iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157, Oeiras, Portugal.
Boehringer Ingelheim RCV GmbH & Co KG, Department of Lead Discovery, 1121, Vienna, Austria.
Biomaterials. 2018 May;163:185-197. doi: 10.1016/j.biomaterials.2018.02.030. Epub 2018 Feb 13.
The tumour microenvironment (TME) shapes disease progression and influences therapeutic response. Most aggressive solid tumours have high levels of myeloid cell infiltration, namely tumour associated macrophages (TAM). Recapitulation of the interaction between the different cellular players of the TME, along with the extracellular matrix (ECM), is critical for understanding the mechanisms underlying disease progression. This particularly holds true for prediction of therapeutic response(s) to standard therapies and interrogation of efficacy of TME-targeting agents. In this work, we explored a culture platform based on alginate microencapsulation and stirred culture systems to develop the 3D-3-culture, which entails the co-culture of tumour cell spheroids of non-small cell lung carcinoma (NSCLC), cancer associated fibroblasts (CAF) and monocytes. We demonstrate that the 3D-3-culture recreates an invasive and immunosuppressive TME, with accumulation of cytokines/chemokines (IL4, IL10, IL13, CCL22, CCL24, CXCL1), ECM elements (collagen type I, IV and fibronectin) and matrix metalloproteinases (MMP1/9), supporting cell migration and promoting cell-cell interactions within the alginate microcapsules. Importantly, we show that both the monocytic cell line THP-1 and peripheral blood-derived monocytes infiltrate the tumour tissue and transpolarize into an M2-like macrophage phenotype expressing CD68, CD163 and CD206, resembling the TAM phenotype in NSCLC. The 3D-3-culture was challenged with chemo- and immunotherapeutic agents and the response to therapy was assessed in each cellular component. Specifically, the macrophage phenotype was modulated upon treatment with the CSF1R inhibitor BLZ945, resulting in a decrease of the M2-like macrophages. In conclusion, the crosstalk between the ECM and tumour, stromal and immune cells in microencapsulated 3D-3-culture promotes the activation of monocytes into TAM, mimicking aggressive tumour stages. The 3D-3-culture constitutes a novel tool to study tumour-immune interaction and macrophage plasticity in response to external stimuli, such as chemotherapeutic and immunomodulatory drugs.
肿瘤微环境（TME）塑造疾病进展并影响治疗反应。大多数侵袭性实体瘤具有高水平的髓系细胞浸润，即肿瘤相关巨噬细胞（TAM）。重现 TME 中不同细胞成分以及细胞外基质（ECM）之间的相互作用，对于理解疾病进展的机制至关重要。这对于预测对标准治疗的治疗反应和研究 TME 靶向药物的疗效尤其重要。在这项工作中，我们探索了基于海藻酸钠微囊化和搅拌培养系统的培养平台，以开发 3D-3 共培养，其中包括非小细胞肺癌（NSCLC）肿瘤细胞球、癌症相关成纤维细胞（CAF）和单核细胞的共培养。我们证明，3D-3 共培养重现了侵袭性和免疫抑制性的 TME，其中细胞因子/趋化因子（IL4、IL10、IL13、CCL22、CCL24、CXCL1）、细胞外基质成分（I 型、IV 型胶原和纤维连接蛋白）和基质金属蛋白酶（MMP1/9）的积累，支持细胞迁移并促进海藻酸钠微胶囊内的细胞-细胞相互作用。重要的是，我们表明单核细胞系 THP-1 和外周血来源的单核细胞都浸润肿瘤组织并转分化为表达 CD68、CD163 和 CD206 的 M2 样巨噬细胞表型，类似于 NSCLC 中的 TAM 表型。3D-3 共培养用化疗和免疫治疗药物进行了挑战，并评估了每种细胞成分对治疗的反应。具体而言，在用 CSF1R 抑制剂 BLZ945 处理后，巨噬细胞表型发生了调制，导致 M2 样巨噬细胞减少。总之，在微囊化 3D-3 共培养物中，ECM 与肿瘤、基质和免疫细胞之间的串扰促进单核细胞向 TAM 的激活，模拟侵袭性肿瘤阶段。3D-3 共培养物构成了一种新的工具，可用于研究肿瘤-免疫相互作用和对外界刺激（如化疗和免疫调节药物）的巨噬细胞可塑性。
