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 monocytes from healthy donors 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-cultures were 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, stroma and immune cells in microencapsulated 3D-3-cultures 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 external stimuli, such as chemotherapeutic and immunomodulatory drugs.

肿瘤微环境（TME）决定疾病的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样细胞浸润，即肿瘤相关巨噬细胞（TAM）。概括TME的不同细胞因子之间的相互作用以及细胞外基质（ECM），对于理解疾病进展的机制至关重要。这对于预测对标准疗法的治疗反应和询问TME靶向剂的功效尤其适用。在这项工作中，我们探索了基于藻酸盐微囊化和搅拌培养系统的培养平台，以开发3D-3-培养，这需要将非小细胞肺癌（NSCLC）肿瘤细胞球体，与癌相关的成纤维细胞进行共培养（CAF）和单核细胞。我们证明3D-3-文化重新创建侵入性和免疫抑制性TME，具有细胞因子/趋化因子（IL4，IL10，IL13，CCL22，CCL24，CXCL1），ECM元素（I型，IV型和纤连蛋白）和基质金属蛋白酶的积累（MMP1 / 9），支持藻类微胶囊内的细胞迁移并促进细胞间的相互作用。重要的是，我们显示单核细胞系THP-1和来自健康供体的单核细胞均浸润肿瘤组织，并跨极化为表达CD68，CD163和CD206的M2样巨噬细胞表型，类似于NSCLC中的TAM表型。用化学和免疫治疗剂攻击3D-3-培养物，并评估每种细胞成分对治疗的反应。具体而言，巨噬细胞表型在接受CSF1R抑制剂BLZ945处理后得到了调节，导致M2类巨噬细胞减少。总之，在微囊化3D-3培养物中，ECM与肿瘤，基质和免疫细胞之间的串扰促进了单核细胞活化为TAM，从而模拟了侵袭性肿瘤阶段。3D-3-培养物是研究肿瘤-免疫相互作用和巨噬细胞可塑性以响应外部刺激（例如化学疗法和免疫调节药物）的一种新颖工具。