PO.MCB09.06 · 分子与细胞生物学
靶向癌症相关成纤维细胞中的糖原代谢可缓解免疫抑制并抑制结直肠癌恶性程度
Targeting glycogen metabolism in cancer-associated fibroblasts alleviates immunosuppression and inhibits colorectal cancer malignancy
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摘要 Abstract
中文摘要
背景：癌症相关成纤维细胞（CAFs）是分泌型细胞因子和趋化因子的主要来源，参与塑造免疫抑制性肿瘤微环境（TME）。糖原代谢失调是癌细胞增殖和转移不可或缺的因素。本研究的目的是确定靶向CAFs中的糖原代谢是否能够缓解免疫抑制并抑制结直肠癌（CRC）生长。
方法：从CRC患者样本中分离人CAFs（hCAFs）。对APC f/f；CDX2-Cre/ERT2小鼠注射他莫昔芬以诱导腺瘤形成。分离并培养小鼠CAFs（mCAFs）。通过实时（RT）-PCR或Western blot测定糖原磷酸化酶肝型（PYGL）、磷酸化PYGL、己糖激酶2（HK2）、GLUT1、IL6、CLCF1、LIF和CXCL6的表达。使用糖原分析试剂盒测定糖原水平。使用WST-1测定细胞增殖。使用Agilent Seahorse XFe96细胞外通量分析仪测定细胞外酸化率（ECAR）。为确定靶向PYGL是否会抑制肿瘤生长，使用了APC f/f；CDX2-Cre/ERT2和MC38小鼠肿瘤模型。
结果：（i）与正常成纤维细胞相比，CAFs中发现葡萄糖代谢升高。（ii）用来源于HCT116 CRC细胞的条件培养基（CM）或TGFbeta处理hCAFs导致糖原代谢激活，表现为p-PYGL/PYGL表达增加和hCAFs中糖原水平降低。此外，CM增加了hCAFs中IL6家族细胞因子如IL6、CLCF1、LIF和CXCL6的表达。（iii）通过敲低PYGL或使用PYG抑制剂CP-91149处理来抑制糖原代谢，显著抑制了hCAFs增殖并降低了hCAFs中这些细胞因子的表达。敲低PYGL降低了糖酵解活性，表现为ECAR降低。（iv）用CP-91149处理显著抑制了肿瘤生长。此外，mCAF细胞与MC38细胞共植入增加了MC38肿瘤生长；这种增加因mCAFs中PYGL的敲低而减弱。
结论：我们的结果表明糖原代谢对于促进CAF免疫抑制功能至关重要。重要的是，我们的发现提示靶向CAFs中的糖原代谢可缓解免疫抑制并抑制CRC恶性程度。
查看英文原文 English abstract
Background : Cancer-associated fibroblasts (CAFs) are the major sources of secreted cytokines and chemokines contributing to the shaping of immunosuppressive tumor microenvironment (TME). Dysregulation of glycogen metabolism is integral to cancer cell proliferation and metastasis. The purpose of this study was to identify whether targeting glycogen metabolism in CAFs could alleviate immunosuppression and inhibit colorectal cancer (CRC) growth.
Methods: Human CAFs (hCAFs) were isolated from CRC patient samples. APC f/f ; CDX2-Cre/ERT2 mice were injected with tamoxifen to induce adenoma formation. Mouse CAFs (mCAFs) were isolated and cultured. The expression of glycogen phosphorylase liver form (PYGL), phospho-PYGL, hexokinase 2 (HK2), GLUT1, IL6, CLCF1, LIF, and CXCL6 was determined by either real time (RT)-PCR or western blot. Glycogen levels were determined using a Glycogen Analysis Kit. Cell proliferation was determined using WST-1. Extracellular acidification rate (ECAR) was determined utilizing an Agilent Seahorse XFe96 extracellular flux analyzer. To determine whether targeting PYGL would inhibit tumor growth, APC f/f ; CDX2-Cre/ERT2 and MC38 mouse tumor models were used.
Results: ( i ) Elevated glucose metabolism in CAFs was found compared to normal fibroblasts. ( ii ) Treatment of hCAFs with either conditioned medium (CM) derived from HCT116 CRC cells or TGFbeta resulted in activated glycogen metabolism as noted by increased expression of p-PYGL/PYGL and the reduced level of glycogen in hCAFs. Moreover, CM increased the expression of IL6 family cytokines such as IL6, CLCF1, LIF and CXCL6 in hCAFs. ( iii ) Inhibition of glycogen metabolism by knockdown of PYGL or treatment with the PYG inhibitor, CP-91149, significantly repressed hCAFs proliferation and reduced the expression of these cytokines in hCAFs. Knockdown of PYGL decreased glycolytic activity as noted by decreased ECAR. ( iv ) Treatment with CP-91149 significantly inhibited tumor growth. In addition, mCAF cells co-implanted with MC38 cells increased MC38 tumor growth; this increase was attenuated by knockdown of PYGL in mCAFs.
Conclusion: Our results demonstrate that glycogen metabolism is crucial for promoting CAF immunosuppressive functions. Importantly, our findings suggest that targeting glycogen metabolism in CAFs alleviates immunosuppression and inhibits CRC malignancy.
利益披露 Disclosure
X. Zhu, None..
Y. Zhou, None..
H. Zhang, None..
Y. Jiang, None..
J. Liu, None..
C. Wang, None..
B. Evers, None..
Q. Wang, None.