Team:UT-Tokyo/CTCD/Content

Please imagine that you are a police officer. You are searching for dangerous criminals who hide in crowd and it is difficult to find them. This situation is the same as finding cancer cells (dangerous criminals in our body) in our blood when they resemble to normal blood cells. In this way picking up cancer cells has been developed around the world, where every year a different method is developed. Don't you think that it is like a dream if there were drugs, which if someone take, they will confess their crime? What about drugs, which if the cancer cells take, they will shine brightly on the blood? Here our team focuses on microRNA (miRNA) and provides a special genetic circuit which exposes cancer cells in blood vessels.(Fig.1)

Circulating tumor cells (CTCs) tumor cells arising from epithelia, flowing through blood vessels and causing metastasis to other tissues. Focusing on these cells, there are ways of early detection of cancer, detecting CTCs in blood of patients with early-stage cancer. The effectiveness of these methods has been ensured through the results of researches especially in breast cancer and colon cancer (ref). These methods of detection are a technology developed in recent years, compared to the traditional methods of picking up the tissue of patients which has been used for a long time. However, these traditional methods produce a hard burden to the patients, because of the several and long procedures. On the other hand, the methods of detecting CTCs produce a non-invasive option for patients compared to traditional ones. From the utility points, CTCs have been used all around the world to develop several detection methods. Most of these methods emphasized utilizing anti-EpCAM antibody, which is an adhesion protein used as marker of cancers, and distinguish CTCs from blood cells by the difference of their cell size. However, because the concentration of CTCs in blood is very low, it is required to develop a new approach for the detection of CTCs.

Here we have developed a new detection method for CTCs focusing on two types of profiles; miRNA and EpCAM. It has given the more specificity for CTCs to utilize the two profiles. miRNA is a special short non-cording RNA and interferes the translation of the complementary mRNA, interacting with some proteins and forming the RNA-induced silencing complex (RISC) in wide-species from virus to human. Focusing on different expression patterns of miRNA in each cell type, we utilized miRNA profiles with a synthetic biological approach for detecting CTCs. The circuit that we have developed has EpCAM promoter, reporter and blood cells-specific miRNA (miR-142-3p/5p) binding sites. Two conditions, which are activation of EpCAM promoter and an absence of blood cells-specific miRNA, are required for translation of the reporter. If we inject this circuit into cells in blood, only in the case of CTCs the reporter is produced.(Fig.2)

[1] Friedlander, Terence W., Gayatri Premasekharan, and Pamela L. Paris. "Looking back, to the future of circulating tumor cells." Pharmacology & therapeutics 142.3 (2014): 271-280.

EGP-2 promoter

EGP-2 promoter is the promoter of Epithelial cell adhesion molecule (EpCAM) protein.EpCAM is a trans membrane glycoprotein which is expressed in epithelial cells and shows high level expression in various type of human epithelial carcinomas[2].Using this promoter, reporter genes can be expressed only in cancer cells. EGP-2 promoter is originally about 3500bp and it is not convenient to make a DNA construct. But, in the previous research, it is known that EGP-2 promoter can work if a part of its sequence is deleted[3]. In this project ,We cloned a part of EGP-2 promoter, and made it useful as a biobrick parts.

miRNA-142

MiRNA is a class of non-coding RNA.MiRNA binds the miRNA-recognition element in the 3' untranslated region of the target gene. MiRNA shows the tissue-specific expression pattern. In our project, we use miRNA-142, which is expressed only in hematopoietic cells[4].After miRNA-142 is transcribed, it is cleaved to miRNA-142-5p and miRNA-142-3p. Adding the recognition element of miRNA-142 in the 3' untranslated region of the reporter genes, leaky expression in non-carcinoma hematopoietic cells can be reduced.

In our project, miRNA is used for degrading mRNA in cells that express miRNA. But if we transfect construct like [pCMV-LacI-miR A binding site] and [pCAG-LacO2-GFP][5], mRNA in cells that does not express miRNA is degraded. This system will makes many application of miRNA in iGEM possible.

We focused on a tissue-specific promoter and miRNA in order to detect CTCs.As well as hematopoietic cells, many other tissues shows specific miRNA expression patterns[3]. Therefore, if by making a circuit that returns an output to a certain miRNA expression pattern, we may identify where a CTC comes from.[5]

In addition to the detection of CTCs, combinations of tissue-specific promoter and miRNA can be applied to many treatments. For example, suicide genes can be expressed in cancer cells in a specific tissue.[6]

Our activities involved in iGEM were all conducted by undergaduates alone!!
Based on fund-raisings and public relations, all team members had a lot of brainstoming, investigations and discussions in order to select project carefully. And we conducted experiments and FINALLY saw results.
We also designed and composed all publish tools, and of course, polished all scripts and presentation by ourselves.

Project

All we had a lot of brainstorming, investigations and discussion when we decide our projects.
σ-ReCounter:
Shunsuke Sumi(idea)
So Nakashima(idea and comformation)
Takefumi Yoshikawa(comformation)

CTCD:
Masayuki Osawa(conformation)
Shigetaka Kobari(investigation and conformation)
Shunsuke Sumi(conformation)
Senkei Hyo(investigation)
Yoshihiko Tomofuji(conformation)
Yshiki Okesaku(investigation)

Experiment

Lab. Leader:
Takefumi Yoshikawa(construction, assay;σ-ReCounter)

Lab. Members:
Atsuki Ito(construction)
Hajime Takemura(construction)
Keisuke Tsukada(construction)
Kentaro Tara(construction)
Kento Nakamura(construction, assay;σ-ReCounter)
Naruki Yoshikawa(construction)
Nobuhiro Hiura(construction)
Shigetaka Kobari(assay;CTCD)
So Nakashima(construction, Assay;σ-ReCounter)
Yoshihiko Tomofuji(assay;CTCD)
Yuto Yamanaka(construction)

Modeling

Keisuke Tsukada, Kentaro Tara, Manabu Nishiura, Masaki Ono

Web

Almost all members wrote drafts of our team wiki.
Cristian David(check our English)
Hiroki Tsuboi(team website, implementation of our team wiki)

App

Naruki Yoshikawa

Design

Cristian David(parker design)
Yoshiki Okesaku(all design, all figure)

Presentation

Kento Nakamura, Manabu Nishiura, Masato Ishikawa, Yumeno Koga, Yuto Yamanaka

Poster

Public Relation

Ding Yuewen, Keisuke Tsukada

Adviser

Kota Tosimitsu

Promega KK.for chamical reagents

Teiyukai, Faculty of Engineering, The University of Tokyofor fund

Integrated DNA Technologies MBL

COSMO BIO Co., Ltd.for fund

Leave a Nest Co., Ltd.(Hiroyuki Takahashi)for advice for public relations & introduction of Promega KK.