SULF1

本文为转化医学网原创，转载请注明出处 作者：Jerry 导读： 肿瘤异质性及其驱动因素影响肿瘤进展和癌症治疗。单细胞RNA测序用于研究肿瘤生态系统的异质性。 近日，浙江大学郭国骥、梁廷波等研究人员在权威期刊《Advanced Science》上发表了题为“Pan-Cancer Single-Nucleus Total RNA Sequencing Using snHH-Seq”的研究论文，本研究中， 研究人员开发了一种高通量、高灵敏度的方法snHH-seq，该方法将随机引物与液滴微流控平台的预索引策略相结合。 这种创新的方法允许从临床冷冻样品中检测单个细胞核中的总RNA。并且建立了一个强大的流水线，以促进全长RNA-seq数据的分析。snHH-seq应用于来自32例不同肿瘤类型患者的73万多个单核。泛癌症研究使其能够全面分析肿瘤转录组数据，包括表达水平、突变、剪接模式、克隆动态等。 https://onlinelibrary-wiley-com.libproxy1.nus.edu.sg/doi/10.1002/advs.202304755 研究背景 01 癌细胞及其驱动因素的异质性在肿瘤发生和恶性进展中起着重要作用，并对癌症治疗有重要影响。单细胞RNA测序(scRNA-seq)能够在单细胞水平上测量转录信息，从而准确地解决肿瘤异质性。 许多研究利用scRNA-seq来阐明肿瘤微环境的多样性，揭示治疗策略的机制，并据此提出新的分子标记和治疗靶点。 然而，scRNA-seq在处理临床肿瘤样本，特别是档案材料(如冷冻组织)方面具有独特的后勤和技术挑战。首先，临床肿瘤样本的scRNA-seq需要实施快速组织解离程序，目前大多数医院的常规病理实验室不存在这种程序。其次，大多数scRNA-seq方法使用oligo-dT引物来捕获和扩增聚腺苷化转录本，这一过程高度依赖于组织样品的质量。此外，寡聚dt捕获导致缺乏对许多生物过程很重要的非聚腺苷化转录本。第三，高通量scRNA-seq方法只能检测转录本3 '或5 '端的短片段，这限制了临床样本的突变和剪接分析，特别是对肿瘤。 为了克服这些挑战，研究人员开发了“高通量高灵敏度单核总RNA测序”(snHH-seq)，将随机引物和预索引策略结合在液滴微流控平台上。单核RNA-seq (snRNA-seq)减少了对样品收集和处理的要求。这也为纵向样本的分析铺平了道路。使用随机引物而不是oligo-dT引物捕获转录本，不仅可以拯救部分降解转录本的档案临床样本，而且具有更高的灵敏度(一个转录本有多个可捕获的位点)和更高的覆盖率(转录本的3 '和5 '端，非聚腺苷化转录本和聚腺苷化转录本)。该预索引策略应用于基于液滴和基于平板的方法，提供至少一个数量级的吞吐量增益，并促进临床样品的多路分析。 研究进展 02 为了能够评估大量临床冷冻样本，减少解离的批次，研究人员建立了snHH-seq，一个基于液滴的高通量、高灵敏度的单核总RNA测序平台。snHH-seq是一种结合随机RT引物和预索引策略优势的强大方法。 snHH-seq具有高通量和高灵敏度，为肿瘤图谱绘制和其他基因组研究提供了新的可能性。 接下来，研究人员使用snHH-seq分析冷冻临床肿瘤样本。研究人员分析了32例患者的735 722个细胞核，涵盖了中国发病率和死亡率最高的肿瘤类型，包括肺腺癌(LUAD)、肝细胞癌(HCC)、肝内胆管癌(ICC)、胶质瘤、结肠腺癌(COAD)、直肠腺癌(READ)、乳腺浸润性癌(BRCA)、食管癌(ESCA)和胃腺癌(STAD)。在单细胞癌症研究中，识别恶性细胞是至关重要的一步，因为它们构成了癌症样本的主要组成部分。为了在研究人员的数据集中识别细胞类型(恶性细胞和非恶性细胞)，研究人员使用了标记基因和推断拷贝数变异(CNV)的组合。考虑到所研究的癌症类型，研究人员使用带注释的非上皮细胞，如内皮细胞、基质细胞和巨噬细胞作为intercnv的参考，以推断每个患者的CNV特征。 为了进一步说明TME(肿瘤微环境)的异质性，研究人员收集了来自不同患者的内皮细胞、骨髓细胞和基质细胞，并进行了亚聚类分析。研究人员获得了15个肿瘤内皮细胞(TEC)亚簇，包括动脉TEC (C4、FBLN5和SULF1)、静脉TEC (C6、ACKR1)、淋巴TEC (C2、PROX1)、毛细血管TEC (C7、BTNL9和CD36)、外周血TEC (C9、PDGFRB)、尖端TEC (C3、COL4A1)、细胞周期TEC (C12、C2orf48和CIT)和免疫样TEC (C10、DOCK2和PTPRC)。Tip TEC (C3)和cell cycle TEC (C12)在多个肿瘤中富集。在泛癌髓系细胞中，研究人员观察到肿瘤和NAT之间共享和特异性的基因特征。此外，研究人员在乳腺肿瘤和NAT样本中观察到显著的异质性。C2和C5主要驻留在乳腺肿瘤中，表达抑癌基因，可能调控乳腺TME中硫酸肝素的表达;C13主要存在于乳腺NAT和高表达的LRP1B中，LRP1B是一种可能抑制肿瘤迁移和侵袭的肿瘤抑制因子。表达C8的TNXB主要存在于NAT中，对多种肿瘤有贡献，可能阻碍肿瘤细胞的侵袭和转移。 总的来说，研究人员对细胞区室的泛癌症分析揭示了肿瘤环境的共享和癌症限制特征。 使用snHH-seq进行泛癌分析 研究结论 03 总之，研究人员证明了高分辨率单细胞全长转录组测序在鉴定新的肿瘤生物标志物方面的价值，并为全长单细胞测序提供了详细的分析。研究人员的综合分析方法包括检查基因表达、体细胞突变、剪接模式和克隆行为。通过利用这些不同的观点，研究人员可以确定与癌症相关基因和细胞类型相关的特定变异。这个项目代表了研究人员理解癌症潜在遗传机制的能力的重大进步。最终，这些发现加深了研究人员对肿瘤生物学的理解，并为更有效的诊断和治疗方法铺平了道路。 参考资料： https://onlinelibrary-wiley-com.libproxy1.nus.edu.sg/doi/10.1002/advs.202304755 注：本文旨在介绍医学研究进展，不能作为治疗方案参考。如需获得健康指导，请至正规医院就诊。 热门·直播/活动 🕓 北京｜12月19日-20日 ▶第四届单细胞测序技术应用研讨会暨单细胞&空间组学研讨会（日程稍后公布） 点击对应文字 查看详情

Scientists have identified a protein known as sulfatase-2 that plays a critical role in the damage caused by rheumatoid arthritis. The researchers' discovery sheds new light on the molecular processes that drive inflammation seen in rheumatoid arthritis. It could also someday lead to improved treatment of the disease, which currently has no cure. Scientists have identified a protein known as sulfatase-2 that plays a critical role in the damage caused by rheumatoid arthritis. A chronic disease in which the immune system attacks the body's own joint tissues, rheumatoid arthritis affects an estimated 1.5 million Americans. Published in the journal Cellular & Molecular Immunology, the discovery sheds new light on the molecular processes that drive inflammation seen in rheumatoid arthritis. It could also someday lead to improved treatment of the disease, which currently has no cure. "Tumor necrosis factor-alpha -- or TNF-alpha for short -- is one of the main inflammatory proteins that drive rheumatoid arthritis and is targeted by many currently available therapies," said senior author Salah-Uddin Ahmed, a professor in Washington State University's College of Pharmacy and Pharmaceutical Sciences. "However, over time patients can develop a resistance to these drugs, meaning they no longer work for them. That is why we were looking for previously undiscovered drug targets in TNF-alpha signaling, so basically proteins that it interacts with that may play a role." Though sulfatases such as sulfatase-2 have been extensively studied for their roles in different types of cancer, Ahmed said no one had looked at how they might be involved in inflammatory or autoimmune diseases such as rheumatoid arthritis. The research team first explored this idea using cells called synovial fibroblasts, which line the joints and keep them lubricated to ensure fluid movement. "In rheumatoid arthritis, these normally quiescent cells get activated by TNF-alpha and other inflammatory molecules, and they take on this aggressive character," said first author Ruby J. Siegel, a PhD graduate in the WSU College of Pharmacy and Pharmaceutical Sciences. "They are not dying when they should, and they proliferate in a way that is almost tumor-like, forming this massive synovial tissue that should not be anywhere near that size and at the same time activating proteins that destroy cartilage and bone." Using the joint-lining cells of rheumatoid arthritis patients, they removed sulfatase-2 from one group of cells before stimulating all cells with the inflammatory TNF-alpha. What they found was that cells lacking sulfatase-2 did not show the same exaggerated inflammatory response to TNF-alpha as cells that were left intact. "Looking at sulfatases for their potential role in inflammation was an educated guess, but once we did we saw a very consistent pattern of increased sulfatase-2 expression throughout different tissues and samples we studied," Ahmed said. "This tells us that TNF-alpha relies on sulfatase-2 to drive inflammation, because as soon as we removed sulfatase-2 the inflammatory effects of TNF-alpha were markedly reduced." Resulting from a series of experiments spanning four years, the researchers' findings open the door to future animal studies to test the effectiveness of inhibiting sulfatase-2 to ease rheumatoid arthritis symptoms. This could someday lead to the development of new combination therapies that along with other inflammatory proteins would also target sulfatase-2 to prevent bone loss, cartilage damage and deformed joints. Such therapies could help address the shortcomings of currently available rheumatoid arthritis drugs, many of which come with significant side effects. "These drugs shut off TNF-alpha in your whole body, but it does have important immune functions," Siegel said, adding that patients who take these types of drugs are more susceptible to infection and have an increased risk of developing cancer with long-term use. She also noted that TNF-alpha inhibitors are not effective in all people and are not recommended for patients with certain other health conditions. Funding support for the study came from the National Institutes of Health and the Rheumatology Research Foundation.