Liver Cancer Institute, Zhongshan Hospital, Fudan University & Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, China.
Department of Radiology, Shanghai Cancer Center, Fudan University, Shanghai, China.
FEBS J. 2021 Jun;288(11):3465-3477. doi: 10.1111/febs.15566. Epub 2020 Oct 6.
Previously, we reported that the secreted lysyl oxidase like 2 (LOXL2) from hepatocellular carcinoma (HCC) cells under higher stiffness stimulation contributed to the formation of lung premetastatic niche. To further clarify whether matrix stiffness also alters LOXL2 expression in other cells within tumor microenvironment, we developed a gel-based culture system combined with a model of macrophage polarization to evaluate the effects of matrix stiffness on the polarization of M2 macrophages and their LOXL2 expression. THP-1 cells cultured on 6KPa, 10KPa, and 16KPa stiffness substrates were first incubated with 100nM phorbol 12-myristate 13-acetate (PMA) for 24 hours and subsequently treated with 20nM interleukin-4 (IL-4) and 20nM interleukin-13 (IL-13) for 48 hours. The polarization states of M2 macrophages under different stiffness stimulation were comparatively analyzed, and their LOXL2 expressions as well as the underlying molecular mechanism were further explored. Our results demonstrated that increased matrix stiffness remarkably strengthened M2 macrophage polarization and promoted their LOXL2 expression. Activation of integrin β5-FAK-MEK1/2-ERK1/2 pathway participated in matrix stiffness-mediated HIF-1α upregulation, and HIF-1α upregulation resulted in a significant improvement in LOXL2 expression. Additionally, M2 macrophage polarization state and LOXL2 expression in HCC tissues with COL1 /LOX were consistent with the results in vitro, further confirming the regulation roles of matrix stiffness in macrophage polarization and LOXL2 expression. The findings about LOXL2 upregulation in the polarized macrophages under higher stiffness stimulation will be helpful to better understand the underlying mechanism of matrix stiffness-induced premetastatic niche formation in HCC.
先前，我们报道了在较高硬度刺激下，来自肝癌（HCC）细胞的分泌赖氨酸氧化酶样 2（LOXL2）有助于形成肺前转移灶生态位。为了进一步阐明基质硬度是否也会改变肿瘤微环境中其他细胞内的 LOXL2 表达，我们开发了一种基于凝胶的培养系统，结合巨噬细胞极化模型，以评估基质硬度对 M2 巨噬细胞极化及其 LOXL2 表达的影响。将 THP-1 细胞培养在 6KPa、10KPa 和 16KPa 硬度基质上，首先用 100nM 佛波醇 12-肉豆蔻酸 13-醋酸盐（PMA）孵育 24 小时，然后用 20nM 白细胞介素 4（IL-4）和 20nM 白细胞介素 13（IL-13）处理 48 小时。比较分析了不同硬度刺激下 M2 巨噬细胞的极化状态，并进一步探讨了其 LOXL2 表达及其潜在的分子机制。结果表明，基质硬度的增加显著增强了 M2 巨噬细胞的极化，并促进了其 LOXL2 表达。整合素 β5-FAK-MEK1/2-ERK1/2 通路的激活参与了基质硬度介导的 HIF-1α 上调，HIF-1α 上调导致 LOXL2 表达显著改善。此外，COL1/LOX 肝癌组织中 M2 巨噬细胞极化状态和 LOXL2 表达与体外结果一致，进一步证实了基质硬度对巨噬细胞极化和 LOXL2 表达的调节作用。在较高硬度刺激下极化巨噬细胞中 LOXL2 上调的发现将有助于更好地理解基质硬度诱导 HCC 前转移灶形成的潜在机制。
