2019 Fiscal Year Annual Research Report
The mechanism of CML leukemia progenitor cells eradication uncovered by Sipa1 deficiency
| Project/Area Number |
18F18406
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| Research Institution | Kyoto University |
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Principal Investigator |
湊 長博 京都大学, 医学研究科, 理事 (40137716)
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| Co-Investigator(Kenkyū-buntansha) |
XU YAN 京都大学, 医学(系)研究科(研究院), 外国人特別研究員
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| Project Period (FY) |
2018-10-12 – 2021-03-31
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| Keywords | scRNA sequencing / MSCs / Aged |
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| Outline of Annual Research Achievements |
I confirmed that Sipa1-deficient mice showed increased resistance to other cancer cells such as MC38 intestinal and IPmN pancreatic cancers in addition to chronic myelogenous leukemia (CML) stem cells. In order to investigate the involvement of the tissue stromal response in the anti-tumor mechanism, single cell RNA sequencing (scRNA-seq) analysis was performed. The analysis revealed that tumor tissue mesenchymal stromal cells (MSCs) consisted of 3 major subtypes, FA, FB, and FC, with distinct gene expression profiles. I was also able to identify the MSC subtypes in tumor tissue quantitatively with FACS analysis using specific cell surface markers. With the analysis, I found that MSCs in the tumor tissue of Sipa1-/- hosts showed preferential increase in FC and FB subtypes, which bore immunoregulatory and extracellular matrix (ECM)-regulatory functions, respectively, as compared to those of wild type (WT) hosts. I further found that the tumor MSCs of Sipa1-/- hosts exhibited enhanced expression of T-cell chemokine gene compared to those of WT hosts. These results have suggested that host Sipa1-deficiency causes altered pattern of MSC development and their gene expression profile in tumor tissues, leading to the enhanced local T-cell immunity against tumor cells.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
1. In WT and Sipa1-/- mice, I proceeded with the investigation of the MSCs and T cells in tumor tissues using various epithelial cancer cell models such as intestinal (MC38) and pancreatic (IPmN) cancers, in addition to CML model.
2. I performed scRNA-seq analysis of MSCs in tumor tissues and was able to identify 3 distinct MSC subtypes, termed FA, FB, and FC. FA cells were characterized by the expression of genes related to cell contraction and angiogenesis, reminiscent of myofibroblasts. FB cells expressed genes related to the regulation of lymphocyte dynamics and function and resembled reticular fibroblasts seen in lymph nodes. FC cells expressed genes involved in controlling the function of ECM such as various proteoglycans.
3. With the use of specific cell markers, I was able to identify the 3 MSC subtypes in the tumor tissue quantitatively by FACS analysis.
4. Using the FACS analysis, I comparatively investigated the patterns of MSC subtypes in tumor tissue of WT and Sipa1-/- hosts. I found that MSCs in Sipa1-/- host showed rather preferential increase in the numbers of FB and FC cells compared to those in WT host. I also found that the Sipa1-/- MSCs exhibited enhanced expression of selective genes including T-cell chemokine genes such as Cxcl9 and Cxcl10 compared to WT MSCs. These changes of MSC in Sipa1-/- host were associated with the significant increase in the numbers of T cells infiltrated in the tumor tissue.
5. The extents of MSC reaction varied remarkably, but the development of 3 MSC subtypes was similarly observed in various tumor models such as MC38 and IPmN.
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| Strategy for Future Research Activity |
1. In addition to FACS analysis, I will perform tissue immunostaining analysis to identify the three MSCs subtypes in situ in various tumor tissues, to verify their topological relation to tumor cells and infiltrated T cells in WT and Sipa1-/- hosts.
2. I will clarify the signaling pathways involved in the differentiation and proliferation of MSCs subtypes in tumor tissues and roles of Sipa1 expression in them. To this end, I will establish an in vitro experimental system to recapitulate the MSCs subtype differentiation/proliferation in response to tumor cells with the use of WT and Sipa1-/- MSCs, such as mouse embryonic fibroblasts (MEFs), mouse adult fibroblasts (MAFs) and MSC line (OP9). I will focus on the expression of MSCs subtype-specific markers and T-cell chemokine genes.
3. I have found that the resistance of Sipa1-/- mice to CML leukemia was remarkably aging sensitive, and aged Sipa1-/- mice allow the rapid expansion of CML unlike young Sipa1-/- mice. My preliminary experiments indicate that Sipa1-/- MSCs in tumor tissue show a marked decrease of T-cell chemokine gene expression. I intend to investigate the changes in the function of tumor MSCs with age.
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