肿瘤组织硬度对巨噬细胞极化及其功能的影响

Tumor microenvironment is not only a chemical microenvironment, but also associated with physical microenvironment. Extracellular matrix increased during the development of solid tumours, and the stiffness of tumour tissue is significantly harder than organizations in adjacent normal tissue. Previous studies have shown that the stiffness of tumour tissue have important influence on tumour cell proliferation, differentiation, motility, invasion and metastasis. But the role of the extracellular matrix stiffness affected the immune cells in the tumor microenvironment is not clear. Tumor-infiltrating macrophages were the largest number of immune cells and mainly of the M2 phenotype in the tumor microenvironment. These macrophages can be tricked by tumor cells to help the cancer grow by promoting angiogenesis, suppressing the anti-tumor immune response, and promoting tumour growth. Our previous study showed that macrophages had different morphology and phenotype when they were grown on different stiffness gels. What impact does tumor tissue stiffness have on M2 phenotype transformation? About this project，two-dimensional polyacrylamide gels coated collagen type I have been used to culture macrophage and to study morphology and phenotype of macrophages on different stiffness gels and find M2 phenotype transformation mechanism. Hoping to provide a new insight for tumor microenvironment intervene and functional switch of M2 phenotype macrophages.

肿瘤微环境不仅只是一个化学的微环境，而且还是具有一定刚性的物理微环境。实体肿瘤的发生、发展常伴随着胞外基质增加，其组织硬度明显高于癌旁正常组织。有研究证明肿瘤微环境的软硬度深刻影响肿瘤细胞的增殖、分化、运动以及转移。但组织硬度对肿瘤微环境中的免疫细胞产生何种影响，现尚不清楚。巨噬细胞作为肿瘤微环境中数量最多的免疫细胞，主要以M2型表型为主，通过促进新生血管形成、抑制抗肿瘤免疫等机制促进肿瘤的生长。我们初步研究发现巨噬细胞受基质软硬度的影响其生长形态和表型均不相同，那么肿瘤的基质硬度是否对巨噬细胞M2型转化具有一定的影响？本项目拟采用胶原蛋白-聚丙烯酰胺二维凝胶模拟不同的基质硬度，研究基质软硬度对巨噬细胞表型和功能的影响，探究肿瘤组织基质硬度对巨噬细胞向M2型转化的重要作用及机理，从物理软硬度这一新的角度，加深对肿瘤微环境的认识，为干预治疗微环境以及逆转M2型肿瘤相关巨噬细胞提供新思路。

本研究阐明了基质硬度可以通过影响巨噬细胞的极化来实现其生物学功能。在较软的基质环境下巨噬细胞更易向M1型转化，而在接近肿瘤的硬度则更易向M2型转化。肿瘤组织硬度通过促进巨噬细胞分泌VEGF等生长因子来加速肿瘤细胞的生长，而通过分泌IL-10等细胞因子来抑制T细胞的活化。在肿瘤组织力学信号的参与下，肿瘤相关巨噬细胞表现出极强的促进肿瘤生长和抑制免疫的作用。巨噬细胞通过整合素Integrin与胞外基质相连。基质硬度以力学信号的形式传递给Integrin蛋白进而激活下游的粘附斑激酶（focal adhesion kinase，FAK），再通过一系列下游信号传递给核转录因子过氧化物酶体增殖物激活受体PPARγ，通过活化这一核转录因子增强Arginase-1，IL-10和VEGF的表达，促进巨噬细胞向M2型转化。在这一过程中，我们发现PPARγ并不完全是随着整合素Integrin的表达越高而活化越强的， rigid上的巨噬细胞PPARγ表达较8kPa上的微微下调，这可能是由于rigid这个硬度已经超出了体内巨噬细胞生长的基质硬度范围。细胞本身的硬度要与其所处的环境硬度相匹配才能维持其正常的生理功能，当基质环境过硬或过软都会导致细胞骨架结构发生变化进而影响其生物学活性。肿瘤组织硬度一般在2k-20kPa不等，胞外基质成分越多肿瘤组织越硬，而巨噬细胞主要位于这些相对较硬的胞外基质部位，在肿瘤微环境中，化学信号协同力学信号一起作用于肿瘤相关巨噬细胞，诱导其向抑制型转化。肿瘤相关巨噬细胞一方面分泌促肿瘤细胞生长的因子，而另一面又抑制T细胞活化，使针对肿瘤的特异性免疫反应减弱，帮助肿瘤细胞逃避免疫系统的监控。