PO.IM02.05 · 免疫学
ROCK2 调控的 LIF-STAT3 驱动胰腺癌免疫抑制
ROCK2-regulated LIF-STAT3 drives immunosuppression in pancreatic cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：免疫抑制是胰腺导管腺癌（PDAC）的一个关键特征，促进转移并导致生存不良。我们的研究已鉴定出肿瘤细胞内在的 Rho 相关卷曲螺旋蛋白激酶-2（ROCK2）是细胞外基质重塑的关键调控因子。在本研究中，我们探讨了 ROCK2 如何通过调节白血病抑制因子（LIF）及其对 STAT3 的影响来调控 PDAC 中的免疫抑制。
方法：使用 TCGA PDAC 患者数据集比较正常组织与 PDAC 组织中 ROCK2 和 LIF 的表达。对 PDAC 数据集进行 CIBERSORT 分析，估算肿瘤浸润免疫细胞亚群的比例。在 LSL-Kras G12D/+; Trp53 R172H/+; Pdx1 Cre/+（KPC）细胞中使用 CRISPR/Cas9 系统进行基因组编辑，生成 KPC Rock2 敲除（Rock2 KO）细胞。对 Rock2 EV 和 Rock2 KO 的条件培养基进行细胞因子芯片分析，并用 ELISA 验证结果。使用流式细胞术分析 KPC 原位肿瘤中不同细胞类型的 LIF 受体（LIFR）表达。用重组 LIF（rLIF）和 LIFR 抑制剂（EC359）处理来自 C57BL/6 小鼠的骨髓来源巨噬细胞，随后通过流式细胞术分析其极化。建立 KPC 原位肿瘤，并在 EC359 治疗后进行免疫细胞分析，以评估免疫细胞亚群的改变。ROCK2 及 ROCK2 调控的 LIF-STAT3 靶向的体外和体内研究结果均通过免疫印迹和免疫组化进行验证。
结果：TCGA 数据集分析显示，人 PDAC 组织中 ROCK2 和 LIF 的表达升高且相互关联。进一步分析表明，巨噬细胞构成免疫细胞浸润的相当大比例。细胞因子芯片和基于 ELISA 的研究显示，ROCK2 敲除后 LIF 分泌减少，为 ROCK2 依赖性调控 LIF 提供了证据。KPC 原位肿瘤显示肿瘤相关巨噬细胞（TAM）中 LIFR 表达更高，且 EC359 治疗降低了这些细胞上 ARG1 和 PD-L1 的表达。此外，EC359 治疗使活化效应及效应记忆 T 细胞群体显著增加。再者，rLIF 处理增加了巨噬细胞中的 pSTAT3 水平，而 EC359 降低了其表达，凸显了 ROCK2 调控的 LIF-LIFR 在 STAT3 激活中的作用。
结论：这些发现表明，肿瘤细胞内在的 ROCK2 调控 LIF-STAT3，后者介导免疫抑制，并可作为 PDAC 的潜在治疗靶点。
查看英文原文 English abstract
Background: Immunosuppression is a key characteristic of pancreatic ductal adenocarcinoma (PDAC), contributing to metastasis and poor survival. Our studies have identified tumor cell intrinsic Rho-associated coiled-coil containing protein kinase-2 (ROCK2) as a key regulator of extracellular matrix remodeling. In this study, we investigated how ROCK2 regulates immunosuppression in PDAC by modulating Leukemia inhibitory factor (LIF) and its effects on STAT3.
Methods: TCGA PDAC patient dataset was used to compare the ROCK2 and LIF expression in normal and PDAC tissues. CIBERSORT analysis of the PDAC dataset estimated the proportion of tumor infiltrating immune cell subsets. Genomic editing using the CRISPR/Cas9-system in LSL-Kras G12D/+; Trp53 R172H/+ ; Pdx1 Cre/+ (KPC) cells was performed to generate KPC Rock2 knockout (Rock2 KO ) cells. Cytokine array was performed on Rock2 EV and Rock2 KO conditioned media and ELISA was used to validate the results. Flow cytometry was used to profile LIF receptor (LIFR) expression in different cell types from KPC orthotopic tumors. Bone marrow-derived macrophages from C57BL/6 mice were treated with recombinant LIF (rLIF) and LIFR inhibitor (EC359), then analyzed for polarization by flow cytometry. KPC orthotopic tumors were generated, and Immune cell profiling was performed to evaluate alterations in immune cell subsets following treatment with EC359. Findings from ROCK2 and ROCK2-regulated LIF-STAT3 targeting, both in vitro and in vivo were validated using Immunoblotting and immunohistochemistry.
Results: Analysis of the TCGA dataset revealed that Human PDAC tissues have increased expression and correlation of ROCK2 and LIF. Further analysis showed that macrophages constitute a substantial proportion of the immune cell infiltrate. Cytokine array and ELISA-based studies revealed decreased LIF secretion with ROCK2 knockout, providing evidence for ROCK2 dependent regulation of LIF. KPC orthotopic tumors demonstrated higher LIFR expression in tumor-associated macrophages (TAMs) and EC359 treatment reduced ARG1 and PD-L1 expression on these cells. Additionally, EC359 treatment led to a significant increase in the activated effector and effector memory T cell populations. Furthermore, rLIF treatment increased pSTAT3 levels in macrophages, while EC359 lowered its expression, highlighting the role of ROCK2-regulated LIF-LIFR in STAT3 activation.
Conclusion: These findings demonstrate that tumor cell-intrinsic ROCK2 regulates LIF-STAT3, which mediates immunosuppression and can serve as a potential therapeutic target for PDAC.
利益披露 Disclosure
V. Krishnamoorthy, None..
S. Jinka, None..
S. Mehra, None..
P. Tao, None..
D. Manrique, None..
R. Khurana, None..
Y. Ban, None..
V. Gupta, None..
A. Dosch, None..
N. Nagathihalli, None.