肾上腺皮质癌(ACC)预后较差，数十年来没有发现新药。药物开发的缺失部分归因于缺乏临床前模型。动物模型和ACC的2D细胞培养均未能准确模拟该疾病，因为动物生理与人类本质上不同，而2D培养无法代表重要的3D结构。器官样体和其他小型的3D体外组织或肿瘤模型可以模拟人体生物学的某些复杂性；然而，这种技术在ACC中几乎尚未应用。在本研究中，我们描述了从已建立的ACC细胞系NCI-H295R中生成3D肿瘤构建物。将NCI-H295R细胞包埋以生成3D ACC构建物。评估肿瘤构建物的生物标志物表达、存活率、增殖和皮质醇产量。此外，通过使用荧光基质金属蛋白酶(MMP)敏感性生物传感器直接评估MMP功能，并通过将NCI-H295R细胞导入微流体芯片平台上的转移-芯片设备中进行验证。ACC肿瘤构建物显示出与ACC相关的生物标志物（包括SF-1、Melan A和抑制素α）的表达。与2D细胞培养相比，对ACC肿瘤构建物进行化疗，显示了药物敏感性的降低。由于大多数肿瘤细胞通过MMPs在组织中迁移以分解细胞外基质，我们通过将荧光MMPs敏感肽生物传感器集成到肿瘤构建物中来验证ACC肿瘤构建物的实用性。最后，在我们的转移-芯片设备中，NCI-H295R细胞成功移植到下游的肺细胞系构建物，但通过MMP抑制减少了对肺细胞构建物的侵袭距离。这些研究，通过使用2D细胞培养无法实现，证明了NCI-H295R细胞分泌活性MMPs，这些MMPs用于3D侵袭。这项工作为ACC的3D肿瘤构建物平台提供了首个证据，可用于未来的机制研究以及干预和治疗新靶点的开发。© 2023. Springer Nature Limited.

Adrenocortical carcinoma (ACC) has a poor prognosis, and no new drugs have been identified in decades. The absence of drug development can partly be attributed to a lack of preclinical models. Both animal models and 2D cell cultures of ACC fail to accurately mimic the disease, as animal physiology is inherently different than humans, and 2D cultures fail to represent the crucial 3D architecture. Organoids and other small 3D in vitro models of tissues or tumors can model certain complexities of human in vivo biology; however, this technology has largely yet to be applied to ACC. In this study, we describe the generation of 3D tumor constructs from an established ACC cell line, NCI-H295R. NCI-H295R cells were encapsulated to generate 3D ACC constructs. Tumor constructs were assessed for biomarker expression, viability, proliferation, and cortisol production. In addition, matrix metalloproteinase (MMP) functionality was assessed directly using fluorogenic MMP-sensitive biosensors and through infusion of NCI-H295R cells into a metastasis-on-a-chip microfluidic device platform. ACC tumor constructs showed expression of biomarkers associated with ACC, including SF-1, Melan A, and inhibin α. Treatment of ACC tumor constructs with chemotherapeutics demonstrated decreased drug sensitivity compared to 2D cell culture. Since most tumor cells migrate through tissue using MMPs to break down extracellular matrix, we validated the utility of ACC tumor constructs by integrating fluorogenic MMP-sensitive peptide biosensors within the tumor constructs. Lastly, in our metastasis-on-a-chip device, NCI-H295R cells successfully engrafted in a downstream lung cell line-based construct, but invasion distance into the lung construct was decreased by MMP inhibition. These studies, which would not be possible using 2D cell cultures, demonstrated that NCI-H295R cells secreted active MMPs that are used for invasion in 3D. This work represents the first evidence of a 3D tumor constructs platform for ACC that can be deployed for future mechanistic studies as well as development of new targets for intervention and therapies.© 2023. Springer Nature Limited.