Kuhn Nicholas F, Zaleta-Linares Itzia, Nyberg William A, Eyquem Justin, Krummel Matthew F
Department of Pathology, University of California, San Francisco, CA 94143, USA.
ImmunoX Initiative, University of California, San Francisco, CA 94143, USA.
bioRxiv. 2024 Jul 16:2024.07.11.603114. doi: 10.1101/2024.07.11.603114.
Discovering the role of fibroblasts residing in the tumor microenvironment (TME) requires controlled, localized perturbations because fibroblasts play critical roles in regulating immunity and tumor biology at multiple sites. Systemic perturbations can lead to unintended, confounding secondary effects, and methods to locally genetically engineer fibroblasts are lacking. To specifically investigate murine stromal cell perturbations restricted to the TME, we developed an adeno-associated virus (AAV)-based method to target any gene-of-interest in fibroblasts at high efficiency (>80%). As proof of concept, we generated single (sKO) and double gene KOs (dKO) of , , and in cancer-associated fibroblasts (CAFs) and investigated how their cell states and those of other cells of the TME subsequently change in mouse models of melanoma and pancreatic ductal adenocarcinoma (PDAC). Furthermore, we developed an knockin-knockout (KIKO) strategy to achieve long-term tracking of CAFs with target gene KO via knocked-in reporter gene expression. This validated gene editing toolbox is fast, affordable, and modular, and thus holds great potential for further exploration of gene function in stromal cells residing in tumors and beyond.
要揭示肿瘤微环境（TME）中驻留的成纤维细胞的作用，需要进行可控的局部扰动，因为成纤维细胞在多个位点调节免疫和肿瘤生物学方面发挥着关键作用。全身扰动可能会导致意外的、混淆性的继发效应，而且缺乏对成纤维细胞进行局部基因工程改造的方法。为了专门研究局限于TME的小鼠基质细胞扰动，我们开发了一种基于腺相关病毒（AAV）的方法，可高效（>80%）靶向成纤维细胞中任何感兴趣的基因。作为概念验证，我们在癌症相关成纤维细胞（CAF）中生成了单基因敲除（sKO）和双基因敲除（dKO）的 、 和 ，并研究了在黑色素瘤和胰腺导管腺癌（PDAC）小鼠模型中，它们的细胞状态以及TME中其他细胞的状态随后如何变化。此外，我们开发了一种敲入-敲除（KIKO）策略，通过敲入报告基因的表达来实现对具有靶基因敲除的CAF进行长期追踪。这个经过验证的基因编辑工具箱快速、经济且具有模块化特点，因此在进一步探索肿瘤及其他部位驻留的基质细胞中的基因功能方面具有巨大潜力。
