anesthetized test animals at each time point of an experiment uses far fewer animals than current methodology

28 Advantages of Molecular imaging 연구개발속도증대 Speed 연구개발비용절감 + + Save 연구의신뢰도제고 Reliability

29 MRI 9.4T bruker MRI Ø phamarcological MRI Ødiffusion tensor imaging (DTI), Dynamic Contrast Enhanced (DCE) MRI magnetic resonance spectroscopy(mrs) Ø Functional MRI(fMRI)

30 광학영상 IVIS spectrum CT IVIS lumina K FMT 4000 Ø 형발광 (orthotopic, Metastasis, Xenograft, Transgenic) 동물모델을이용한신약유효성평가 유효성평가용생체영상프로브개발

31 Micro CT Quantum FX Ø Skeletal Imaging (trabecular analysis and BMD measurement) Ø Phenotyping Ø SafetyandPerformance(medicalDevices)

32 Longitudinal Studies Require Calibration in Physical Units Five 4T1-luc2-1A4 cells subcutaneously injected on day 0 in a nu/nu mouse

33 항암제유효성평가모델

34 I 마우스종양동물모델 Ⅱ 유방암동물모델 Ⅲ Ⅲ Ⅳ 생체영상기법을대장암동물모델활용한유효성평가생체영상기법을유효성평가활용한유효성평가

35 Xenograft Models of Breast Cancer Vargo-Gogola& Rosen Nat Review Cancer 2007

36 ONCOLOGY LETTERS 15: , 2018

37 ONCOLOGY LETTERS 15: , 2018

38 Experimental protocol for the orthotopic breast cancer model ONCOLOGY LETTERS 15: , 2018

39 Orthotopic Breast Cancer Mouse Model - Surgery Tools - 1. Stainless scalpel 2. Forceps 3. Stainless scissors 4. Autoclip wound clip applier 5. Autoclip wound clip Paschall, A.V., Liu, K. J. Vis. Exp. (114), e54040 (2016)

40 An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis Paschall, A.V., Liu, K. J. Vis. Exp. (114), e54040 (2016)

41 Orthotopic Breast Cancer Mouse Model - Surgery Tools - 1. Scissors 2. Angled forceps 3. Straight forceps 4. Mosquito forceps Kocatürk, B., Versteeg, H.H. J. Vis. Exp. (96), e51967 (2015)

42 Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth Kocatürk, B., Versteeg, H.H. J. Vis. Exp. (96), e51967 (2015)

43 Mammary Fat Pad of Mouse Current Protocols in Mouse Biology (2013) Dunphy K.A., Tao L., Jerry D.J. (2010). Mammary Epithelial Transplant Procedure.

44 Mammary Fat Pad of Mouse BoneKEy Reports 5, Article number: 804 (2016)

45 동소이식수술

46

47 Derivation of PDX models of human breast cancer in mice Whittle et al. Breast Cancer Research (2015) Zhang & Lewis. Current Protocols in Mouse Biology (2013)

48 발광유방암세포 Orthotopic Xenograft 마우스모델

49 발광유방암세포 Orthotopic Syngeneic 마우스모델

50 유방암전이모델 ( 심장주입 ) 발광유방암세포 xenograft 마우스전이모델 Low cell number High cell number

51 발광유방암세포 xenograft 마우스전이모델

52 발광유방암세포 xenograft 전이모델 유방암전이모델 ( 미정맥주입 ) Low cell number High cell number

53 I 마우스종양동물모델 Ⅱ 유방암동물모델 Ⅲ Ⅲ Ⅳ 생체영상기법을대장암동물모델활용한유효성평가생체영상기법을유효성평가활용한유효성평가

54 Mouse and human abdominal anatomy

55 55. Abdominal viscera displayed. Male.

56 57. Alimentary canal removed and displayed. Oesophagus, 4 cm; duodenum, jejunum and ileum, 35 cm; colon and rectum, 14 cm.

57 Cecum

58 Cecum Written By: The Editors of Encyclopaedia Britannica

59 대장암

60 Summary of comparisons between the novel orthotopic CRC model and classic subcutaneous CRC model Oncotarget, 2017, Vol. 8, (No. 33), pp:

61 Orthotopic Cancer Mouse Model - Surgery Tools - 1. Stainless scalpel 2. Angled Forceps 3. Stainless scissors 4. Autoclip wound clip applier 5. Autoclip wound clip

62 Orthotopic Colorectal Cancer Xenograft Mouse Model - Materials and Reagents ~31 G syringes 2. Phosphate-buffered saline (PBS), sterile 3. Buprenorphine 4. Betadine 5. 70% alcohol % sodium chloride ( 생리식염수 ) 7. 멸균거즈 8. 멸균면봉 9. 흡수성봉합사 (VICRYL, 4-0) 10. 흡입마취제 (isoflurane)

63 Orthotopic Colorectal Cancer Xenograft Mouse Model - Equipment - 1. Forceps (sterilize before use) : curved forceps, straight forceps, splinter forceps, mosquito forceps 2. Surgical scissors (sterilize before use) : sharp-sharp microscissors, 10cm curved spring scissors 3. Small needle holder 4. 흡입미취기 5. 수술용현미경 6. Reflex wound closure clip applier 7. Reflex wound closure clips 8. Reflex wound clip remover 9. Heat lamp 10. Heating pad

64 Injection of human colon cancer cells into the cecal wall of a 4-week-old athymic nude mouse Int. J. Cancer: 39, (1987)

65 OCMI into the cecum of immunosuppressed mice AJP March 2007, Vol. 170, No. 3

66 Tumor Staging and Survival Time in Animals Bearing Tumors Derived from OCMI-Implanted Human Colorectal Cancer Cell Lines AJP March 2007, Vol. 170, No. 3

67 Transplantable models for colorectal cancer Cancer Letters (2016)

68 Intracecal injections of cancer cells into the outer wall tissue layer of the cecum Dis Colon Rectum Jun;61(6):

69 Nature Protocols volume 13, pages (2018)

70 Nature Protocols volume 13, pages (2018)

71 Nature Protocols volume 13, pages (2018)

72 surgical procedure to transplant CRC organoids into the caecal wall of recipient mice (Video) Nature Protocols volume 13, pages (2018)

73 Establishment and Validation of an Orthotopic Metastatic Mouse Model of Colorectal Cancer PLoS ONE 6(12): e

74 Utilization of Murine Colonoscopy for Orthotopic Implantation of Colorectal Cancer - Orthotopic induction of CRC using the mouse colonoscopy system - PLoS ONE 6(12): e

75 Utilization of Murine Colonoscopy for Orthotopic Implantation of Colorectal Cancer - Sub-mucosal injections of PBS-/- and India ink- PLoS ONE 6(12): e

76 Utilization of Murine Colonoscopy for Orthotopic Implantation of Colorectal Cancer - Endoscopic live imaging of sub-mucosal injection of India ink dye - PLoS ONE 6(12): e

77 Utilization of Murine Colonoscopy for Orthotopic Implantation of Colorectal Cancer - Endoscopic live imaging of the progressive localized growth of a CRC-tumor following its orthotopic implantation - PLoS ONE 6(12): e

78 Utilization of Murine Colonoscopy for Orthotopic Implantation of Colorectal Cancer - Orthotopic induction of distinct genetically manipulated CRC tumors - PLoS ONE 6(12): e

79 발광대장암세포 Spheroid xenograft 모델 Day 0 Day 4 Day 10 Day 18 Day 28 Day 47 Low cell number Middle cell number High cell number 2D 3D

80 발광대장암세포 Spheroid xenograft 모델 (MRI T1 영상 ) Day 18 JSP JSP

81 발광대장암세포 xenograft 복막암전이모델 IP 패치의대장암유래복막암증식억제효능평가 2 주후

82 발광대장암세포 Orthotopic Xenograft 마우스모델

83 I 마우스종양동물모델 Ⅱ 유방암동물모델 Ⅲ Ⅲ Ⅳ 생체영상기법을대장암동물모델활용한유효성평가생체영상기법을유효성평가활용한유효성평가

84 In Vivo metastasis model A B C D DN10764-meidated suppression of breast cancer metastasis JS Park et al. (2016) Oncotarget

85 Efficacy of DN10764 on Breast Cancer Metastasis Prevention Model

86 Reduction of Total Metastasis Burden by DN10764 Vehicle DN10764 (10 mg/kg) DN10764 (10mg/kg) Veh DN10764 (20 mg/kg) DN10764 (20mg/kg) DN10764 can exert its anti-angiogenic activity without drastic reduction of cell viability. JS Park et al. (2016) Oncotarget

87 A B C D E F JS Park et al. (2016) Oncotarget

88 Reduction of mbc-induced Osteolysis by DN10764 (CT Imaging) Veh-1 Veh-2 DN10764 (10mg/kg) JS Park et al. (2016) Oncotarget

89 Bioluminescence Imaging and MRI unpublished

90 Efficacy in 4T1 Syngeneic Mouse Model LAC LAC Ref. LAC LAC Ref. LAC LAC Ref. LAC LAC Ref.

91 Survival Analysis in 4T1 Syngeneic Mouse Model Survival of tumor-bearing mice Percent survival Vehicle 1mpk 5mpk 10mpk 5mpk(3w1) Days after tumor injection

92 [Lac2] Lead Optimization Reference 100 mpk HX mpk HX mpk HX mpk HX mpk Reference 100 mpk HX mpk HX mpk HX mpk

93 [Lac2] Lead Optimization In-vivo Efficacy (SW-620 orthotopic model) HX mpk HX mpk HX---92 (100 mpk)

94 항암제개발을위한전임상플랫폼구축 항암제개발을위한수요자맞춤형전임상유효성평가지원

95 Conclusions 1. Human & rodent neoplasms are not Identical BUT can be: relevant to identify carcinogens.. relevant to model mechanisms & treatments.. 2. Know the options. 3. Know your model: Similarities to and differences from conditions being modeled. Know expected findings in your model - Including genetic backgrounds & expected tumors - Including the environment and factors that can complicate your project Use Sufficient and relevant concurrent controls 4. Unexpected findings can be important! modify/improve experimental design

96 대한민국의료산업의꿈을실현합니다! 감사합니다.

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