RNF43 p.G659fs 通过 PI3K/AKT/mTOR 信号通路和 HLA-E 上调导致 MSI-high 结直肠癌中 NK 细胞功能障碍
RNF43 p.G659fs leads to natural killer cell dysfunction in MSI-high colorectal cancer through PI3K/AKT/mTOR signaling and HLA-E up-regulation.
CELL INTELLIGENCE · 肿瘤细胞治疗研究
肿瘤细胞治疗研究
垂体神经内分泌肿瘤免疫微环境监测的高维流式细胞术方法学
英文原题：Methodology of high-dimensional flow cytometry in monitoring immune microenvironment of pituitary neuroendocrine tumors.
查看英文原题
Methodology of high-dimensional flow cytometry in monitoring immune microenvironment of pituitary neuroendocrine tumors.
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中文摘要
垂体神经内分泌肿瘤（PitNETs）的肿瘤免疫微环境（TIME）特征对于理解不同类型PitNETs的行为以及识别其侵袭性、快速生长和治疗耐药的可能原因至关重要。高维流式细胞术（FC）是研究TIME的一项有前景的技术，但存在独特的技术挑战，尤其是应用于实体组织和PitNETs时。本文通过解决整个工作流程各阶段的方法学难题并提出解决方案，评估了FC用于分析PitNETs中TIME的潜力。
我们开发了一种从PitNET组织制备用于FC的单细胞悬液的方案。这涉及酶消化的优化，并将其与机械组织解离进行比较，评估细胞产量、活力和目标抗原表达。
我们设计了四个多色FC panel，用于分析主要淋巴细胞和髓系细胞亚群，包括确定T、B、NK细胞亚群及其活化和细胞毒性潜力、中性粒细胞、单核细胞、M2和M1亚型的CD68 + CD64 + CD11b low巨噬细胞，以及两种类型的髓系抑制细胞——PMN-MDSC和M-MDSC。讨论了多色panel设计原则、扩散误差以及电压平衡对正确设置流式细胞仪的重要性。这些panel经过验证，并证明了其在垂体肿瘤手术组织上同时使用以进行全面TIME表征的可行性。
我们比较了血液、PitNETs和三个连续PitNET洗脱液中的淋巴细胞频率，以确定PitNET样本被血液白细胞污染的程度。为解决技术挑战，我们提出了一种逻辑数据门控策略，该策略可去除聚集体、死细胞和亚细胞碎片产生的干扰分析的虚假信号。
我们的结果表明，尽管存在所有技术困难，多参数FC仍能有效表征不同类型的PitNETs。对免疫浸润的这种增强理解为了解PitNETs生物学提供了宝贵见解，并推动了临床诊断的进步。
展开英文摘要原文
Characterization of the tumor immune microenvironment (TIME) of pituitary neuroendocrine tumors (PitNETs) is crucial for understanding the behavior of different types of PitNETs and identification of possible causes of their aggressiveness, rapid growth, and resistance to therapy.
High-dimensional flow cytometry (FC) is a promising technology for studying TIME but poses unique technical challenges, especially when applied to solid tissues and PitNETs, in particular. This paper evaluates the potential of FC for analyzing TIME in PitNETs by addressing methodological difficulties across all stages of the workflow and proposing solutions.
We developed a protocol for preparing single-cell suspensions from PitNET tissues for FC. This involved optimization of enzymatic digestion and comparison of it with mechanical tissue dissociation assessing cell yield, viability, and target antigen expression.
We designed four multicolor FC panels to analyze major lymphocyte and myeloid cell subsets including determination of subpopulations of T, B, NK cells and their activation and cytotoxic potential, neutrophils, monocytes, CD68 + CD64 + CD11b low macrophages of M2 and M1 subtypes, and two types of myeloid suppressor cells - PMN-MDSC and M-MDSC.
Principles of multicolor panel design, spreading error, and importance of voltage balance for proper flow cytometer setting are discussed. The panels were validated and demonstrated the feasibility of their simultaneous use on pituitary tumor surgical tissue for comprehensive TIME characterization.
We compared lymphocyte frequencies in blood, PitNETs, and three sequential PitNET eluates to find out the contamination level of PitNET samples with blood leukocytes. To address technical challenges, we propose a strategy of logical data gating that removes spurious signals from aggregates, dead cells, and subcellular debris that can interfere with analysis.
Our results indicate that despite all technical difficulties, multiparametric FC can effectively characterize different types of PitNETs. This enhanced understanding of the immune infiltrate provides valuable insights into PitNET biology and advances clinical diagnostics.
论文信息
- 作者
- Loguinova MY、Mazeeva VV、Lisina DV、Zakharova EN、Sorokina AV、Dzhemileva LU、Grigoriev AY、Shutova AS
- 单位
- Endocrinology Research Centre, Moscow, Russia.Russia
- 期刊
- Cytometry. Part B, Clinical cytometry2025 May
- 原文标识
- PubMed 40223192 · DOI 10.1002/cyto.b.22235