细胞培养的结构（二维 (2D) 与三维 (3D)）显着影响各种细胞因素，包括细胞间相互作用、营养和氧气梯度、代谢活动和基因表达谱。这可能会导致癌症药物治疗期间不同的细胞反应，3D 培养的细胞通常对化疗药物表现出更高的耐药性。虽然各种遗传和蛋白质组分析已被用来研究这种耐药性增加的潜在机制，但提供空间分子分析数据实验证据的补充技术是有限的。受激拉曼散射 (SRS) 显微镜已证明其能够测量细胞内药物摄取和生长抑制。在这项工作中，我们将三波段（C-D、C-H 和指纹区域）SRS 成像应用于 2D 和 3D 细胞培养物，并对药物摄取和反应进行比较分析，目的是了解药物摄取的差异是否可以解释药物与 2D 培养相比，3D 培养中的耐药性。我们的研究表明，尽管拉帕替尼治疗期间 2D 和 3D A549 细胞的细胞内药物水平相似，但 3D 球体的生长受到的影响较小，支持 3D 微环境中药物耐受性的增强。我们进一步阐明了药物渗透模式以及由此产生的跨不同球体层的异质细胞反应。此外，我们研究了细胞外基质在调节药物递送和细胞反应中的作用，并发现 3D 中有限的药物渗透也可能导致药物反应降低。我们的研究为癌症药物治疗期间 3D 肿瘤模型中耐药性增加的复杂机制提供了宝贵的见解。

The architecture of cell culture, two-dimensional (2D) versus three-dimensional (3D), significantly impacts various cellular factors, including cell-cell interactions, nutrient and oxygen gradients, metabolic activity, and gene expression profiles. This can result in different cellular responses during cancer drug treatment, with 3D-cultured cells often exhibiting higher resistance to chemotherapeutic drugs. While various genetic and proteomic analyses have been employed to investigate the underlying mechanisms of this increased resistance, complementary techniques that provide experimental evidence of spatial molecular profiling data are limited. Stimulated Raman scattering (SRS) microscopy has demonstrated its capability to measure both intracellular drug uptake and growth inhibition. In this work, we applied three-band (C-D, C-H, and fingerprint regions) SRS imaging to 2D and 3D cell cultures and performed a comparative analysis of drug uptake and response with the goal of understanding whether the difference in drug uptake explains the drug resistance in 3D culture compared to 2D. Our investigations revealed that despite similar intracellular drug levels in 2D and 3D A549 cells during lapatinib treatment, the growth of 3D spheroids was less impacted, supporting an enhanced drug tolerance in the 3D microenvironment. We further elucidated drug penetration patterns and the resulting heterogeneous cellular responses across different spheroid layers. Additionally, we investigated the role of the extracellular matrix in modulating drug delivery and cell response and discovered that limited drug penetration in 3D could also contribute to lower drug response. Our study provides valuable insights into the intricate mechanisms of increased drug resistance in 3D tumor models during cancer drug treatments.