Langerman Justin, Baghdasarian Sevana, Cheng Rene Yu-Hong, James Richard G, Plath Kathrin, Di Carlo Dino
Department of Biological Chemistry, David Geffen School of Medicine at the University of California Los Angeles, Los Angeles, CA, USA.
Department of Chemical and Biomolecular Engineering, University of California Los Angeles, Los Angeles, CA, USA.
Nat Protoc. 2025 Feb 20. doi: 10.1038/s41596-024-01112-w.
Cells secrete numerous proteins and other biomolecules into their surroundings to achieve critical functions-from communicating with other cells to blocking the activity of pathogens. Secretion of cytokines, growth factors, extracellular vesicles and even recombinant biologic drugs defines the therapeutic potency of many cell therapies. However, gene expression states that drive specific secretory phenotypes are largely unknown. We provide a protocol that enables the secretion amount of a target protein encoded (SEC) by oligonucleotide barcodes to be linked with transcriptional sequencing (seq) for thousands of single cells. SEC-seq leverages microscale hydrogel particles called Nanovials to isolate cells and capture their secretions in close proximity, oligonucleotide-labeled antibodies to tag secretions on Nanovials and flow cytometry and single-cell RNA-sequencing (scRNA-seq) platforms for readout. Cells on Nanovials can be sorted on the basis of viability, secretion amount or other surface markers without fixation or permeabilization, and cell- and secretion-containing Nanovials are directly introduced into microfluidic droplets-in-oil emulsions for single-cell barcoding of cell transcriptomes and secretions. We have used SEC-seq to link T cell receptor sequences to the relative amount of associated cytokine secretions, surface marker gene expression with a highly secreting and potential regenerative population of mesenchymal stromal cells and the transcriptome with high immunoglobulin secretion from plasma cells. Nanovial modification and cell loading takes <4 h, and once the desired incubation time is over, staining, cell sorting and emulsion generation for scRNA-seq can also be completed in <4 h. Compared to related techniques that link secretions to a cell's surface, SEC-seq provides a general solution across any secretion target because of the ease with which biotinylated Nanovials can be modified. By linking gene expression and secretory strength, SEC-seq can expand our understanding of cell secretion, how it is regulated and how it can be engineered to make better therapies.
细胞向周围环境分泌大量蛋白质和其他生物分子，以实现关键功能——从与其他细胞通信到阻断病原体的活性。细胞因子、生长因子、细胞外囊泡甚至重组生物药物的分泌决定了许多细胞疗法的治疗效力。然而，驱动特定分泌表型的基因表达状态在很大程度上尚不清楚。我们提供了一种方案，可将寡核苷酸条形码编码的靶蛋白（SEC）的分泌量与数千个单细胞的转录测序（seq）联系起来。SEC-seq利用称为纳米管的微型水凝胶颗粒来分离细胞并在其附近捕获它们的分泌物，用寡核苷酸标记的抗体标记纳米管上的分泌物，并利用流式细胞术和单细胞RNA测序（scRNA-seq）平台进行读出。纳米管上的细胞可以根据活力、分泌量或其他表面标志物进行分选，无需固定或透化处理，并且将含有细胞和分泌物的纳米管直接引入油包微流体液滴中，用于细胞转录组和分泌物的单细胞条形码分析。我们已经使用SEC-seq将T细胞受体序列与相关细胞因子分泌的相对量、表面标志物基因表达与间充质基质细胞的高分泌和潜在再生群体以及浆细胞的高免疫球蛋白分泌的转录组联系起来。纳米管修饰和细胞加载耗时不到4小时，一旦达到所需的孵育时间，用于scRNA-seq的染色、细胞分选和乳液生成也可在不到4小时内完成。与将分泌物与细胞表面联系起来的相关技术相比，SEC-seq由于生物素化纳米管易于修饰，因此为任何分泌靶点提供了一种通用解决方案。通过将基因表达与分泌强度联系起来，SEC-seq可以扩展我们对细胞分泌、其调控方式以及如何通过工程改造以实现更好治疗效果的理解。
