非小细胞肺癌（NSCLC）是一种高度致命的疾病，具有复杂且异质的肿瘤微环境。目前，基于皮下接种癌细胞悬浮液的常见动物模型并不能重现非小细胞肺癌的肿瘤微环境。在此，我们描述了一种采用三维多细胞球体（MCS）肺内接种的小鼠原位肺癌异种移植模型。具体而言，将荧光人NSCLC细胞（A549-iRFP）在含有胶原蛋白的低贴壁96孔微孔板中培养3周形成MCS，然后将其接种到无胸腺裸鼠的左肺肋间以建立原位肺癌模型。与原始A549细胞系相比，A549-iRFP细胞系的MCS对抗癌药物的反应相似。 A549-iRFP 细胞的长波长荧光信号与癌细胞生长的常见标志物密切相关，包括球体体积、细胞活力和细胞蛋白水平，从而允许通过荧光成像动态监测体内癌症生长。接种小鼠后，A549-iRFP MCS 异种移植物可靠地经历了与 NSCLC 临床阶段非常相似的阶段，包括原发肿瘤的扩张、邻近继发肿瘤的出现以及癌细胞转移到对侧右肺和远程器官。此外，该模型对基准抗肺癌药物顺铂做出了反应，具有预期的毒性和较慢的癌症进展。因此，这种小鼠非小细胞肺癌原位异种移植模型将作为一个平台来概括疾病的进展并促进潜在抗癌药物的开发。

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models based on subcutaneous inoculation of cancer cell suspensions do not recapitulate the tumor microenvironment in NSCLC. Herein we describe a murine orthotopic lung cancer xenograft model that employs the intrapulmonary inoculation of three-dimensional multicellular spheroids (MCS). Specifically, fluorescent human NSCLC cells (A549-iRFP) were cultured in low-attachment 96-well microplates with collagen for 3 weeks to form MCS, which were then inoculated intercostally into the left lung of athymic nude mice to establish the orthotopic lung cancer model. Compared with the original A549 cell line, MCS of the A549-iRFP cell line responded similarly to anticancer drugs. The long-wavelength fluorescent signal of the A549-iRFP cells correlated strongly with common markers of cancer cell growth, including spheroid volume, cell viability, and cellular protein level, thus allowing dynamic monitoring of the cancer growth in vivo by fluorescent imaging. After inoculation into mice, the A549-iRFP MCS xenograft reliably progressed through phases closely resembling the clinical stages of NSCLC, including the expansion of the primary tumor, the emergence of neighboring secondary tumors, and the metastases of cancer cells to the contralateral right lung and remote organs. Moreover, the model responded to the benchmark antilung cancer drug, cisplatin with the anticipated toxicity and slower cancer progression. Therefore, this murine orthotopic xenograft model of NSCLC would serve as a platform to recapitulate the disease's progression and facilitate the development of potential anticancer drugs.