LBPO.IM02 · 免疫学 · Late-Breaking
用于免疫肿瘤学候选药物评估的肿瘤类器官-基质微环境中的定量MDSC浸润测定法
A quantitative MDSC infiltration assay in a tumor organoid-stromal microenvironment for immuno-oncology candidate evaluation
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：髓源性抑制细胞（MDSC）是癌症中免疫抑制和治疗耐药的关键驱动因素，但用于量化MDSC功能和迁移的实用、贴近人类的检测方法仍然有限。
方法：我们采用细胞因子驱动的分化条件建立了一个THP-1来源的MDSC样细胞平台，并通过流式细胞术和qRT-PCR验证其表型和免疫相关基因表达。使用PBMC/T细胞聚集测定（包括直接共培养和条件培养基暴露）检验其免疫抑制功能。为模拟迁移，我们开发了一种Matrigel包被的Transwell浸润测定（5 μm孔径），将MDSC样细胞接种于上室，将结直肠癌（CRC）类器官和/或癌症相关成纤维细胞（CAF）置于下室。通过基于成像的计数和流式细胞术对浸润细胞进行定量。最后，使用CellTiter-Glo 3D对候选MDSC靶向药物的选择性效应进行分析，并在Transwell系统中评估其对MDSC浸润的抑制作用。
结果：THP-1来源的MDSC样细胞表现出与MDSC一致的表型和免疫调节基因特征，并与传统THP-1巨噬细胞分化条件相区别。在功能上，这些细胞通过接触依赖性和可溶性因子介导的机制抑制T细胞聚集。Transwell测定能够定量测量MDSC向类器官、CAF或两者组合的浸润，并通过成像和FACS提供正交读出。药物分析揭示了在类器官、MDSC样细胞和CAF之间的差异活性，多个候选药物以浓度依赖性方式调节MDSC浸润的程度。
结论：我们提出了一个整合的、基于类器官的工作流程，将MDSC样细胞生成、免疫抑制功能测定以及在CRC类器官-CAF微环境中的定量浸润读出相结合。该平台可在贴近人类的环境中支持基于机制的筛选和MDSC调节策略的临床前评估。
查看英文原文 English abstract
Background: Myeloid-derived suppressor cells (MDSCs) are key drivers of immunosuppression and therapy resistance in cancer, but practical, human-relevant assays to quantify MDSC function and trafficking remain limited.
Methods: We established a THP-1-derived MDSC-like cell platform using a cytokine-driven differentiation condition and validated phenotype and immune-related gene expression by flow cytometry and qRT-PCR. Immunosuppressive function was tested using PBMC/T-cell clustering assays, including direct co-culture and conditioned-medium exposure. To model migration, we developed a Matrigel-coated transwell infiltration assay (5-µm pore) in which MDSC-like cells were seeded in the upper chamber and colorectal cancer (CRC) organoids and/or cancer-associated fibroblasts (CAFs) were placed in the lower chamber. Infiltrated cells were quantified by imaging-based counting and flow cytometry. Finally, candidate MDSC-targeting agents were profiled for selective effects using CellTiter-Glo 3D and for inhibition of MDSC infiltration in the transwell system.
Results: THP-1-derived MDSC-like cells showed an MDSC-consistent phenotype and immune-regulatory gene signatures, and were distinct from conventional THP-1 macrophage differentiation conditions. Functionally, these cells suppressed T-cell clustering through both contact-dependent and soluble-factor-mediated mechanisms. The transwell assay enabled quantitative measurement of MDSC infiltration toward organoids, CAFs, or their combination, and provided orthogonal readouts by imaging and FACS. Drug profiling revealed differential activity across organoids, MDSC-like cells, and CAFs, and several candidates modulated the extent of MDSC infiltration in a concentration-dependent manner.
Conclusions: We present an integrated organoid-based workflow that combines MDSC-like cell generation, immunosuppressive function assays, and quantitative infiltration readouts in a CRC organoid-CAF microenvironment. This platform can support mechanism-informed screening and preclinical evaluation of MDSC-modulating strategies in a human-relevant setting.
利益披露 Disclosure
B. Lee,
Lambda biologics ).
ORGANOIDSCIENCES Copyright.
W. Yang,
ORGANOIDSCIENCES ).
Y. Go,
ORGANOIDSCIENCES ).
P. Nguyen,
VOS Discovery ).
D. Lim,
ORGANOIDSCIENCES ).