Annual Report 2020
Division of Cancer Differentiation
Koji Okamoto, Daisuke Shiokawa, Yuuki Obata, Hirokazu Ohata, Hiroaki Sakai, Yutaro Mori, Yusuke Kanda, Seiko Ogawa, Ayumi Yumura, Kohei Yamawaki, Mariko Niwa, Saki Kato
Introduction
Clinical cancer shows wide intratumoral heterogeneity. The refractory traits of cancer, such as chemoresistance and the ability to metastasize, have been profoundly associated with the heterogeneity of cancer, in which cancer cell subpopulations survive chemotherapty and lead to fatal outcomes. While genetic and epigenetic alterations contribute to cancer heterogeneity, our lab has focused on cancer heterogeneity associated with epigenetic diversity. In order to understand molecular basis of cancer heterogeneity, we developed the in vitro spheroid culture system that employs clinical specimens of several solid tumors including colon, ovarian, and lung tumors. We subsequently the established spheroid cells to generate mouse tumor xenografts, which were then applied to evaluate cancer heterogeneity. By performing single-cell gene expression analyses of the xenograft models or clinical specimens, we stratify cancer and non-cancer cells into distinct subgroups and establish a clear picture of cancer heterogeneity. Using the same specimens, we perform spatial transcriptomics and demonstrate the spatial organization of tumors at near-single-cell levels. By combining the single-cell analyses and spatial transcriptomics, we are in the process of obtaining detailed information on the spatial organization of chemoresistant areas in refractory cancer. In addition, we use the established spheroids and xenograft tumors for drug-sensitivity experiments to understand the mechanism of chemoresistance.
The Team and What We Do
We mainly conduct basic research to understand the refractory nature of cancer and to devise effective diagnoses and therapies. We specialize in the 3D culture of clinical specimens and its PDX models and mainly perform single-cell analyses and spatial transcriptomics to investigate the cellular heterogeneity of cancer.
Research activities
Understanding of cancer heterogeneity and chemoresistance via single-cell analyses of xenografted colon tumors
We have performed single-cell gene expression analyses of xenografted tumors derived from human colon cancer spheroids. Stratification of cancer cells in the xenografted tumors has revealed that the formed tumors harbored slow-cycling and fast-cycling populations of Lgr5-positive cancer stem-like cells. We have demonstrated that the identified slow-cycling CSC-like cells are responsible for chemoresistance and specifically expressed the signature genes including the PROX1 transcription factor. The identified slow-cycling CSC-like cells will be a promising target for novel cancer chemotherapy in combination with conventional chemotherapy agents.
Understanding of cancer heterogeneity and chemoresistance via single-cell nuclear analyses of clinical specimens of clear cell carcinoma
Ovarian clear cell carcinoma is regarded as the deadliest type of ovarian cancer. In order to identify cell populations associated with the chemoresistance of clear cell carcinoma, we have performed single-cell nuclear RNA-seq analyses of frozen specimens from resistant and sensitive cases of this cancer and stratified them into distinct cell populations. Through comparison of resistant and sensitive cases, we have identified a cell population associated with chemoresistance. Examination of genes specifically expressed in each population has revealed that the chemoresistant population expressed high levels of genes associated with the extracellular matrix. We are in the process of identifying crucial genes that can serve as therapeutic targets.
Elucidation of the spatial architecture of chemoresistant regions in cancer via spatial transcriptomics
We have examined the spatial architecture of the gene expression of cancer tissues by performing spatial transcriptomic analyses (Visium, 10x Genomics). We used the same cancer specimens analysed by single-cell gene expression analyses (colon cancer, ovarian cancer) and stratified the cancer tissues into distinct locations based on the gene expression profiles. We subsequently combined the data from the spatial transcriptomics with single-cell analyses, therby identifying the regions associated with cancer chemoresistance. We thereby identified the chemoresistant regions in cancer tissues. This information will aid understanding cellular networks responsible for cancer chemoresistance.
Mislocalization of cancer-causing mutant molecules and organelle signaling
Using immunofluorescence assay, we found that cancer-causing mutants, such as EGFR
(⊿ex19), KIT(D816V), and K-RAS(G13D), mislocalize to endosomes, lysosomes, and Golgi apparatus. We have previously reported that a mutant KIT in leukemia is auto-phosphorylated preferentially on the Golgi apparatus, where it can activate downstream molecules. Furthermore, we have recently showed that FLT3 mutants in acute myelogenous leukemia also transduce growth signals during its biosynthetic trafficking before reaching the plasma membrane. Establishment of a new strategy for the inhibition of oncogenic signals through the blocking of intracellular trafficking is currently underway.
Education
Teaching students (3 graduate students)
Future Prospects
We intend to identify the biological weaknesses of refractory cancer by single-cell analyses and to translate the acquired knowledge into clinical purposes by targeting the biological weaknesses of these cells to eradicate refractory cancer.
List of papers published in 2020
Journal
1. Yamawaki K, Shiina I, Murata T, Tateyama S, Maekawa Y, Niwa M, Shimonaka M, Okamoto K, Suzuki T, Nishida T, Abe R, Obata Y. FLT3-ITD transduces autonomous growth signals during its biosynthetic trafficking in acute myelogenous leukemia cells. BioRxiv, doi: https://doi.org/10.1101/2021.01.01.424454, 2021
2. Ueda H, Mori Y, Yamawaki K, Ishiguro T, Ohata H, Sato A, Sugino K, Yachida N, Yamaguchi M, Suda K, Tamura R, Yoshihara K, Okamoto K, Enomoto T. Establishment of in vitro 3D spheroid cell cultivation from human gynecologic cancer tissues. STAR Protoc, 2:100354, 2021
3. Murayama T, Takeuchi Y, Yamawaki K, Natsume T, Li M, Marcela RN, Nishimura T, Kogure Y, Nakata A, Tominaga K, Sasahara A, Yano M, Ishikawa S, Ohta T, Ikeda K, Horie-Inoue K, Inoue S, Seki M, Suzuki Y, Sugano S, Enomoto T, Tanabe M, Tada KI, Kanemaki MT, Okamoto K, Tojo A, Gotoh N. MCM10 compensates for Myc-induced DNA replication stress in breast cancer stem-like cells. Cancer Sci, 112:1209-1224, 2021
4. Takada K, Aizawa Y, Sano D, Okuda R, Sekine K, Ueno Y, Yamanaka S, Aoyama J, Sato K, Kuwahara T, Hatano T, Takahashi H, Arai Y, Nishimura G, Taniguchi H, Oridate N. Establishment of PDX-derived salivary adenoid cystic carcinoma cell lines using organoid culture method. Int J Cancer, 148:193-202, 2021
5. Miyagawa Y, Nagasaka K, Yamawaki K, Mori Y, Ishiguro T, Hashimoto K, Koike R, Fukui S, Sugihara T, Ichinose T, Hiraike H, Kido K, Okamoto K, Enomoto T, Ayabe T. Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-like Cells using the Three-dimensional Co-culture System, NICO-1. J Vis Exp, 2020
6. Namekawa T, Kitayama S, Ikeda K, Horie-Inoue K, Suzuki T, Okamoto K, Ichikawa T, Yano A, Kawakami S, Inoue S. HIF1α inhibitor 2-methoxyestradiol decreases NRN1 expression and represses in vivo and in vitro growth of patient-derived testicular germ cell tumor spheroids. Cancer Lett, 489:79-86, 2020
7. Shiokawa D, Sakai H, Ohata H, Miyazaki T, Kanda Y, Sekine S, Narushima D, Hosokawa M, Kato M, Suzuki Y, Takeyama H, Kambara H, Nakagama H, Okamoto K. Slow-Cycling Cancer Stem Cells Regulate Progression and Chemoresistance in Colon Cancer. Cancer Res, 80:4451-4464, 2020
8. Sekine K, Ogawa S, Tsuzuki S, Kobayashi T, Ikeda K, Nakanishi N, Takeuchi K, Kanai E, Otake Y, Okamoto S, Kobayashi T, Takebe T, Taniguchi H. Generation of human induced pluripotent stem cell-derived liver buds with chemically defined and animal origin-free media. Sci Rep, 10:17937, 2020
9. Sekine K, Tsuzuki S, Yasui R, Kobayashi T, Ikeda K, Hamada Y, Kanai E, Camp JG, Treutlein B, Ueno Y, Okamoto S, Taniguchi H. Robust detection of undifferentiated iPSC among differentiated cells. Sci Rep, 10:10293, 2020