암세포주와 공동 배양된 인간 지방 조직 유래 중간엽 줄기 세포의 유전자 발현 분석 Analysis of Global Gene Expression Profile of Human Adipose Tissue Derived Mesenchymal Stem Cell Cultured with Cancer Cells원문보기
중간엽 줄기 세포는 다분화능을 가지고 있으며 골수, 지방, 태반, 치아속질, 윤활막, 편도 및 가슴샘 등 인체의 다양한 조직에서 분리된다. 중간엽 줄기세포는 조직의 항상성을 조절하며 다분화능, 분리와 조작의 용이함, 암세포로의 화학주성 및 면역 반응 조절 등의 특징을 가지고 있어서 재생 의학, 암 치료 및 식대주 질환(GVHD) 등에 이용할 수 있는 세포치료제로 주목 받고 있다. 하지만 주위 세포와 조직을 지지하고 조절하는 특징과 관련하여 중간엽 줄기세포가 혈관 생성을 촉진하고 성장인자를 분비하며 암세포를 공격하는 면역 반응을 억제함으로써 암의 진행을 촉진시킨다는 사실 또한 보고 되고 있다. 이러한 사실들로 인해 중간엽 줄기세포의 임상 적용이 제한되고 있다. 본 연구에서는 어떠한 기전을 통해서 중간엽 줄기세포가 암의 진행을 촉진하는 지지 세포로 기능하는지를 밝히기 위해서 인체 지방 조직에서 유래한 중간엽 줄기세포를 두 개의 암세포주(H460, U87MG)와 각각 공동 배양하고 microarray를 이용해서 암세포와 공동 배양되지 않은 중간엽 줄기세포와 유전자의 발현을 비교하였다. 두 암세포주와 공동배양에서 공통적으로 2배 이상 차이 나는 유전자를 DAVID (Database for Annotation, Visualization and Integrated Discovery)와 PANTHER (Protein ANalysis THrough Evolutionary Relationships)를 이용해 분석하였으며 생물학적 과정, 분자적 기능, 세포의 구성 성분, 단백질의 종류, 질병과 인체 조직 그리고 신호전달에 관련된 정보를 획득하였다. 이를 통해서 암세포는 중간엽 줄기세의 분화, 증식, 에너지 대사, 세포의 구조 및 분비기능을 조절하여 유전자의 발현 양상을 암 연관 섬유모세포(cancer associated fibroblast)와 유사한 세포로 변형 시킨다는 사실을 알 수 있었다. 본 연구의 결과는 중간엽 줄기세포를 이용한 임상 치료제의 효과와 안정성을 개선하는데 응용될 수 있을 것이다.
중간엽 줄기 세포는 다분화능을 가지고 있으며 골수, 지방, 태반, 치아속질, 윤활막, 편도 및 가슴샘 등 인체의 다양한 조직에서 분리된다. 중간엽 줄기세포는 조직의 항상성을 조절하며 다분화능, 분리와 조작의 용이함, 암세포로의 화학주성 및 면역 반응 조절 등의 특징을 가지고 있어서 재생 의학, 암 치료 및 식대주 질환(GVHD) 등에 이용할 수 있는 세포치료제로 주목 받고 있다. 하지만 주위 세포와 조직을 지지하고 조절하는 특징과 관련하여 중간엽 줄기세포가 혈관 생성을 촉진하고 성장인자를 분비하며 암세포를 공격하는 면역 반응을 억제함으로써 암의 진행을 촉진시킨다는 사실 또한 보고 되고 있다. 이러한 사실들로 인해 중간엽 줄기세포의 임상 적용이 제한되고 있다. 본 연구에서는 어떠한 기전을 통해서 중간엽 줄기세포가 암의 진행을 촉진하는 지지 세포로 기능하는지를 밝히기 위해서 인체 지방 조직에서 유래한 중간엽 줄기세포를 두 개의 암세포주(H460, U87MG)와 각각 공동 배양하고 microarray를 이용해서 암세포와 공동 배양되지 않은 중간엽 줄기세포와 유전자의 발현을 비교하였다. 두 암세포주와 공동배양에서 공통적으로 2배 이상 차이 나는 유전자를 DAVID (Database for Annotation, Visualization and Integrated Discovery)와 PANTHER (Protein ANalysis THrough Evolutionary Relationships)를 이용해 분석하였으며 생물학적 과정, 분자적 기능, 세포의 구성 성분, 단백질의 종류, 질병과 인체 조직 그리고 신호전달에 관련된 정보를 획득하였다. 이를 통해서 암세포는 중간엽 줄기세의 분화, 증식, 에너지 대사, 세포의 구조 및 분비기능을 조절하여 유전자의 발현 양상을 암 연관 섬유모세포(cancer associated fibroblast)와 유사한 세포로 변형 시킨다는 사실을 알 수 있었다. 본 연구의 결과는 중간엽 줄기세포를 이용한 임상 치료제의 효과와 안정성을 개선하는데 응용될 수 있을 것이다.
Mesenchymal stem cells (MSC) are multipotent and can be isolated from diverse human tissues including bone marrow, fat, placenta, dental pulp, synovium, tonsil, and the thymus. They function as regulators of tissue homeostasis. Because of their various advantages such as plasticity, easy isolation a...
Mesenchymal stem cells (MSC) are multipotent and can be isolated from diverse human tissues including bone marrow, fat, placenta, dental pulp, synovium, tonsil, and the thymus. They function as regulators of tissue homeostasis. Because of their various advantages such as plasticity, easy isolation and manipulation, chemotaxis to cancer, and immune regulatory function, MSCs have been considered to be a potent cell source for regenerative medicine, cancer treatment and other cell based therapy such as GVHD. However, relating to its supportive feature for surrounding cell and tissue, it has been frequently reported that MSCs accelerate tumor growth by modulating cancer microenvironment through promoting angiogenesis, secreting growth factors, and suppressing anti-tumorigenic immune reaction. Thus, clinical application of MSCs has been limited. To understand the underlying mechanism which modulates MSCs to function as tumor supportive cells, we co-cultured human adipose tissue derived mesenchymal stem cells (ASC) with cancer cell lines H460 and U87MG. Then, expression data of ASCs co-cultured with cancer cells and cultured alone were obtained via microarray. Comparative expression analysis was carried out using DAVID (Database for Annotation, Visualization and Integrated Discovery) and PANTHER (Protein ANalysis THrough Evolutionary Relationships) in divers aspects including biological process, molecular function, cellular component, protein class, disease, tissue expression, and signal pathway. We found that cancer cells alter the expression profile of MSCs to cancer associated fibroblast like cells by modulating its energy metabolism, stemness, cell structure components, and paracrine effect in a variety of levels. These findings will improve the clinical efficacy and safety of MSCs based cell therapy.
Mesenchymal stem cells (MSC) are multipotent and can be isolated from diverse human tissues including bone marrow, fat, placenta, dental pulp, synovium, tonsil, and the thymus. They function as regulators of tissue homeostasis. Because of their various advantages such as plasticity, easy isolation and manipulation, chemotaxis to cancer, and immune regulatory function, MSCs have been considered to be a potent cell source for regenerative medicine, cancer treatment and other cell based therapy such as GVHD. However, relating to its supportive feature for surrounding cell and tissue, it has been frequently reported that MSCs accelerate tumor growth by modulating cancer microenvironment through promoting angiogenesis, secreting growth factors, and suppressing anti-tumorigenic immune reaction. Thus, clinical application of MSCs has been limited. To understand the underlying mechanism which modulates MSCs to function as tumor supportive cells, we co-cultured human adipose tissue derived mesenchymal stem cells (ASC) with cancer cell lines H460 and U87MG. Then, expression data of ASCs co-cultured with cancer cells and cultured alone were obtained via microarray. Comparative expression analysis was carried out using DAVID (Database for Annotation, Visualization and Integrated Discovery) and PANTHER (Protein ANalysis THrough Evolutionary Relationships) in divers aspects including biological process, molecular function, cellular component, protein class, disease, tissue expression, and signal pathway. We found that cancer cells alter the expression profile of MSCs to cancer associated fibroblast like cells by modulating its energy metabolism, stemness, cell structure components, and paracrine effect in a variety of levels. These findings will improve the clinical efficacy and safety of MSCs based cell therapy.
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제안 방법
To analysis the influence of cancer cells to global gene expression of ASCs, CM-Dil labeled ASCs were co-cultured with GFP-positive H460 or U87MG. After three days of co-culture, ASCs were sorted by FACS (Fig. 2) and gene expression was quantified using microarray analysis in comparison with control ASCs, which were not co-cultured with cancer cells. We only focused on the genes, which had more than 2-fold difference in expression, compared to control and had same direction of expressional changes in both ASCs co-cultured with H460 (ASC-H) and ASCs co-cultured with U87MG (ASC-U) to study the general characteristics of expression profile.
In this study, to get insight how cancer cells manipulate MSCs to utilize its tumor promoting activity, we obtained gene expression data from adipose tissue derived MSC (ASC) co-cultured with cancer cells through whole genome expression analysis using gene chip assay. Differentially expressed transcripts compared to those of ASC cultured alone were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.
In this study, we used microarray and comparative gene expression analysis by a differential clustering approach to investigate the influence of cancer cells on phenotypic changes of MSCs leading promotion of malignancy.
Balb/C nude mice at 7 weeks of age were purchased from the Laboratory Animal Center of Seoul, Korea. The animals were housed in micro-isolator cages under sterile conditions, and observed for at least 1 week in order to ensure proper health prior to the initiation of the study. Lighting, temperature, and humidity were centrally controlled and recorded daily.
데이터처리
Comparisons between groups were analyzed via Student’s t-tests.
이론/모형
In this study, to get insight how cancer cells manipulate MSCs to utilize its tumor promoting activity, we obtained gene expression data from adipose tissue derived MSC (ASC) co-cultured with cancer cells through whole genome expression analysis using gene chip assay. Differentially expressed transcripts compared to those of ASC cultured alone were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.7 and Protein ANalysis THrough Evolutionary Relationships (PANTHER) version 7 [18,31]. Expression profiles of ASCs were categorized to functionally related groups in diverse level of cell and molecular biology and its significances were interpreted.
성능/효과
In conclusion, our comparative expression data analysis show that cancer cells change the behavior of MSCs into cancer supportive cells by affecting its energy metabolism, stemness, cellular structure components, proliferation and secretory function in variety levels. Our understanding from this study could not only be applied to CAFs targeting cancer therapy but also improve safety of MSCs utilizing cancer treatment and regenerative medicine.
Biological processes of up-regulated genes with high fold enrichment included tryptophan catabolic process to acetyl-CoA, lipid and cholesterol metabolism, amino acid metabolism, macrophage activation, angiogenesis, carbohydrate transport, response to toxin, and CDC42 signal transduction. In terms of down-regulated genes, mesoderm and embryonic development, which is characteristics of stemness, membrane raft formation, potassium ion import, PDGF receptor pathway, response to progesterone, angiogenesis, microtubule polymerization, nitric oxide synthesis, and cell-matrix adhesion, were significantly enriched. It is noteworthy that angiogenesis belongs to both up and down-regulated gene groups (Fig.
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