폐암 세포 유래 인터루킨-23에 의한 선천성 림프구 세포 1형에서 3형으로의 변환이 종양 성장에 미치는 영향 연구

학위논문(박사)--서울대학교 대학원 :의과대학 의학과,2019. 8. 김지은.Due to the recent success of immunotherapy in non-small cell lung cancer (NSCLC) patients, there has been a growing interest in the role of immune cells in tumor microenvironment (TME). Innate lymphoid cell (ILC) is one kind of immune subsets which might contribute to the TME yet relatively unexplored. ILC has three subsets classified according to their transcription factor and cytokine expression, however, the plasticity between ILC subsets has been reported in the inflammatory bowel disease by in vitro experiments. Up to now, the effect of ILC subsets and their plasticity with changes in cytokines on TME has not been fully investigated. This study aimed to reveal the role of ILC plasticity and significance for potential therapeutic target in human lung TME. ILC subsets and cytokine expression profiles were evaluated in 80 cases of NSCLC each consisting of 40 squamous cell carcinoma (SqCC) and 40 adenocarcinoma (ADC) with matched normal lung tissue, which were freshly collected in Seoul National University Hospital. Experimental conversion of ILC1 into ILC3 under co-culture with lung cancer cells was explored. The contribution of IL-23 on tumor growth and regulation of immune components in TME was investigated using in vivo mouse tumor model. Verification and prognostic effects of converted ILC3 and related pathways were evaluated by tissue microarray from retrospective cohort composed of 875 lung cancer patients. As a result, IL-17RA expression on tumor cells and IL-17A expression on immune cells from TME were significantly higher in SqCC than ADC, which suggests that IL-17 has pro-tumoral effects in pulmonary SqCC. Among IL-17+ cells, the percentages of ILCs were higher in SqCC than ADCs, and also ILC3s were more frequent that ILC1s in SqCC in contrast to ADC or normal lung tissue. Among cytokines which were involved in plasticity of ILC1 and ILC3, only IL-23 mRNA or protein expression from tumor cells was significantly correlated with the number of ILC1 and ILC3. And the percentage of ILC3 was associated with IL-23 expression in tumor cells but not immune cells. Based on these findings, it was suggested that the conversion of ILC1 from ILC3 in TME could be caused by IL-23 expression from tumor cells. The polarization of ILC1 into ILC3 was observed in experiment of in vitro co-culture model using sorted-ILC1 or total ILCs and SqCC which expresses high level of IL-23, but not in co-culture of sorted ILC1 or total ILCs and ADCs. In in vivo murine tumor model, tumor growth was dependent on IL-23 expression from tumor cells via IL-17 from converted ILC3 from ILC1. In retrospective cohort using immunohistochemistry on TMA, the numbers of CD3-RORγt+ ILC3, IL-17 expression level, and IL-23- or IL-17RA-expressing tumor cells were more frequently observed in SqCC. These factors were also associated with short survival of patients with SqCC but not ADC. In conclusion, these results indicate that IL-23 produced by tumor cells drives differentiation ILC1 toward ILC3 and the interaction of IL-17 expression from ILC3 and IL-17R on tumor cells promotes tumor proliferation. Therefore, the IL-23 – ILC3 – IL-17 axis affects to poor prognosis in lung cancer patients, this axis may suggest potential immunotherapy targets for patients with IL-23-producing lung cancers.최근 PD-1을 비롯한 면역 치료가 환자에게 좋은 반응을 보이면서 종양 미세환경에 존재하는 면역세포에 대한 이해가 그 어느 때보다 중요해지고 있다. 선천성 림프구 세포는 선천성 면역 세포에 가깝지만 도움 T세포에 상응하는 역할을 담당하는 특징을 가진 비교적 최근에 밝혀진 세포군이다. 선천성 림프구 세포는 각 발현하는 전사인자 및 분비하는 사이토카인을 통해 3가지 아형으로 나뉜다. 선천성 림프구 세포 아형 간 형질 전환은 대장염 실험 모델에서 보고된 바 있으나 종양에서는 아직까지 밝혀진 내용이 없고, 선천성 림프구 세포의 아형별 종양 미세환경에서의 역할에 대하여 아직까지 정립되지 않았다. 또한, 선천성 림프구 세포 3형을 비롯한 여러 종류의 면역 세포가 분비할 수 있는 물질 중 하나인 인터루킨-17이 종양에 미치는 영향에 대해서는 연구마다 다른 결과를 제시하고 있어 아직까지 더 많은 연구가 필요하다. 이번 연구는 선천성 림프구 세포가 폐암 조직 내에 존재 및 형질 전환 발생 여부, 종양 내에서의 역할을 파악해 봄으로써 새로운 면역 치료 타겟을 발굴하고자 계획하게 되었다. 2015년부터 2017년까지 서울대학교병원에서 비소세포폐암으로 절제술을 받은 환자 80명의 신선 상태의 종양과 정상 폐 조직을 대상으로 각 조직 내 존재하는 면역 세포의 분포 및 발현 물질을 파악하였다. 또한 신선 종양 조직에서 선천성 림프구 세포를 얻은 뒤 비소세포 폐암 세포주 혹은 환자 조직으로부터 얻은 종양 세포와 함께 배양하여 나타나는 변화를 파악하였고, 마우스 종양 모델을 구축하여 생체 내에서의 종양 성장 변화를 확인하였다. 또한 2001년부터 2012년까지 동일 기관에서 비소세포폐암으로 폐엽 절제술을 시행한 환자들의 파라핀 블록에서 종양 부위만을 획득하여 조직 미세배열 블록을 제작 및 면역조직화학 염색을 시행하여 임상 지표와의 연관성 및 생존 분석을 시행하였다. 우선 종양 세포에서의 인터루킨-17수용체 발현과 주변 면역 세포들의 인터루킨-17의 발현을 조사하였을 때 선암에 비해 편평상피세포암에서 두 인자 모두 높게 발현하고 있음을 확인하였다. 인터루킨-17을 분비할 수 있는 여러 면역 세포군 중에서는 유일하게 선천성 림프구 세포 3형이 폐 편평상피세포암에서 선암이나 정상 폐 조직에 비해 많이 존재함을 관찰하였고 선천성 림프수 세포 아형의 분포를 파악하였을 때 편평상피세포암에서 3형이 1형보다 종양 내에 많이 분포하고 있었으나 선암에서는 정상 조직과 큰 차이를 보이지 않았다. 이러한 분포의 차이는 아형별 전환을 유도할 수 있는 사이토카인 분비에 의할 것이라는 가정 하에 사이토카인 분비와 선천성 림프구 세포 1형, 3형과의 연관성을 확인하였다. 그 중 종양 세포에서 발현되는 인터루킨-23이 선천성 림프구 1형과 역상관관계를, 3 형 과 정상관관계를 보였다. 공생배양 실험을 하였을 때 편평상피세포암 세포와 선천성 림프구 전체 혹은 1 형을 함께 배양한 경우에 3 형 의 증가를 보였고, 종양 세포의 증식 증가를 확인하였다. 추가적으로 마우스 및 마우스 편평상피세포암 세포주를 이용하여 실험을 하였을 때에도 인터루킨-23 과발현 종양 세포를 투여한 마우스에서 종양 세포의 증식 증가 및 선천성 림프구 세포 1 형 이 3 형으로 전환되는 현상과 인터루킨-17의 증가를 확인하여 생체 내에서도 같은 현상을 보임을 증명하였다. 마지막으로 종양 조직에서 면역조직화학염색을 통해 종양 세포에서 분비되는 인터루킨-23의 발현 증가와 선천성 림프구 세포 3 형 의 증가가 편평상피암에서 관찰됨을 확인하였다. 또한 편평상피암 환자에서만 생존 분석에서 두 인자 모두 불량한 예후와 연관됨을 확인함으로써 종양이 자가 생존 및 성장을 위해 주변 면역세포, 그 중에서도 선천성 림프구 세포의 형질 전환을 유도할 수 있는 물질을 분비하여 자가 증식에 이용함을 최초로 보고하였다. 결론적으로 본 연구자는 편평상피세포폐암에서 발현하는 인터루킨-23이 종양 내 미세환경에 존재하는 선천성 림프구 세포 1 형을 3 형으로 전환시켜 종양 내 인터루킨-17 축적의 증가를 유도하고, 이로 인해 인터루킨-17 기전에 의한 종양 성장 촉진이 환자의 불량한 예후로 이어지게 된다는 사실을 밝혔다. 본 연구는 선천성 림프구 세포를 이용한 종양의 생존 전략을 규명함으로써 폐암, 그 중에서도 편평상피세포암과 같이 인터루킨-23을 많이 분비하는 종양에 대해 인터루킨-23/ 선천성 림프구 세포 3/ 인터루킨-17 축의 한 부분을 차단함으로써 환자의 생존률 향상이 가능한 치료 타겟을 제시하는 근거로 사용될 수 있을 것으로 기대된다.Contents List of Tables List of Figures 1. Introduction 1.1 Different two subtypes of non-small cell lung cancer 1.2 Various strategies for tumor survival 1.3 IL-17 pathway in tumor microenvironment 1.4 Innate lymphoid cells and regulation of tumor immunity 2. Materials and Methods 2.1 The cohorts of patients with NSCLC 2.2 Preparation of fresh tissues obtained from NSCLC patients and tissue microarray 2.3 Human and mouse lung cancer cell lines 2.4 Antibodies for immune subset analysis using flow cytometry 2.5 Cell sorting 2.6 Lentiviral transduction and transfection study 2.7 Mice and tumor inoculation in vivo 2.8 Immunohistochemistry 2.9 Immunofluorescence 2.10 Quantitative real-time PCR 2.11 Proliferation assay 2.12 In vitro culture of ILCs 2.13 Co-culture experiments using ILCs 2.14 RNAseq analysis 2.15 TCGA data analysis 2.16 Statistics 3. Results 3.1 The IL-17- IL-17 receptor axis is enhanced in pulmonary SqCCs but not ADCs 3.2 IL-17-IL-17R axis is associated with short survival of SqCC patients 3.3 Higher numbers of ILC3 among IL-17-producing immune cells in SqCCs 3.4 SqCC exhibit an inverse correlation between ILC1 and ILC3 compared to ADCs 3.5 Conversion from ILC1 into ILC3 is promoted by IL-23-producing SqCCs, rather than immune cells, in the TME 3.6 SqCCs rather than ADCs drive the conversion from ILC1 into ILC3 subsets by producing IL-23 3.7 IL-23-producing SqCCs promote tumor growth by inducing ILC3-mediated IL-17 production in in vitro 3.8 IL-23-producing SqCCs promote tumor growth by inducing ILC3-mediated IL-17 production in in vivo 3.9 ILC3 contributes to the poor prognosis of patients with SqCC 4. Discussion 4.1 The conversion of ILC1 into ILC3 by tumor cells exists in TME 4.2 IL-23 mediated novel tumor survival strategy 4.3 IL-23/ILC3/IL-17 axis: a critical pathway of tumor progression in SqCCs 4.4 Conclusive remarks Bibliography 국문초록Docto

Computational Experimentation of Information Dissemination in a Communication Network

본 연구에서는 통신네트워크에서 로컬브로드캐스팅 방식에 따른 정보전달 방법을 다루었다. 나무형 구조의 경우 최적기법에 대해 고찰한 다음 퍼스널 컴퓨터용 패키지를 개발하였고 일반적 구조의 경우 발견적 해법을 제시하고 전산모의실험을 하였다.Information dissemination by local broadcasting in a communication network is studied. In the case of a tree-type network, an optimal technique is reviewed and a program package for a personal computer is developed. In the case of a general-type network, a heuristic algorithm is suggested and computational experimentation is performedInformation dissemination by local broadcasting in a communication network is studied. In the case of a tree-type network, an optimal technique is reviewed and a program package for a personal computer is developed. In the case of a general-type network, a heuristic algorithm is suggested and computational experimentation is performe

Minimum Broadcasting Time in Tree Network

본 논문에서는 라인형 조직에서 한 구성원이 알고 있는 정보를 다른 모든 구성원에 전달하는 시간을 최소화하는 기법을 개발하였다. 한 구성원은 한번에 한 구성원에게만 전달할 수 있으며 각 구성원 사이의 전달 시간은 구성원의 짝에 따라 다르다고 가정하였다. 여기서 개발된 기법은 석유 수송 파이프 문제와 전산망 문제 등에 응용될 수 있다.In this paper, a algorithm minimizing the broadcasting time in a tree network has been developed. It is assumed that a node may be a participant in at most one call during any given time unit and transmission times are different among arcs. The algorithm may be applied to oil pipe system and computer communication system.In this paper, a algorithm minimizing the broadcasting time in a tree network has been developed. It is assumed that a node may be a participant in at most one call during any given time unit and transmission times are different among arcs. The algorithm may be applied to oil pipe system and computer communication system

A Broadcasting Center in a Tree-type Network

본 논문은 일반적인 나무 구조 네트워크에서 각 edge에서의 정보전달 시간이 주어졌을 때 로칼 브로드 케스팅 방식에 따라 전체 전달 시간을 최소화하는 문제를 다루었다. 정보의 최적 출발점과 각 vertex 에서의 전달 순서를 결정하는 해법을 개발하고 이에 대한 타당성을 입증하였다.This paper deals with the problem of minimizing the broadcasting time in a tree-type network given edge transmission by local broadcasting. An algorithm determining a broadcasting center and the call sequence at each vertex is suggested and its correctness is given.This paper deals with the problem of minimizing the broadcasting time in a tree-type network given edge transmission by local broadcasting. An algorithm determining a broadcasting center and the call sequence at each vertex is suggested and its correctness is given

A Survey on the Level of Informationizedness in Ulsan

본 연구에서는 울산지역 주민과 제조업체를 대상으로 정보화에 대한 인식 및 정보화수준에 대해 설문지 조사를 하였다. 정보화사회에 대한 인식, 정보통신기기의 보급 및 활용, 정보통신서비스의 이용, 기업정보화의 수준, 지역정보센타의 인식 등에 대한 설문지를 작성하여 지역주민에 대해서는 면접조사를 하였고, 제조업체에 대해서는 우편조사를 실시하였다. 그 결과 대체적으로 정보화사회에 대?? 높은 인식을 가지고 있으나, 정보통신서비스에 대한 지식을 충분히 갖지 못한 것으로 나타났다. 또 지역정보의 필요성을 인식하고 여건이 조성되면 지역정보서비스를 이용할 것으로 나타났다. 이에 대한 효과적인 방안으로서 지역정보센타의 설립을 많은 사람들이 원하고 있는 것으로 나타났다.This paper studied residents' and industry's views on informationizedness in Ulsan with the field survey. Questionnaires were made on the opinion on information society, the usage of communication equipment, information services, the level of firm-informationizedness, and the regional information center. Individual interviews were taken for the residents and postal survey was made for the industry. As results, they have high perception of information society, but have no enough knowledge of information services. They recognize the necessity of regional information and intend to subscribe to the regional information services if possible. One of the efficient policies for their desire is the establishment of the regional information center.This paper studied residents' and industry's views on informationizedness in Ulsan with the field survey. Questionnaires were made on the opinion on information society, the usage of communication equipment, information services, the level of firm-informationizedness, and the regional information center. Individual interviews were taken for the residents and postal survey was made for the industry. As results, they have high perception of information society, but have no enough knowledge of information services. They recognize the necessity of regional information and intend to subscribe to the regional information services if possible. One of the efficient policies for their desire is the establishment of the regional information center

Computerization of the Network Model Using the Object-Oriented Programming

본 논문에서는 개체지향 프로그래밍 개념을 이용하여 네트워크 모형의 전산 구현 통합패키지를 개발하였다. 네트워크 모형을 표현하기 위하여 내부적으로 단일 연결리스트를 채택하였다. 풀-다운 메뉴 방식을 이용하여 파일, 자료 입출력, 그리기, 문제선택 메뉴를 구성하였다. 자료입력의 경우 마우스를 이용한 입력, 고밀도 네트워크에 대한 파일 입력, 저밀도 네트워크에 대한 파일 입력의 3가지 방식을 제시하였다. 현실감을 높이기 위해 모형을 그래픽으로 나타낼 수 있도록 하였다. 또한 사용자가 자신의 환경에 따라 선택이 가능하도록 고밀도 및 저밀도 자료형식으로 저장이 가능하도록 하였다. 응용 예를 위하여 최단경로나무문제, 최소걸침나무문제, 최대흐름문제의 해법을 첨가하였으며, 사용자는 자신의 문제에 대한 해법을 서브루틴으로 추가할 수 있도록 ?臼눼?. 개체지향개념을 이용하였기 때문에 네트워크 모형을 쉽게 확장할 수 있으며 프로그램을 재사용할 수도 있다. 따라서 사용자는 네트워크 모형을 전산구현하는데 드는 노력을 대폭 경감시킬 수 있다.An integrated computer-package for the network model is developed, where the network model is implemented using object-oriented programming. Node object, arc object and network object are defined a user-defined objects. Singly linked list is adopted as the internal data structure for network representation. All works are designed to be implemented through the pull-down menu. The main menu consists of FILE, INPUT TYPE, DRAWING, and PROBLEM SELECTION. Three methods are suggested for data input: mouse input, file input for dense-type data, and file input for sparse-type data. The model can be displayed graphically for the sense of reality. It can be stored in both dense-type and sparse-type data format, which enables the user to fit the model to his/her environment. Shortest path tree problem, minimum spanning tree problem, and maximal flow problem are applied for illustrations. Since the network model is realized using the object-oriented concept, the user can apply any problem with its own subroutine. The object-oriented programming gives the benefit of modifying or expanding the model easily and reusing the program. As a result, the user can dramatically save time and efforts for implementing the network model.An integrated computer-package for the network model is developed, where the network model is implemented using object-oriented programming. Node object, arc object and network object are defined a user-defined objects. Singly linked list is adopted as the internal data structure for network representation. All works are designed to be implemented through the pull-down menu. The main menu consists of FILE, INPUT TYPE, DRAWING, and PROBLEM SELECTION. Three methods are suggested for data input: mouse input, file input for dense-type data, and file input for sparse-type data. The model can be displayed graphically for the sense of reality. It can be stored in both dense-type and sparse-type data format, which enables the user to fit the model to his/her environment. Shortest path tree problem, minimum spanning tree problem, and maximal flow problem are applied for illustrations. Since the network model is realized using the object-oriented concept, the user can apply any problem with its own subroutine. The object-oriented programming gives the benefit of modifying or expanding the model easily and reusing the program. As a result, the user can dramatically save time and efforts for implementing the network model

Software Development of Generalized Linear/Goal Programming for Microcomputer

The propose of this study is to presnet a generalized linear/goal programming software, which has been developed to run on mickrocomputers with at least 512 K bytes of memory. The main characteristics of our algorithm for solving LP/GP problems are outlined as follows ; First, it uses the revised simplex algorithm, which is the most efficient computational procedure for computers. Second, it employs the sparse matrix technique to overcome the limited memory of microcomputers. Last, it uses the modified product form of invers (MPFI) to reduce round-off errors. The test runs with our code written in FORTRAN show that it can be used as an effective tool for solving linear/goal programming problems of considerable size

(A) heuristic approach to the design of a multipoint linkage teleprocessing network under capacity option

학위논문(석사) - 한국과학기술원 : 산업공학과, 1981.2, [ iii, 62 p. ]This thesis deals with the problem of the design of the minimum-cost centralized network with multipoint linkage where options are available as to discrete link capacities. In this thesis, the concept of the ``main path`` which is the path from a node to the center is introduced. Information requests, link capacity, and link cost are considered as important factors. It is assumed that the rate and manner in which information is requested at terminals is fixed. It is shown that the problem can be formulated as a mixed integer program. Many heuristic methods as well as the optimization technique for one capacity were developed, of which Esau-Williams algorithm is known to be the best. This thesis develops a heuristic algorithm for the case of capacity options, based on Esau-Williams algorithm. This algorithm essentially applies the link exchange technique, and in each link exchange, the flow change with the associated capacity change on the main path is checked. The motivation for such efforts also stems from a need of an advanced procedure that can be applied to the case of discrete cost functions.한국과학기술원 : 산업공학과

통신망에서 정보전파에 관한 연구

학위논문(박사) - 한국과학기술원 : 경영과학과, 1990.2, [ iv, 95 p. ]This thesis is concerned with the information dissemination process in a communication network, whereby a message, originated by one vertex, is transmitted to all vertices of the network. It is assumed that a vertex can either transmit or receive a message and an informed vertex can transmit it to only one of its neighbors at a time. For the case of uniform edge transmission times, a moderate amount of work has been done to date. However, for the nonuniform case, few results have been found in the literature. The main objective of this thesis is to investigate the case of nonuniform edge transmission times. In a tree-type network, it is shown that a simple ordering policy achieves the minimum broadcast time under the minimax criterion, and based on this, an O(NlogN) algorithm is derived to determine the broadcast center. Two extended models are considered. One is the model with an ACK scheme and the other is the model with due dates. Broadcasting under the minimum criterion is also considered. The necessary and sufficient conditions is established for the optimal call sequencing in a tree. It is shown that the optimal call sequence is determined by edge transmission times and the sizes of the corresponding subtrees. An O (NlogN) algorithm is designed to determine the broadcast center in a tree under the criterion. In a general-type network, some heuristics are designed based on the results of the tree-type network and vertex degrees. Through the computer simulation, their performances are evaluated. It is observed that in a sparse that in a sparse network, it is good to transmit a message first to the vertex with the largest number of uniformed neighbors. But it is also observed that in a dense network, one must take account of the eccentricity in determining call sequences.한국과학기술원 : 경영과학과