结直肠癌（CRC）具有相当大的异质性；因此，疾病模型也必须反映多种成分。与传统的 2D 模型相比，3D 细胞模型（例如肿瘤球体）可用于确定潜在疗法的药效。众所周知，单一培养球体可以概括无血管肿瘤的基因表达、细胞信号传导和病理生理梯度。然而，它们无法模拟结直肠癌中存在的基质细胞影响，众所周知，结直肠癌会干扰药物疗效，并与转移性晚期结直肠癌相关。本研究旨在开发一种使用癌和非癌成纤维细胞的共培养球体模型。我们使用气相分级分离的数据独立采集来表征共培养球体与单一培养球体的蛋白质组学特征。具体来说，我们确定与单一培养的球体相比，共培养的球体中与翻译和 mTOR 信号传导相关的蛋白质组差异显着增加。与成纤维细胞功能相关的蛋白质，例如包被囊泡的胞吐作用和生长因子的分泌，在共培养的球体中表达显着差异。最后，我们将单一培养和共培养球体的蛋白质组谱与来自实体结直肠癌的公开数据集进行了比较。我们发现共培养球体的蛋白质组与患者样本的蛋白质组更加相似，表明它们具有作为肿瘤模拟物的潜力。

Colorectal cancer (CRC) contains considerable heterogeneity; therefore, models of the disease must also reflect the multifarious components. Compared to traditional 2D models, 3D cellular models, such as tumor spheroids, have the utility to determine the drug efficacy of potential therapeutics. Monoculture spheroids are well-known to recapitulate gene expression, cell signaling, and pathophysiological gradients of avascularized tumors. However, they fail to mimic the stromal cell influence present in CRC, which is known to perturb drug efficacy and is associated with metastatic, late-stage colorectal cancer. This study seeks to develop a cocultured spheroid model using carcinoma and noncancerous fibroblast cells. We characterized the proteomic profile of cocultured spheroids in comparison to monocultured spheroids using data-independent acquisition with gas-phase fractionation. Specifically, we determined that proteomic differences related to translation and mTOR signaling are significantly increased in cocultured spheroids compared to monocultured spheroids. Proteins related to fibroblast function, such as exocytosis of coated vesicles and secretion of growth factors, were significantly differentially expressed in the cocultured spheroids. Finally, we compared the proteomic profiles of both the monocultured and cocultured spheroids against a publicly available data set derived from solid CRC tumors. We found that the proteome of the cocultured spheroids more closely resembles that of the patient samples, indicating their potential as tumor mimics.