| 研究実績の概要 |
"Purpose of the research"
The primary objective of this research is to better understand how cancer cells acquire drug resistance state, in particular through adoption of dormant and cancer stem-cell like phenotypic state. Many recent studies have demonstrated the existence and relevancy of quiescence, dormant cells in the form of ‘persister’ cells that survive therapies and responsible for relapse, sometimes decades later.
"Research implementation plan and Achievements so far"
Using pancreatic ductal adenocarcinoma (PDAC) as a model system, I am attempting to address this challenge from two angles. First, using a fluorometric stem cell activity reporter (aka ‘OSCAR’) recently published by my previous lab to demonstrate that, even under artificial in vitro culturing condition, there exists phenotypic heterogeneity in an otherwise genetically uniform cell line. Crucially, I was able to detect, a subpopulation of cells (< 1%) which display ‘dormant’ properties based in the readout of the OSCAR reporter. The abundancy of which varies between cell lines. Second, I initiated the investigation in PDAC, the proteins TFEB and TFE3, members of the same family of protein as MITF which we and other have extensively showed regulates melanoma cells plasticity and heterogeneity including the dormant and cancer stem-like state. Preliminary results so far support my proposed hypothesis of TFEB and TFE3 involvement in PDAC cells response to microenvironmental stress (nutrient limitation) commonly observed in tumour.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
Unforeseen drawback: The progress of the research is rather delay from the planned timeline for a number of reasons. The primary reason is due to mycoplasma contamination of all the cell lines. The sources of this contamination is from patient derived organoid lines developed in the lab. Inadequate cautionary steps meant these unscreened samples were kept and maintained in the share clean space which inevitably led to cross-contamination to all of the cell lines used in this study. Since all high profile journals require certificate/statement of mycoplasma contamination free and it is well known that mycoplasma contamination lead to alter cellular phenotype I had no choice but to trash all the tools/sample generated in the first 6 months and repeat the experiment in new, clean cell line.
Current Status: I have generated resources (inducible knockdown and knock-out cell lines) for further studies on TFEB/TFE3 roles in PDAC adaptive response to microenvironmental stress (nutrient and oxygen limitation) and in the process of generating inducible constitutively active TFEB/TFE3 in a number of PDAC cell lines which I plan to use, initially for in vitro phenotypic characterisation (e.g. drug sensitivity and proliferation) and molecular characterisation (RNA-seq, part of the samples submitted, funded by an external grant) and potentially ChIP-seq (fund to be sourced). A number of TFEB/3 knock-out cell lines have also been engineered to contain luciferase reporter for future in vivo studies to assess their roles in PDAC progression (growth, number of metastases and persister cells).
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| 今後の研究の推進方策 |
The overarching aim of this study is to characterise dormant and cancer-stem like population with the hope of identifying new cell surface biomarker of dormancy in PDAC which can be used to isolate dormant cells for identification of novel therapeutic opportunities as well as assess their prognostic value for identification of patient with high risk of relapse for routine check-up.
Having shown that different cell lines demonstrated different abundancy of dormant population (as conferred from the OSCAR reporter), the short term future work is to assess whether this population can repopulate the initial heterogeneity as confer from the OSCAR activity. This will be a proof of cellular plasticity. The next step is to optimise single cell isolation and single cell sequencing of different population as classified from the OSCAR reporter. Subsequent bioinformatics will aim to shortlist potential cell surface biomarkers that can segregate these distinct population.
We and others have demonstrated the use of cell lines panel covering distinct phenotypic states (e.g. epithelial vs mesenchymal) as ‘simplified’ model to investigate how distinct subpopulation in the heterogeneous tumour may responses differently to microenvironmental stress as well as potential population specific TFEB/TFE3 contribution towards cellular response to microenvironmental stress such as nutrient deprivation. I will expand this initial studies to more cell lines with different phenotypes. If clear different is observed, comprehensive transcriptomics will be carried out on these different cell lines.
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