用于表征成人型弥漫性浸润性胶质瘤微环境的数字空间剖析
Summary
蛋白质组学失调在弥漫性浸润性胶质瘤的扩散中起重要作用，但几种相关蛋白质仍未鉴定。数字空间处理（DSP）提供了一种高效、高通量的方法，用于表征可能导致浸润性胶质瘤侵袭和迁移的候选蛋白的差异表达。
Abstract
弥漫性浸润性胶质瘤由于肿瘤扩散的浸润性而与高发病率和死亡率相关。它们是形态复杂的肿瘤，在肿瘤本身及其异质微环境中具有高度的蛋白质组学变异性。这些肿瘤的恶性潜力通过参与几个关键途径的蛋白质失调而增强，包括维持细胞稳定性和保持微环境结构完整性的过程。尽管已经进行了大量和单细胞胶质瘤分析，但这些蛋白质组学数据的空间分层相对缺乏。了解内在肿瘤、侵袭边缘和微环境之间致瘤因子和免疫细胞群的空间分布差异，为肿瘤增殖和增殖的机制提供了有价值的见解。数字空间剖析 （DSP） 代表了一种强大的技术，可以为这些重要的多层分析奠定基础。
DSP是一种有效定量组织标本中用户指定空间区域内蛋白质表达的方法。DSP是研究区分区域内和跨区分区域多种蛋白质差异表达的理想选择，可实现多层次的定量和定性分析。DSP协议是系统且用户友好的，允许对蛋白质组学数据进行定制的空间分析。在该实验中，组织微阵列由存档的胶质母细胞瘤核心活检构建。接下来，选择一组抗体，靶向样品中感兴趣的蛋白质。然后将预偶联到紫外光切割DNA寡核苷酸的抗体与组织样品一起孵育过夜。在抗体的荧光显微镜可视化下，用样品定义用于定量蛋白质表达的感兴趣区域（ROI）。然后将紫外线照射到每个ROI，切割DNA寡核苷酸。微吸寡核苷酸在每个ROI内计数，在空间基础上量化相应的蛋白质。
Introduction
弥漫性浸润性胶质瘤是成人中最常见的恶性脑肿瘤类型，并且总是致命的。神经胶质瘤细胞在大脑中广泛迁移的倾向是一个主要的治疗挑战。它们的传播机制包括定向迁移和不受控制的入侵。浸润性胶质瘤细胞已被证明沿白质束1 表现出嗜性和迁移性，最近的研究表明这些束脱髓鞘是一种活跃的促肿瘤特征2。侵袭由上皮到间充质转化介导，其中胶质瘤细胞通过减少编码细胞外基质蛋白和细胞粘附分子的基因的表达来获得间充质特性，放大迁移并促进通过肿瘤微环境的繁殖3，4，5。
在分子水平上，已经证明了几种赋予细胞稳定性并与免疫原性成分界面的蛋白质的破坏6。已知浸润性胶质瘤会抑制具有抗凋亡（例如PTEN）特性的蛋白质7。它们还过表达促进逃避宿主免疫反应的蛋白质（例如，PD1/PDL1）8。这些复杂途径的失调增强了致瘤性并增加了恶性潜力。
在侵袭性胶质瘤样本中，目的是评估对细胞生长、存活和增殖以及侵袭性和非侵入性成分之间的微环境结构完整性至关重要的蛋白质的差异表达。此外，我们试图研究具有活性免疫原性作用的蛋白质的差异调节，从而深入了解受损的宿主免疫防御可能增强胶质瘤的增殖和侵袭潜力的机制。鉴于最近广泛的研究表明，恶性肿瘤中的免疫标志物和失调的驱动因素如何成为免疫治疗的靶标，这一点尤其重要。在参与免疫监视和反应性的众多蛋白质中确定可行的治疗靶点需要一种高度灵敏和全面的方法。
鉴于可以研究的候选蛋白质种类繁多，我们寻求一种类似于免疫组织化学但具有增强数据处理效率的方法。在癌症生物学领域，DSP已成为一项强大的技术，与蛋白质组学分析和定量的替代工具相比具有重要优势。DSP的特点是其高通量多重检测能力，允许同时研究样品中的几种不同蛋白质，这标志着与免疫组织化学（IHC）等标准但低重技术的重要区别9，10。DSP的多重功能不会影响其作为定量和分析工具的保真度，正如比较DSP与IHC的研究所证明的那样。例如，当用于非小细胞肺癌标本的蛋白质组学定量时，DSP已被证明具有与IHC11相似的结果。此外，DSP还提供可定制的区域规范，用户可以在其中手动定义要执行蛋白质组学分析的区域。这比全断面多重方法10，12具有优势。因此，在单轮处理中，DSP通过调查多个感兴趣区域的多个蛋白质靶标来提供多层分析。
DSP在几种不同的病理环境中都有应用。DSP在肿瘤学分析中特别有利，因为空间变化可以与细胞转化和差异蛋白表达相关。例如，DSP已被用于将乳腺癌的蛋白质组学特征与邻近的肿瘤微环境进行比较。这对于了解该肿瘤的自然史及其进展以及对治疗的潜在反应具有重要意义13。说明DSP多功能性的其他背景包括前列腺癌中蛋白质多样性的空间定量14，免疫细胞标志物表达与头颈部鳞状细胞癌疾病进展的关联15，以及蛋白质表达的上皮 – 间充质梯度的证明，以区分转移性和原发性透明细胞卵巢癌16.通过实施DSP，我们表征了可能影响胶质瘤肿瘤发生和侵袭的蛋白质的空间地形。
Protocol
Representative Results
Discussion
鉴于可能影响胶质瘤侵袭性的蛋白质的多样性，以及其中一些蛋白质仍未被发现的概念，高通量蛋白质定量方法是一种理想的技术方法。此外，鉴于肿瘤样品中的空间数据通常与差异表达相关18，将空间分析纳入蛋白质定量方法可以实现更有效的分析。
DSP的高通量方法还使其能够用于类似霰弹枪的方法，这是发现疾病和治疗反应的潜在新型生物标志物的理想…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢达特茅斯希区柯克卫生系统病理学和实验室医学系临床基因组学和先进技术实验室的支持。作者还承认达特茅斯癌症中心的病理学共享资源与NCI癌症中心支持补助金5P30 CA023108-37。
Materials
| BOND Research Detection System | Leica Biosystems, Wetzlar, Germany | DS9455 | Open detection system containing open containers in a reagent tray |
| BOND Wash | Leica Biosystems, Wetzlar, Germany | AR950 | 10X concentrated buffer solution for washing fixed tissue |
| Buffer W | NanoString, Seattle, WA | contact company | Blocking reagent |
| Cy3 conjugation kit | Abcam, Cambridge, UK | AB188287 | Cy3 fluorescent antibody conjugation kit |
| GeoMx Digital Spatial Profiler (DSP) | NanoString, Seattle, WA | contact company | System for imaging and characterizing protein and RNA targets |
| GeoMx DSP Instrument BufferKit | NanoString, Seattle, WA | 100471 | Buffer kit for GeoMX DSP (including buffers for sample processing and preparation) |
| GeoMx Hyb Code Pack_Protein | NanoString, Seattle, WA | 121300401 | Controls for running GeoMX DSP experiemtns |
| GeoMx Immune Cell Panel (Imm Cell Pro_Hs) | NanoString, Seattle, WA | 121300101 | Protein module with targets for human immune cells and immuno-oncologic targets |
| GeoMx Pan-Tumor Panel (Pan-Tumor_Hs) | NanoString, Seattle, WA | 121300105 | Protein module with targets for multiple human tumor types and for markers of epithelial-mesenchymal transition |
| GeoMx Protein Slide Prep FFPE | NanoString, Seattle, WA | 121300308 | Sample preparation reagents for GeoMX DSP protein analysis |
| IDH1-R132H antibody | Dianova, Hamburg, Germany | DIA-H09 | Monoclonal antibody against human IDH1 R132H |
| LEICA Bond RX | Leica Biosystems, Wetzlar, Germany | contact company | Fully automated IHC stainer |
| Master Kit–12 reactions | NanoString, Seattle, WA | 100052 | Materials and reagents for use with the nCounter Analysis system |
| nCounter Analysis System | NanoString, Seattle, WA | contact company | Automated system for multiplex target expression quantification (to be used with GeoMx DSP) |
| TMA Master II | 3DHistech Ltd., Budapest, Hungary | To create the tissue microarray block |
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