Synergism Induced by Combination of Farnesyl Transferase Inhibitor SCH66336 and Insulin Like-Growth Factor Binding Protein-3 in apoptosis of Non-Small Cell Lung Cancer Cell Lines

Background : Insulin-like growth factor binding protein (IGFBP)-3 regulates non-small cell lung cancer(NSCLC) cell proliferation in vitro and in vivo by inhibiting IGF-mediated signaling pathways. To have better strategies for the treatment of lung cancer, we analyzed the combining effects of adenovirus expressing IGFBP-3 (Ad5CMV-BP3) and SCH66336, a farnesyl transferase inhibitor (FTI) designed to block Ras-mediated proliferative signaling pathways. Methods : To measure the combining effects of Ad5CMV-BP3 and SCH66336 on the proliferation of NSCLC cells,human NSCLC cell lines (H1299, H596, A549, H460, and H358), SCH66336, recombinant adenovirus expressing IGFBP-3 (Ad5CMV-BP3) and athymic nude mice were used in these experiments. Results : The combination of Ad5CMV-BP3 and SCH66336 produced a synergistic enhancement in antiproliferative effects over a range of clinically achievable concentrations in a variety of NSCLC cell lines. Furthermore, we observed a significant reduction in growth of NSCLC xenograft induced in athymic nude mice. Conclusion : In conclusion, this study demonstrated for the first time that the FTI SCH66336 synergizes with IGFBP-3 and enhances its apoptotic activity in NSCLC cells in vitro and in vivo. The combined treatment of Ad5CMV-BP3 and SCH66336 raises the possibility of using this regimen in clinic for the treatment of NSCLC.ope

환자맞춤형 치료를 위한 체외 항암제 스크리닝용 바이오칩

학위논문 (박사) -- 서울대학교 대학원 : 공과대학 전기·정보공학부, 2020. 8. 권성훈.정밀의학(Precision Medicine) 혹은 개인맞춤의학(Personalized Medicine)은 개개인의 최적화된 치료방법을 결정하는 것을 목표로 하는 의학의 패러다임이다. 특히, 임상종양학에서는 차세대염기서열분석(NGS), 전사체서열분석, 그리고 질량분석법들을 통한 환자의 분자 프로파일(molecular profile) 방법이 발전해오고 있으며, 이를 바탕으로 환자를 세분화하여 맞춤형 치료를 구현하려고 노력해오고 있다. 하지만, 여전히 현 수준에서 이해되지 못하는 수준의 종양 이질성(tumor heterogeneity)과 오랜 처방기록을 가진 환자군들의 항암제 획득내성(acquired resistance) 등의 원인으로 맞춤형 환자 처방은 쉽지 않은 경우가 많다. 이러한 경우 환자로부터 얻어진 암세포, 조직으로부터 얻어진 일차세포 혹은 체외 배양된 세포, 스페로이드, 장기유사체 등을 이용하여 고속다중약물스크리닝기술을 통한 맞춤형 항암제를 선별해내는 체외 약물진단 기술을 생각해낼 수 있는데, 이는 기존의 유전체 기반의 시도와 병행되어 개개의 환자들에게 더욱 적합한 치료방법을 찾는 것이 가능하게 한다. 하지만 이러한 목적의 고속다중약물스크리닝기술은 높은 활용가능성에도 불구하고, 광범위한 보급과 활용이 되기에는 제약점이 많았다. 기존의 고속다중약물스크리닝기술은 많은 양의 샘플이 소모되고, 값비싼 시약의 소모량도 적지 않았다. 게다가, 수천 가지 이상의 서로 다른 물질들을 탐색하기 위해 반드시 필요한 고가의 자동화된 액체 운반기(liquid handler) 등이 필요하였는데, 이러한 문제로 대형 제약사, 연구소 등을 제외하고는 도입이 쉽지가 않아 기술접근성이 제한되어 있었다. 본 연구에서는 반도체공정에서의 노광기술을 이용하여 개개의 식별할 수 있는 코드를 가지고 있는 코드화된 하이드로젤 기반의 광경화성폴리머 미세입자를 만들어, 이를 원하는 암세포에 약물 스크리닝을 해보고자 하는 다양한 약물라이브러리를 이용 각각의 코드화된 미세입자에 흡수시켜 약물-미세입자 라이브러리를 제작한다. 그후, 값비싼 어레이 제작용 스포터 혹은 디스펜서 장비없이 간단한 자기조립을 통해 대규모의 다양한 약물-하이드로젤 어레이를 제작할 수 있는 기술을 개발하였다. 또한, 소량의 세포들 만으로도 미세우물(microwell) 기반의 세포칩에 도포하는 방식을 개발하였으며, 이를통해 약물-하이드로젤 어레이와 미세우물기반의 세포칩의 결합으로 수백-수천의 다양한 어세이를 적은 수의 샘플만으로도 한번에 수행할 수 있는 고속다중약물스크리닝 기술을 수행할 수 있게 만들었다. 본 연구에서 제시한 소형화된 체외 항암제 스크리닝용 약물플랫폼은 다음과 같은 의의를 가진다. 적은 수의 환자세포 혹은 샘플의 양에 적용할 수 있는, 사용하기 손쉬운 기술로서, 기존의 값비싼 장비, 시약의 사용량을 획기적으로 줄일 수 있는 기술이다. 본 연구에서 제안된 기술을 통해 기존의 장비를 사용할 때 시약의 값이 비싸거나, 장비의 가격이 비싸서, 혹은 다루고자 하는 샘플의 양이 제한적이어서 기존에 접근하기 힘들었던 다양한 학술연구에 적용할 수 있으며, 병원에서의 임상연구 및 실제 환자맞춤형 치료에 사용 될 수 있는 접근성을 획기적으로 높일 수 있다. 특히, 비교적 중,소 규모의 연구환경에서도 다양한 희귀한 환자유래세포 혹은 환자유래오가노이드 등과 접목하여 사용된다면 본 플랫폼의 가능성을 더욱 극대화 할 수 있을 것으로 기대한다.Precision or Personalized Medicine is a medical paradigm aimed to determine optimal therapy for individual patient. In particular, clinical oncology has been using methods of molecular profiling for each patient through next-generation sequencing (NGS), mRNA-sequencing, and mass spectrometry, and has been trying to implement personalized treatment. However, personalized treatment based on molecular profiling to each patient is not always possible due to the high level of heterogeneity of tumor that is still not fully understood at the current level and acquired resistance of anti-cancer drug due to cumulative targeted therapy. In such cases, in vitro drug testing platform using primary cells obtained from patients, or patient-derived cells, spheroids, and organoids can make it possible to find a more appropriate treatment for each individual patient. However, though high-throughput drug screening technology for this purpose is of the utmost importance in saving lives, there were many limitations to its wide use in many hospitals. The existing high-throughput drug combination screening technology consumes a large number of samples and consumes a considerable amount of expensive reagents. In addition, expensive automated liquid handlers, which were essential for exploring thousands of different pipetting, were not easy to introduce except for large-sized pharmaceutical companies and research institutes, which limited access to technology. In this study, I construct a heterogeneous drug-loaded microparticle library by fabricating encoded photocurable polymer particle that has individually identifiable codes to track loaded drug. and I load various drug molecules, which I want to test to target cells, into each coded microparticle. Then, I developed to produce heterogeneous drug-laden microparticle arrays through simple self-assembly without the need for a microarray spotter or dispensing machine for generating microarray. I also have developed cell seeding method of seeding small-volume samples into the microwell-based cell chip. By utilizing the drug-laden microparticle hydrogel array and microwell-based cell chip technology, hundreds to thousands of different assays can be done at once with just a small number of samples and low cost. Through the implemented platform, the anti-cancer drug sequential combination screening was conducted on the triple-negative breast cooler (TNBC) cells, which are generally known to be difficult to treat due to lack of known drug target, and the results of screening were analyzed by establishing a library of drugs in the EGFR inhibitory type and drugs in the genotoxin type. In addition, another study was conducted to find optimal drug combinations using patient-derived cells derived from tumors in patients with non-small cell lung cancer that have obtained acquired resistance. Finally, as the growing need for three-dimensional culture, such as spheroid and organoid for having a similar response to in vivo drug testing, it was also developed that microwell-based cell chip that is capable of 3D culture with low-cost and small-volume of cells. The miniaturized in vitro anticancer drug screening platform presented in this study has the following significance. An easy-to-use technique that can be applied to a small number of patient cells or samples, which can dramatically reduce the use of conventional expensive equipment, reagents. The proposed technology in this study can be applied to a variety of academic studies previously inaccessible to high-throughput screening due to the high cost of reagents, the high price of equipment, or the limited amount of samples in conventional drug screening. and this platform can also dramatically increase access to clinical research in hospitals for personalized treatments. In particular, it is expected that the possibility of this platform will be further maximized if it is used in a relatively small and medium-sized research environment by the combined use of various rare samples such as patient-derived cells or patient-derived organoids.Chapter 1 Introduction １ 1.1 Motivation of this research ２ 1.2 Competing technologies and Previous works ８ 1.3 Main Concept: In vitro drug testing using miniaturized encoded drug-laden hydrogel array technology １５ Chapter 2 Platform Development of Drug Releasing Hydrogel Microarray ２０ 2.1 Encoded Drug-Laden Hydrogel & Library construction ２１ 2.2 Array generation of heterogenous drug-laden microparticles. ３４ 2.3 Cell Culturing on Cell Chip and bioassay ３６ Chapter 3 Sequential Drug Combination Screening Assy on TNBC ４０ 3.1 Background : Sequential Drug Combination as promising therapeutic option ４１ 3.2 Experimental design with sequential drug treatment assay ４３ 3.3 Technical Issue & its engineering solution ４４ 3.4 Assay Result ４９ Chapter 4 Drug Combination Assay on Patient-Derived Cells ５８ 4.1 Background : Simultaneous Combination Treatment using Patient-Derived Cells ５９ 4.2 Improvement of Platform for facilitating translational study ６２ 4.3 Study Design for small-volume drug combinatorial screening with NSCLC patient derived cell ６５ 4.4 Assay Result ６９ Chapter 5 Development of platform for 3D culture model ７２ 5.1 3D culturable platform ７３ 5.2 Development of 3D culture platform based Matrigel scaffold. ７８ 5.3 Advantage over conventional 3D culture-based drug testing platform. ８５ Chapter 6 Conclusion ８７ Bibliography ９０ Abstract in Korean ９７Docto