SELENOP

Immunity-related microenvironment of glioblastoma.Glioblastoma (GBM) is an extremely aggressive primary brain tumor. Currently, the standard treatment involves neurosurgery, adjuvant radiotherapy, and chemotherapy using temozolomide (TMZ). Despite this rigorous regimen, the likelihood of disease recurrence remains high, and survival rates post-recurrence are dismally low. Consequently, there's a pressing demand for novel treatment tactics for GBM. Immunotherapy has marked significant milestones in multiple cancer types. Unfortunately, GBM patients' response has been limited. This limitation can be attributed to the unique immunosuppressive microenvironment of GBM, characterized by a high population of tumor-associated macrophages (TAMs). TAMs interact uniquely with T cells, influencing their activation state, functional anti-tumor effects, and fostering tumor growth for pro-tumor effects. Efforts to regulate TAMs to enhance anti-tumor immune responses have been largely unsatisfactory, possibly due to the absence of ideal TAM targets. The new target on TAMsRecently, a study published in Nature Cancer identified a molecule called Siglec-9 that acts as an immune checkpoint molecule on the surface of TAMs in GBM, significantly affecting the tumor's immune environment and immunotherapy response. Siglec-9 is a relatively new target for the pharmaceutical industry, as evidenced by the fact that there is only one small molecule drug targeting it listed in the Synapse database: In the Nature Cancer study, the research team analyzed samples from 24 glioblastoma (GBM) patients with primary, recurrent, and new adjuvant therapy with anti-PD-1 antibodies. They used single-cell RNA sequencing and spatial transcriptome analysis to find that patients who did not respond to anti-PD-1 treatment had many tumor-associated macrophages (TAMs) in the tumor with high expression of the Siglec-9 gene. Although anti-PD-1 treatment was found to change the tumor immune environment in some GBM patients, it could not change the overall immunosuppressive state of the intra-tumor environment, especially the immunosuppressive function of Siglec-9+ TAMs. The research team further identified two types of Siglec-9+ TAMs: Siglec-9+ MARCO+ TAMs and Siglec-9+ SEPP1+ TAMs. They found that these cells were differentiated from CD14+ monocytes that migrated into the tumor and differentiated in a stepwise manner. The team also discovered that knocking out SiglecE (the mouse functional homolog of human Siglec-9) resulted in a significant increase in the number of activated T cells and a decrease in tumor growth. This suggests that Siglec-9 on TAMs plays a crucial role in restricting T cell priming and promoting tumor growth. Siglec-9 acts as an immune-checkpoint molecule on macrophages in glioblastoma, restricting T-cell priming and immunotherapy response In addition, treatment with an anti-Siglec-9 antibody combined with anti-PD-1 antibody could significantly improve the response rate of GBM patients to immunotherapy, suggesting that Siglec-9 may be a potential target for improving the efficacy of immunotherapy in GBM patients. These findings provide new insights into the unique immunosuppressive microenvironment of GBM and identifies Siglec-9 as a potential target for improving the efficacy of immunotherapy in GBM patients. However, further studies are needed to confirm these findings and to evaluate the safety and efficacy of anti-Siglec-9 therapy in GBM patients. The sialic acid/Siglec-9 axisApart from TAMs, the expression of Siglec-9 on other immune cells also presents challenges. Another study, published in the journal Frontiers in Immunology, showed that sialic acid regulates immune evasion by engaging inhibitory Siglec receptors on myeloid and other immune cells. Inhibitory Siglecs have immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their intracellular domain, which become phosphorylated upon ligand binding and trigger immunosuppressive signaling. Disruption of the sialic acid/Siglec-9 axis improves antibody-mediated neutrophil cytotoxicity towards tumor cellsThe researchers found that neutrophils express Siglec-9, and that EGFR and HER2 positive breast tumor cells express ligands for Siglec-9. Treatment of tumor cells with neuraminidases or a sialyl transferase inhibitor significantly reduced binding of a soluble recombinant Siglec-9-Fc fusion protein, while EGFR and HER2 expression remained unchanged. The cytotoxic activity of neutrophils driven by therapeutic EGFR or HER2 antibodies in vitro was increased by blocking the sialic acid/Siglec interaction, either by reducing tumor cell sialylation or by a Siglec-9 blocking antibody containing an effector silenced Fc domain. In an in vivo study using a short-term xenograft mouse model, the improved therapeutic efficacy of EGFR antibodies against tumor cells depleted of sialic acid, achieved through the use of a sialyltransferase inhibitor, was confirmed. This was in comparison to the efficacy observed in untreated cells. α2,3 linked sialic acid on tumor cells can be reduced by neuraminidase treatment or by sialyltransferase inhibition.The team's findings demonstrated that sialic acid/Siglec interactions between tumor cells and myeloid cells can impair antibody-dependent tumor cell killing, and that Siglec-9 on polymorphonuclear cells (PMN) is critically involved. Considering that PMN are often a highly abundant cell population in the tumor microenvironment, Siglec-9 constitutes a promising target for myeloid checkpoint blockade to improve antibody-based tumor immunotherapy.  Similar to the situation with Siglec-9, there’s also only one entry for drug targeting sialic acid in Synapse database: The rarity of drugs targeting the newly-discovered sialic acid/Siglec-9 axis in cancer treatment may signify a new frontier with abundant opportunities opened up. The studies discussed here provide a foundation for the development of novel approaches to improve the efficacy of cancer immunotherapy.  References: 1.Mei, Y., Wang, X., Zhang, J. et al. Siglec-9 acts as an immune-checkpoint molecule on macrophages in glioblastoma, restricting T-cell priming and immunotherapy response. Nat Cancer (2023).  2.Lustig M, Chan C, Jansen JHM, et al. Disruption of the sialic acid/Siglec-9 axis improves antibody-mediated neutrophil cytotoxicity towards tumor cells. Front. Immunol. 14:1178817(2023).

胶质母细胞瘤（GBM）是最凶险的原发性脑肿瘤类型。现行的标准治疗策略是综合神经外科手术切除、辅助放疗和替莫唑胺（temozolomide，TMZ）化疗，但疾病复发的可能性极高，复发后的整体生存时间只有24至44周。针对GBM的新治疗策略迫在眉睫。近年来，尽管免疫疗法在多种癌症中取得了显著突破， 但GBM患者的响应却非常有限。在III期临床试验（CheckMate-143）中，肿瘤无法手术切除的GBM患者使用纳武单抗（Nivolumab，抗PD-1）单药治疗后的总生存期并未比使用贝伐单抗（Bevacizumab，抗VEGF）单药治疗的患者有所提高。其中重要的原因可能是GBM特有的免疫抑制微环境包含了大量的肿瘤相关巨噬细胞（TAMs）。巨噬细胞是GBM肿瘤微环境中主要的免疫细胞，由两种不同的细胞群体构成：一种是来自胚胎期卵黄囊、存在于脑实质中的组织驻留小胶质细胞；另一种是来源于骨髓细胞、通过脑血管迁移到肿瘤内的边界相关巨噬细胞。在胶质瘤病理进程中，TAMs以独特的方式与T细胞相互作用，影响其活化状态和功能产生抗肿瘤效应，也可以通过促进肿瘤内在生长过程（包括炎症、血管生成和侵袭）产生促肿瘤效应。在肿瘤中，这些功能的平衡被打破，促肿瘤效应占了上风。尽管有研究显示调控TAMs的策略可以增强抗肿瘤免疫应答，但这些策略在临床试验中并未取得令人满意的结果，这可能是现有的TAMs靶点不够理想。因此，科学家们仍在努力寻找新的基于TAMs调控的治疗策略及理想的新靶点。2023年7月17日，陈俊及合作团队合作在Nature Cancer发表了题为Siglec-9 acts as an immune checkpoint molecule on macrophages in glioblastoma, restricting T cell priming and immunotherapy response的研究成果，发现了一种被称为Siglec-9的分子，它在GBM中充当巨噬细胞表面的免疫检查点分子，对肿瘤内免疫环境和免疫治疗效果有显著影响。研究团队通过对24例原发、复发和接受了抗PD-1抗体新辅助治疗的GBM患者样本进行单细胞RNA测序和空间转录组分析，发现在抗PD-1治疗无效的患者中，肿瘤内高表达Siglec-9基因的肿瘤相关巨噬细胞（TAMs）特别多。更具挑战性的是，研究者发现，尽管抗PD-1治疗能够在一些GBM患者中改变肿瘤免疫环境，使T细胞激活，增加某些化学因子基因和干扰素-γ相关基因的表达，诱导前体样耗竭T细胞的产生，并激活传统树突状细胞，但这并不能改变瘤内免疫环境的整体抑制状态，特别是无法抑制Siglec-9高表达的巨噬细胞（Siglec-9+ TAMs）的免疫抑制功能。研究团队进一步发现，Siglec-9+ TAMs可以区分为连续分化的Siglec-9+MARCO+ TAMs和Siglec-9+SEPP1+ TAMs。空间转录组分析显示，这两类TAMs是由CD14+ 单核细胞以分步方式迁移到肿瘤中并分化而来的，Siglec-9+MARCO+ TAMs可能代表了循环中的单核细胞分化为Siglec-9+SEPP1+ TAMs的过渡状态。值得一提的是，与已发表的GBM单细胞测序数据交叉比较显示，Siglec-9+ TAMs与接受免疫检查点阻断（ICB）治疗的GBM患者的与共表达GPNMB、CSTB、MRC1的巨噬细胞相似，而Siglec-9在仅包括复发GBM患者的数据集中没有富集。因此，抗PD1治疗可能导致Siglec-9+ TAMs的重编程，这一研究反映了新辅助抗PD1治疗产生的响应和非响应效果的原因。Siglec-9分子属于免疫球蛋白样凝集素（Siglec）家族成员，是表达于巨噬细胞等髓系免疫细胞表面的受体。这些受体与含有唾液酸残基的宿主/癌细胞膜上的糖蛋白结合时，会作为免疫检查点分子传递免疫抑制信号8。通过细胞和基因敲除小鼠颅内肿瘤模型的研究，研究团队发现，SiglecE（人Siglec-9的鼠功能同源基因）的敲除可以抑制GBM肿瘤的生长，改变巨噬细胞的免疫抑制表型，使其转变为更具有抗肿瘤活性的表型，并且显著增强T细胞的活化和增殖。进一步，研究者通过多种小鼠体内、体外和人临床样本实验，证明表达Siglec-9+/SiglecE+ TAMs是SiglecE敲除小鼠中抑制肿瘤生长的主要细胞群体，并且直接和T细胞相互作用，靶向Siglec-9是通过T细胞活化发挥肿瘤杀伤作用的。为了进一步明确靶向Siglec-9的临床前景，研究团队发现，SiglecE敲除小鼠接受抗PD-1治疗时，生存时间可以进一步延长。研究团队在小鼠GBM模型，使用具有靶向Siglec-9功能的竞争性蛋白治疗，结果发现这种治疗可以显著延长小鼠的生存时间。这项研究的发现为胶质母细胞瘤的治疗提供了新的思路，即 Siglec-9是一种新颖的巨噬细胞免疫检查点分子，可以作为靶点以提高针对GBM治疗的抗PD-1/PD-L1疗效。这一研究还显示，将人类样本的单细胞分析与小鼠转化研究相结合，可以有效地发现新的基于免疫检查点的免疫疗法。会议推荐会议详情会议名称：2023（第14届）细胞与基因治疗国际研讨会暨抗体工程与创新免疫治疗技术论坛暨2023细胞免疫治疗专业委员会年会主办单位：生物谷、梅斯医学、同济大学附属同济医院、南京可缘大会时间：9月22-23日大会地点：上海 大会规模：1500人2010年至今，生物谷联合各合作单位已经顺利召开了13届细胞与基因治疗国际研讨会，与全球致力于细胞治疗行业同仁们一同成长沉淀，在提供行业信息交流平台的同时，也为基础研究提供了技术/产品等产业化的孵化平台。基于此，本届会议将继续以转化医学为切入点，以基础研究与临床应用相结合，针对细胞治疗的临床前沿研究、临床监管、治疗规范、细胞治疗安全性，免疫细胞新型疗法、实体瘤治疗、干细胞与癌症、抗体药物、核酸药物、基因编辑、肿瘤免疫治疗等热门议题进行讨论，诚邀国内外产学研医专家共聚，共绘产业发展新蓝图！长按识别二维码立即报名参会

一、摘要|导读：许多生物过程离不开动态的蛋白质磷酸化调节，因此监测循环生物体液中与疾病相关的磷酸化事件意义非凡，然而，由于磷酸酶的存在使得监测生物体液中磷酸化蛋白的变化极具挑战性。近期，东南大学W. Andy Tao教授团队在Angewandte Chemie在线发表了题为Profiling Phosphoproteome Landscape in Circulating Extracellular Vesicles from Microliters of Biofluids through Functionally Tunable Paramagnetic Separation的研究文章。 该研究介绍了一种功能可调的材料和方法：从细胞外囊泡到磷酸化蛋白-------EVTOP(Extracellular Vesicles To Phosphoproteins)，它实现了一步法分离、提取和消化EV蛋白，并富集了磷酸化肽段，而且只需微量的起始生物液。用钛(IV)离子功能化的磁珠和穿膜肽八精氨酸（ R8+）有效地分离EV，同时R8+提供了亲水表面以在裂解过程中保留EV蛋白。随后磁珠上消化的同时将EVTOP转化为纯Ti(IV)离子表面，有效地富集了磷酸化肽。这个简化的超灵敏平台能够仅用几个μL血浆就能定量500个独特的EV磷酸化肽，用100μL脑脊液（CSF）就能定量1,200多个磷酸化肽。该团队还探讨了小容量CSF监测原发性中枢神经系统淋巴瘤(PCNSL，Primary Central Nervous System Lymphoma)化疗效果的临床应用，为临床的广泛应用提供了有力工具。二、实验|设计和方法 图1. 实验流程示意图上图简要展示了该团队从微量体液中分离肠道病毒并原位富集磷酸化肽用于深度磷酸化蛋白质组图谱的研究方法。三、结果|创新点图2. 关于EVTOP磁珠制备及EV分离的表征EVTOP设计充分利用了EV的双层磷脂层结构，包括自组装的疏水尾部和亲水性磷酸盐头部，并考虑了直接在珠上的样品的下游工艺。该方法特殊在：(I)磁珠被Ti(IV)离子和 R8+功能化，为有效分离EV提供协同亲和力；(Ⅱ)带正电的Ti(IV)离子和 R8+通过疏水-亲水(HILIC)相互作用提供亲水表面，在EV裂解过程中保留EV蛋白；(Ⅲ)珠上消化产生多肽并同时将 R8+从珠子上切割下来；(Ⅳ)EVTOP只在表面吸附了Ti(IV)离子，实现了磷酸化肽在LC-MS分析中的特异性富集化。EVTOP的主要特点是能够通过同一磁珠上官能团的相互作用和转化，实现从生物液体到浓缩EV磷酸化肽的所有样品制备步骤。一步法在MS分析前不需要任何清理或除盐步骤，最大限度地减少了样品损失，从而能够利用微升临床样品实现极其灵敏和定量的磷酸化蛋白质组学。该团队考察了三种功能化磁珠以及超速离心法对EV的捕获。根据三种EV标志物(CD9、Flotillin 1和β-Actin)的WB结果，EV分离效率的顺序是EVTOP>仅R8+>仅Ti(IV)>超速离心法，其中EVTOP最有效。图3. 胰酶处理EVTOP磁珠对EV捕获率的影响经胰酶消化后的EVTOP磁珠的捕获EV效率明显下降，同时Zeta电位发生变化。WB结果和质谱学鉴定进一步证实了R8+和Ti(IV)离子的重要作用。通过在胰酶消化过程中同时切割R8+，EVTOP被转化为纯Ti(IV)离子磁珠。PCNSL是一种罕见的仅限于中枢神经系统的非霍奇金淋巴瘤亚型，作为一种具有复杂发病机制的高度异质性疾病，PCNSL的分子特征至关重要，CSF是准确评估疾病的理想微创生物液体。以前使用大量CSF样本的尝试只发现了少量的磷酸化蛋白。为了展示EVTOP的临床应用，该团队首先使用EVTOP方法分析了21例PCNSL患者和21例匹配的非PCNSL对照CSF样本的EV磷酸化蛋白质组学定量。图4.临床CSF样本本分析过程及结果。该团队总共从CSF样本 EVs中鉴定出3,344个磷酸化肽，代表689个磷酸化蛋白，其中160个磷酸化蛋白在PCNSL患者中上调。GO、KEGG途径和PPI分析表明，上调的磷酸化蛋白参与神经肽激素活性、透明质酸结合和多种信号途径，如Wnt信号和PI3K-Akt途径。其中一些上调的磷酸化蛋白被报道为PCNSL和其他癌症的潜在生物标记物，如骨桥蛋白(SPP1)，组织蛋白B(CTSB)，纤维连接蛋白(FN1)，CD44，Tenascin-X(TNX)，肉豆蔻酰化丙氨酸C-激酶底物(Marcks)和Testican-3(SPOCK3)。图5.已识别的EV磷酸化蛋白作为潜在的生物标志物评估PCNSL化疗结果的可行性。该团队选择了11名PCNSL患者的独立队列，这些患者最初接受大剂量甲氨蝶呤治疗，总体临床反应良好。分别于化疗前和化疗后采集CSF标本100ug。然后对CSF样本进行EVTOP程序以获得EV磷酸化肽。使用TimeTOF Pro仪上的平行反应监测和平行累积-连续碎裂(PRM-FASEF)相结合的方法来监测化疗前后这些上调的磷酸化蛋白的强度变化。排除了重复性相对较差、产物离子覆盖率较低、缺乏生物学意义(无疾病或相关生物途径关联)的磷酸化肽。发现10个磷酸化蛋白【SPP1、促甲状腺激素释放激素原(TRH)、Tenascin-C(TNC)、硒蛋白P(SEPP1)、纤维蛋白原β链(FGB/FIBB)、层粘连蛋白β1(Lamb1)、TNX、富组氨酸糖蛋白(HRG)、高尔基相关蛋白1B(GAK1B)和胰岛素样生长因子结合蛋白5(fbp5/IBP5)】在化疗后显示出明显的信号减少。此外，Reatcome/KEGG通路分析和PPI分析表明，这其中6个属于脑组织，这些生物标记物大多参与PI3K-Akt、PI3K-Akt-mTOR和其他重要的信号通路，据报道与PCNSL的发生和发展有关，并成为各种癌症和神经退行性疾病的治疗靶点。磷酸化蛋白SPP1、TRH、TNC、SEPP1、IBP5和TNX与神经系统疾病相关，其中SPP1是PCNSL筛查的生物标志物。该团队能够从治疗前和治疗后的100μL CSF样本中定量它们的事实，突显了EVTOP方法的强大和未来应用的巨大潜力。四、总结|讨论该研究介绍的EVTOP磁珠有希望用于处理大样本批次、有限样本材料和具有挑战性的生物样本的临床筛查和生物标记物发现。该研究展示了EVTOP用于使用微量生物流体进行EV磷酸化蛋白质组谱分析的优势，未来改进该方法可以扩展到含有其他翻译后修饰的EV多肽的富集化，例如糖基化、乙酰化和其他修饰。EVTOP由于其超高灵敏度和稳定性，可以作为一种临床程序来筛选或靶向循环EVs中的功能蛋白，用于疾病诊断。参考文献：Jie Sun, et al, Profiling Phosphoproteome Landscape in Circulating Extracellular Vesicles from Microliters of Biofluids through Functionally Tunable Paramagnetic Separation, Angew. Chem. Int. Ed. 2023, e202305668DOI: 10.1002/anie.202305668https://doi.org/10.1002/anie.202305668