内因性tenascin-Cはグリオブラストーマ浸潤を促進し、周囲正常脳組織に反応性変化を惹起する

京都大学0048新制・課程博士博士(医学)甲第15268号医博第3468号新制||医||981(附属図書館)27746京都大学大学院医学研究科医学専攻(主査)教授 笹井 芳樹, 教授 稲垣 暢也, 教授 武藤 誠学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA

ERK Activity Imaging During Migration of Living Cells In Vitro and In Vivo

Extracellular signal-regulated kinase (ERK) is a major downstream factor of the EGFR-RAS-RAF signalling pathway, and thus the role of ERK in cell growth has been widely examined. The development of biosensors based on fluorescent proteins has enabled us to measure ERK activities in living cells, both after growth factor stimulation and in its absence. Long-term imaging unexpectedly revealed the oscillative activation of ERK in an epithelial sheet or a cyst in vitro. Studies using transgenic mice expressing the ERK biosensor have revealed inhomogeneous ERK activities among various cell species. In vivo Förster (or fluorescence) resonance energy transfer (FRET) imaging shed light on a novel role of ERK in cell migration. Neutrophils and epithelial cells in various organs such as intestine, skin, lung and bladder showed spatio-temporally different cell dynamics and ERK activities. Experiments using inhibitors confirmed that ERK activities are required for various pathological responses, including epithelial repair after injuries, inflammation, and niche formation of cancer metastasis. In conclusion, biosensors for ERK will be powerful and valuable tools to investigate the roles of ERK in situ

脳転移肺がん細胞の薬剤応答と初期耐性のキネティクス解析に基づく新規治療法の開発

金沢大学がん進展制御研究所 / 金沢医科大学肺がん脳転移マウスモデルの病理組織学的解析により、EGFR阻害剤に対して初期耐性を示すがん細胞は脳内微小環境によって一時的に守られていることが明らかとなった。この初期耐性に関わるシグナル伝達経路の同定を目的として1細胞遺伝子発現解析を行い、ある特定のサイトカインシグナルが初期薬剤耐性に関与していることを突き止めた。またEGFR阻害剤に対する初期薬剤耐性微小環境の分子基盤解明を目的として、がん細胞と初代培養グリア細胞の新規in vitro共培養系を確立した。この共培養系を用いた薬剤スクリーニングにより、脳微小環境のがん促進性・抑制性を規定するシグナル伝達経路の同定に成功した。Epidermal growth factor receptor (EGFR) inhibitors show certain effects in the treatment of lung cancer brain metastasis with EGFR mutation, however, the problem of drug resistance still remains to be solved. PC9-BrM3 human lung cancer cells form aggressive brain metastasis within one month after intra-cardiac injection in nude mice. The brain metastases respond to gefitinib, an EGFR inhibitor, very well and the drug induces quiescent phenotype in the surviving cells. Intriguingly, we find that the survived cells have not acquired any intrinsic resistance but seems to be temporally tolerating the drug in the brain microenvironment. Single cell transcriptome analysis reveals cytokine signaling as an alternative pathway for the surviving cells to tolerate the drug. We established a novel in vitro co-culture system of cancer cells and primary glial cells and now investigating the mutual interactions between these cell types, which can cause the initial resistance to EGFR inhibitors.研究課題/領域番号:17K07181, 研究期間(年度):2017-04-01 - 2020-03-31出典：「脳転移肺がん細胞の薬剤応答と初期耐性のキネティクス解析に基づく新規治療法の開発」研究成果報告書　課題番号17K07181 (KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-17K07181/17K07181seika/)を加工して作

Extrinsic factors can mediate resistance to BRAF inhibition in central nervous system melanoma metastases

Here, we retrospectively review imaging of 68 consecutive unselected patients with BRAF V600-mutant metastatic melanoma for organ-specific response and progression on vemurafenib. Complete or partial responses were less often seen in the central nervous system (CNS) (36%) and bone (16%) compared to lung (89%), subcutaneous (83%), spleen (71%), liver (85%) and lymph nodes/soft tissue (83%), P < 0.001. CNS was also the most common site of progression. Based on this, we tested in vitro the efficacy of the BRAF inhibitors PLX4720 and dabrafenib in the presence of cerebrospinal fluid (CSF). Exogenous CSF dramatically reduced cell death in response to both BRAF inhibitors. Effective cell killing was restored by co-administration of a PI-3 kinase inhibitor. We conclude that the efficacy of vemurafenib is variable in different organs with CNS being particularly prone to resistance. Extrinsic factors, such as ERK- and PI3K-activating factors in CSF, may mediate BRAF inhibitor resistance in the CNS

Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin β1/FAK signaling.

SummaryIntravital imaging of BRAF-mutant melanoma cells containing an ERK/MAPK biosensor reveals how the tumor microenvironment affects response to BRAF inhibition by PLX4720. Initially, melanoma cells respond to PLX4720, but rapid reactivation of ERK/MAPK is observed in areas of high stromal density. This is linked to “paradoxical” activation of melanoma-associated fibroblasts by PLX4720 and the promotion of matrix production and remodeling leading to elevated integrin β1/FAK/Src signaling in melanoma cells. Fibronectin-rich matrices with 3–12 kPa elastic modulus are sufficient to provide PLX4720 tolerance. Co-inhibition of BRAF and FAK abolished ERK reactivation and led to more effective control of BRAF-mutant melanoma. We propose that paradoxically activated MAFs provide a “safe haven” for melanoma cells to tolerate BRAF inhibition

Cell-compatible isotonic freezing media enabled by thermo-responsive osmolyte-adsorption/exclusion polymer matrices

Abstract During the long-term storage of cells, it is necessary to inhibit ice crystal formation by adding cryoprotectants. Non-cell-permeable cryoprotectants have high osmotic pressure which dehydrates cells, indirectly suppressing intracellular ice crystal formation. However, the high osmotic pressure and dehydration often damage cells. Emerging polymer-type non-cell-permeable cryoprotectants form matrices surrounding cells. These matrices inhibit the influx of extracellular ice nuclei that trigger intracellular ice crystal formation. However, these polymer-type cryoprotectants also require high osmotic pressure to exert an effective cryoprotecting effect. In this study, we designed a poly(zwitterion) (polyZI) that forms firm matrices around cells based on their high affinity to cell membranes. The polyZI successfully cryopreserved freeze-vulnerable cells under isotonic conditions. These matrices also controlled osmotic pressure by adsorbing and desorbing NaCl depending on the temperature, which is a suitable feature for isotonic cryopreservation. Although cell proliferation was delayed by the cellular matrices, washing with a sucrose solution improved proliferation