Title Page

ABSTRACT

Contents

CHAPTER 1. Therapeutic Strategy for Metastatic Malignant Fibrous Histiocytoma with Mesenchymal Stromal Cell-mediated TRAIL Production 14

1.1. INTRODUCTION 14

1.2. MATERIALS AND METHODS 16

1.2.1. Construction of replication-defective adenoviral vectors 16

1.2.2. Transduction of adenovirus into hMSCs 16

1.2.3. Cells 16

1.2.4. In vitro co-culture experiments 17

1.2.5. Cytotoxicity and caspase-8 activation assays 17

1.2.6. ELISA 18

1.2.7. Flow Cytometry 18

1.2.8. Mice 18

1.2.9. Local tumorigenesis murine model 19

1.2.10. Isolation and characterization of the MFH-ino xenograft explants 19

1.2.11. Karyotyping of the xenograft explants 19

1.2.12. Wound filling assay 20

1.2.13. Lung metastasis murine model 20

1.2.14. Liver metastasis murine model 20

1.2.15. Positron emission tomography (PET) 20

1.2.16. Histological analysis 21

1.2.17. Statistical analysis 21

1.3. RESULTS 22

1.3.1. Optimization of soluble TRAIL protein production in human MSCs 22

1.3.2. MFH-ino-specific cytotoxicity of MSC-TRAIL via caspase-8 activation 24

1.3.3. Inhibition of local tumorigenesis by MSC-TRAIL in vivo 27

1.3.4. In vivo inhibition of xenograft explants tumorigenesis by MSC-TRAIL 29

1.3.5. Anti-metastatic effects of MSC-TRAIL in malignant tumorigenesis 32

1.3.6. MSC-TRAIL reduces pre-established metastasis in the liver 38

1.4. DISCUSSION 43

1.5. REFERENCES 47

CHAPTER 2. Therapeutic Strategy for De-differentiated Liposarcoma with Mesenchymal Stromal Cell Expressing Herpes Simplex Virus-Thymidine Kinase 53

2.1. INTRODUCTION 53

2.2. MATERIALS AND METHODS 56

2.2.1. Human adipose tissue-derived Mesenchymal stem cells (hAD-MSCs) isolation 56

2.2.2. Human De-differentiated liposarcoma (DDLPS) cell lines 56

2.2.3. Adenoviral vectors 57

2.2.4. Transduction of adenovirus into hMSCs 57

2.2.5. ELISA 58

2.2.6. Reverse transcription PCR 58

2.2.7. In vitro co-culture experiments 59

2.2.8. Cell viability 59

2.2.9. Flow Cytometry 59

2.2.10. Gap junction intracellular communication 60

2.2.11. Mice 60

2.2.12. Maximum Tolerate dose (MTD) of GCV in NSG mice 60

2.2.13. Metastasis murine model 61

2.2.14. Locoregional recurrence murine model 61

2.2.15. Histological analysis 62

2.2.16. Positron emission tomography (PET) 63

2.2.17. Statistical analysis 63

2.3. RESULTS 65

2.3.1. LPS cell lines have different sensitivities in response to rhTRAIL 65

2.3.2. Engineered hAD-MSCs successfully express TRAIL and HSV-TK 67

2.3.3. LPS cell lines are highly sensitive to MSC-TK, followed by GCV treatment 71

2.3.3. MSC-TK induces apoptosis in LPS by bystander effects via gap junction intracellular communication 74

2.3.4. Maximum tolerate dose (MTD) of GCV was optimized in severely immune-deficient NSG mice 77

2.3.5. MSC-TK significantly inhibits pulmonary metastasis of LPS 83

2.3.6. Sarcoma recurrent model was established following macroscopically complete resection and verified by macroscopic and microscopic evaluations 91

2.3.7. MSC-TK significantly reduces locoreginal recurrence of LPS following resection 97

2.4. DISCUSSION 101

2.5. REFERENCES 105

국문초록 111

Table 2.1. Kidney creatinine and liver enzyme levels in NSG mice. Mice treated a various dose of GCV for 2 cycles of 5 consecutive days and measured levels of kidney creatinine and Liver enzyme, AST and ALT at 2 days and 2 weeks post-GCV treatment. 80

Table 2.2. Summary of locoregional recurrence model. 100

Figure 1.1. The engineered MSCs efficiently secrete soluble TRAIL proteins under optimized adenovirus conditions. 23

Figure 1.2. MSC-TRAIL induces caspase-8–mediated apoptosis in MFH-ino cells. 26

Figure 1.3. MSC-secreted TRAIL reduces local MFH-ino tumorigenesis. 28

Figure 1.4. Characteristics of the xenograft explants. 30

Figure 1.5. MSC-secreted TRAIL eliminates local tumorigenesis of xenograft explants. 31

Figure 1.6. Wounds filling assay. 34

Figure 1.7. The anti-tumor effects of MSC-TRAIL in lung metastases. MFH-ino and MSC-TRAIL or MSC-Mock cells were intravenously implanted into NSG mice. 35

Figure 1.8. MSC-secreted TRAIL reduces metastatic tumorigenesis in the liver. 36

Figure 1.9. Histology of liver metastasis model. The presence of MSCs with SP-DiOC18(3) (green) and MFH-ino cells with CM-DiI (red) in sections of spleen (A, bar, 50 μm) and liver (B, bar, 25 μm).... 37

Figure 1.10. Distribution of CM-DiI-labeled MFH-ino cells in the spleen and liver. CM-DiI-labeled MFH-ino cells (5x106) were implanted into the spleens of NSG mice. A and B, A histological analysis was performed at days 1, 3, 5, and 7 after transplantation....(이미지참조) 39

Figure 1.11. MSC-secreted TRAIL inhibits previously established metastatic tumorigenesis in the liver. 40

Figure 1.12. Histology of pre-established metastasis model. Photomicrographs showing the presence of MSCs with SP-DiOC18(3) (green) and MFH-ino cells with CM-DiI (red) in sections of spleen (A, bar, 50 μm) and liver (B, bar, 25 μm).... 41

Figure 1.13. No influence of hAD-MSCs on either local tumorigenesis or liver metastases. 42

Figure 2.1. Sarcoma cell lines showing different susceptibility to recombinant human TRAIL (rhTRAIL). MFHino-xeno, LPS246, and LPS863 cells were plated and incubated with serial dilutions of rhTRAIL proteins (0-100 ng/ml).... 66

Figure 2.2. hAD-MSCs are engineered to secret TRAIL (MSC-TRAIL). 69

Figure 2.3. hAD-MSCs are engineered to express HSV-TK (MSC-TK) and induced cell death by ganciclovir (GCV). 70

Figure 2.4. Engineered MSCs induce apoptosis in LPS246 cells. Dot plots of apoptosis in LPS246 cells gated on DiIC18(5)-DS negative cells that were cocultured with DiIC18(5)-DS-labeled MSC-TRAIL, MSC-TK or MSC-Mock with 100 μM of GCV for 48 hours (A) and 72... 72

Figure 2.5. Engineered MSCs induces apoptosis in LPS863 cells. Dot plots of apoptosis in LPS863 cells gated on DiIC18(5)-DS negative cells that were cocultured with DiIC18(5)-DS-labeled MSC-TRAIL, MSC-TK or MSC-Mock with 100 μM of GCV for 48 hours (A) and 72... 73

Figure 2.6. Gap junction intracellular communication (GJIC) occurred between MSC-TK and LPS246 cells. MSC-TK and MSC-Mock were labeled with green fluorescence calceinAM, which can be transferred through gap junction.... 76

Figure 2.7. Determination of maximum tolerate dose (MTD) of GCV in NSG mice. 79

Figure 2.8. Levels of kidney creatinine and liver enzymes in NSG mice. Serum were obtained from NSG mice treated an increasing dose of GCV for 2 cycles of 5 consecutive days at 2 days and 2 weeks after the final intraperitoneal GCV treatment.... 81

Figure 2.9. Histology of nephrotoxicity in NSG mice treated with GCV. Representative H&E staining of section of kidney at 2 days and 2 weeks after the final GCV injection showing normal glomerulars and tubules (bar, 100 μm). 82

Figure 2.10. MSC-TK Inhibits an early stage of lung metastasis. 86

Figure 2.11. Histology of the LPS246 metastatic organs. Representative H&E staining of tumor-bearing lungs, livers, and kidneys of mice injected with MSC-TRAIL or MSC-Mock at 1 week post-tumor implantation (bar, 100 μm). 87

Figure 2.12. Lung migration of LPS246 in metastatic mouse model. 88

Figure 2.13. MSC-TK significantly reduces an advanced stage of lung metastasis. 89

Figure 2.14. Histology of the metastatic organs in an advanced stage of metastasis model. 90

Figure 2.15. Locoregional recurrence mice modeling. 93

Figure 2.16. Hisotology of a LPS246 xenograft tumor and a recurrent xenograft tumor. 94

Figure 2.17. Macroscopic evaluation of R0/R1 resection by PET/CT. 95

Figure 2.18. Microscopic evaluation of R0/R1 resection by histological study. 96

Figure 2.19. Implantation of MSC-TK following macroscopically complete resection prevents local recurrence of LPS. 99

 본 연구는 육족암을 대상으로 항암물질인 TRAIL과 HSV-TK 를 발현하는 엔지니어링 줄기세포의 항암효과 검증을 목적으로 한다.

육종암은 근육, 결합조직, 지방, 뼈, 연골, 혈관 등 온 몸에서 발생 가능한 악성암으로 수술 후 화학치료 및 방사선치료를 받아도 재발율 및 전이율이 높아 5년 생존율이 36~63% 로 예후가 매우 나쁜 암이다. 하지만 육종암은 근본적인 치료 및 예방 연구가 거의 전무 한 상태로, 생존율과 직결되는 재발 및 전이를 줄일 수 있는 새로운 패턴의 표적치료가 요구된다. 본 연구에서는 육종암 표적치료를 위해 줄기세포를 도입하였다. 줄기세포는 몸 속 암세포로 이동하는 성질 (tropism) 과 이식 후 낮은 면역거부반응 (hypoimmunogenicity), 항암치료유전자의 엔지니어링이 용이한 장점을 가지기 때문이다. 따라서 본 연구에서 줄기세포에 TRAIL 이라는 정상세포는 피하고 암세포만 특이적으로 죽이는 항암유전자와, HSV-TK 라는 빠르게 증식하는 암세포를 타겟으로 세포사멸을 유도하는 항암유전자를 엔지니어링하고 육종암의 대표적인 악성섬유성조직구종 및 지방조직에의 치료효과를 분석하였다.

시험관에서 TRAIL과 HSV-TK를 발현하도록 엔지니어링한 줄기세포 (MSC-TRAIL과 MSC-TK) 가 악성섬유성조직구종 (MFHino) 및 지방육종 (LPS246, LPS863) 세포주와 함께 배양한 후 세포사멸이 유도되는지 분석하였다. 전임상 단계에서 악성섬유성조직구종의 폐 전이 및 간 전이암 동물모델을 통해 MSC-TRAIL의 항 전이효과를 확인하고, 지방육종의 폐 전이암 및 재발암 동물모델을 통해 MSC-TK 의 항전이 및 재발 예방효과를 확인하였다.

그 결과, 육종암은 암세포주 종류에 따라 TRAIL에 다른 민감성을 보였다. 특히 MSC-TRAIL은 악성섬유성조직구종의 세포주에 매우 높은 항암 효과를 보였고 이는 엔지니어링 줄기세포에서 분비하는 TRAIL이 암세포의 표면에서 발현하는 세포사멸수용체와 결합하여 세포사멸신호전달 메카니즘을 통한 것임을 확인하였다. 반면 지방육종의 한 세포주는 MSC-TRAIL에 저항성을 보였지만 MSC-TK에는 높은 민감성을 보였다. 이 MSC-TK의 항암효과는 엔지니어링 줄기세포에서 발현하는 HSV-TK에 의해 변환된 독성의 간사이클로비르 (ganciclovir) 가 세포간 신호전달통로인 갭졍션 (gap junction) 을 통해 암세포로 전달되어 세포사멸을 유도했음을 확인하였다. 시험관 항암효과 실험과 메커니즘 연구를 바탕으로 한 전임상 동물실험에서 MSC-TRAIL이 악성섬유성조직구종의 폐 전이암뿐 아니라 간 전이암을 강력히 줄인 것을 확인하였다. 이는 폐 및 간으로 암을 따라 이동한 엔지니어링 줄기세포가 TRAIL을 분비시켜 전이암을 세포사멸에 유도한 것을 시사한다. MSC-TK은 TRAIL에 저항성을 가지는 지방육종 세포주의 단계별 폐 전이암 모델에서 초기뿐 아니라 암세포의 증식이 상당히 진행된 단계에서도 높은 항전이 효과를 보였다. 주목할 부분은, 임상에서 수술 후에도 80% 이상의 재발율을 보이는 지방육종을 대변할 수 있는 지방육종 재발동물모델이 확립되었고, MSC-TK의 효과를 확인 한 결과 통계학적 유의성 있는 재발 억제 효과를 확인하였다. 이는 MSC-TK가 암세포 제거 수술 후 잔여하고 있던 암세포를 공격하여 다시 재발되는 확률을 낮춘 것으로, 임상에서 재발을 막아 생존율을 높이는 전략에 있어서 매우 의미 있는 결과라고 사료된다.

결론적으로, 본 전임상 연구는 엔지니어링 줄기세포의 악성 육종암 전이동물모델 및 재발동물모델에서 효과적인 항암효과를 확인함으로써 줄기세포 기반의 임상치료가 육종암 환자를 위한 새로운 치료전략으로서의 가능성을 제시한다고 사료된다.