BACKGROUND Spread through air spaces (STAS) is a method of invasion in lung adenocarcinoma, associated with tumor recurrence and poor survival. The spatial orientation of STAS cells/clusters to the lung alveolar parenchyma is not known. The aim of this study was to utilize high resolution and high-quality three-dimensional (3D) reconstruction of images from immunohistochemistry (IHC) and multiplex immunofluorescence (IF) experiments to understand the spatial architecture of tumor cell clusters by STAS in the lung parenchyma. METHODS Four lung adenocarcinomas: 3 micropapillary (MIP) predominant and 1 solid (SN) predominant adenocarcinoma subtypes, were investigated. A 3D reconstruction image was created from the formalin fixed paraffin-embedded (FFPE) blocks. 350 serial sections were obtained and stained with hematoxylin and eosin (H&E) (100 slides), IHC (200 slides), and multiplex IF (50 slides) with the following antibodies: CD31, collagen type 4, TTF-1 and E-Cadherin. Whole slide images (WSIs) were reconstructed into 3D images for evaluation. RESULTS Serial 3D image analysis by H&E as well as IHC and IF showed the MIP clusters and SN nests of STAS focally attached to alveolar walls away from the main tumor. CONCLUSION Our 3-D reconstructions demonstrated STAS tumor cells can attach to alveolar walls rather than appearing free floating as seen on 2D sections. This suggests that tumor cells detach from the main tumor, migrate through air spaces and reattach to alveolar walls through vessel co-option allowing them to survive and grow. This may explain the higher recurrence rate and worse survival for STAS positive tumors undergoing limited resection compared to lobectomy.

中文翻译：

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肺腺癌气腔 (STA) 传播动态血管合作的三维组织学、免疫组织化学和多重免疫荧光分析

背景 气腔播散（STAS）是肺腺癌的一种侵袭方法，与肿瘤复发和生存率低相关。STAS 细胞/簇到肺泡实质的空间方向尚不清楚。本研究的目的是利用免疫组织化学 (IHC) 和多重免疫荧光 (IF) 实验图像的高分辨率和高质量三维 (3D) 重建，通过 STAS 了解肺实质中肿瘤细胞簇的空间结构。方法 对四种肺腺癌进行了研究：3 种以微乳头状 (MIP) 为主的腺癌亚型和 1 种以实性 (SN) 为主的腺癌亚型。从福尔马林固定石蜡包埋 (FFPE) 块创建 3D 重建图像。获得 350 个连续切片，并使用苏木精和伊红 (H&E)（100 张载玻片）、IHC（200 张载玻片）和多重 IF（50 张载玻片）以及以下抗体进行染色：CD31、4 型胶原蛋白、TTF-1 和 E-钙粘蛋白。将整个幻灯片图像 (WSI) 重建为 3D 图像以进行评估。结果 H&E 以及 IHC 和 IF 进行的系列 3D 图像分析显示，STAS 的 MIP 簇和 SN 巢集中附着在远离主肿瘤的肺泡壁上。结论 我们的 3D 重建表明 STAS 肿瘤细胞可以附着在肺泡壁上，而不是像 2D 切片上看到的那样自由漂浮。这表明肿瘤细胞与主肿瘤分离，通过空气空间迁移并通过血管共选择重新附着到肺泡壁上，从而使它们能够生存和生长。这可能解释了与肺叶切除术相比，接受有限切除的 STAS 阳性肿瘤的复发率更高和生存率更差的原因。