Hypoxia drives the formation of lung micropapillary adenocarcinoma through hypoxia-inducible factor-1a (RNA-seq)

Micropapillary adenocarcinoma (MPC) is an aggressive histologic subtype of lung adenocarcinoma (LUAD). MPC is composed of small clusters of cancer cells exhibiting inverted polarity. However, the mechanism underlying its formation is poorly understood. Here we show that hypoxia is involved in MPC formation. Spatial transcriptome analysis with human LUAD tissue suggested that hypoxia-inducible factor (HIF)-1 signaling is activated in MPC. Hypoxia induced the formation of MPC-like structures (MLSs) with inverted polarity in a three-dimensional culture system using A549 human LUAD cells, and HIF-1a was indispensable for MLS formation. RNA sequencing analysis demonstrated that A549 cells forming MLSs exhibited a gene expression signature similar to that of lung MPC. Moreover, MLS formation enhanced the resistance of A549 cells to natural killer cell cytotoxicity. Our findings suggest that hypoxia drives lung MPC formation through HIF-1a and that immune escape from natural killer cells might underlie the aggressiveness of MPC. Overall design: To characterize the hypoxia-induced MLSs in detail, we performed RNA-seq analysis in A549 cells cultured in the usual 2D culture system or the 3D culture system under normoxia or hypoxia.

微乳头状腺癌（Micropapillary adenocarcinoma, MPC）是肺腺癌（Lung adenocarcinoma, LUAD）的侵袭性组织学亚型。MPC由表现出极性倒置的小癌细胞簇构成，但其形成机制尚不清楚。
本研究证实缺氧参与了MPC的形成过程。针对人肺腺癌组织的空间转录组分析（spatial transcriptome analysis）显示，缺氧诱导因子（hypoxia-inducible factor, HIF）-1信号通路在MPC中被激活。利用人肺腺癌细胞系A549开展的三维培养系统实验表明，缺氧可诱导形成具有极性倒置特征的MPC样结构（MPC-like structures, MLSs），且缺氧诱导因子-1α（HIF-1α）对该结构的形成不可或缺。RNA测序（RNA sequencing）分析显示，形成MLSs的A549细胞其基因表达特征与肺MPC高度相似。此外，MLSs的形成可增强A549细胞对自然杀伤细胞细胞毒性的抵抗能力。本研究结果提示，缺氧通过HIF-1α驱动肺MPC的形成，而逃避免疫系统中自然杀伤细胞的攻击可能是MPC侵袭性的潜在机制。
整体实验设计：为详细表征缺氧诱导的MLSs，我们分别在常氧或缺氧条件下，于常规二维培养系统或三维培养系统中培养A549细胞，并开展RNA测序分析。