PO.TB02.02 · 肿瘤生物学
多柔比星在胰腺导管腺癌中肿瘤特异性地诱导表面钙网蛋白
Tumor specific induction of surface calreticulin by doxorubicin in pancreatic ductal adenocarcinoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：胰腺导管腺癌（PDAC）占大多数胰腺癌病例。尽管在外科手术和化疗方面取得了进展，5年生存率仅为13%。PDAC的侵袭性本质加之诊断延迟，凸显了对新型靶向诊断和治疗策略的需求。钙网蛋白（CALR）是一种内质网伴侣蛋白，在化疗等应激条件下可易位至细胞表面。我们假设全身性多柔比星可在PDAC中诱导CALR的肿瘤特异性表面易位。
方法：将小鼠2838c3 PDAC细胞皮下注射入小鼠。一旦肿瘤可触及，即以间隔48小时的两次腹腔注射多柔比星（10 mg/kg）或生理盐水处理小鼠，然后处死。采集肿瘤和主要器官（心、肺、肾、胰腺、结肠、小肠、肝和脾），制备用于流式细胞术或免疫荧光分析。对于流式细胞术，将肿瘤和器官解离为未透化、固定的单细胞悬液，并用可固定的活/死染料和抗CALR抗体染色。免疫荧光染色在未透化条件下使用抗CALR抗体和麦胚凝集素（WGA）膜标记进行。在Fiji中将信号强度定量为积分密度/面积。采用带Welch校正的非配对t检验进行比较。
结果：与生理盐水处理的对照相比，多柔比星处理显著增加了PDAC肿瘤中表面CALR的表达，经流式细胞术测量（增加3.0倍；p < 0.05）和免疫荧光测量（增加1.3倍；9623 AU ± 522.6 对 7341 AU ± 485.9，均值 ± SEM，p = 0.0021）。表面CALR的增加在各生物学重复间一致。重要的是，经流式细胞术或免疫荧光分析，多柔比星诱导后，在肝、脾、心、肺、肾、胰腺、结肠或小肠等非肿瘤组织中CALR表达无显著差异。
查看英文原文 English abstract
Background Pancreatic ductal adenocarcinoma (PDAC) accounts for most pancreatic cancer cases. Despite advances in surgery and chemotherapy, the 5-year survival rate is only 13%. The aggressive nature of PDAC combined with delayed diagnosis highlights the need for new targeted diagnostic and treatment strategies. Calreticulin (CALR) is an endoplasmic reticular chaperone protein that can translocate to the cell surface under stress conditions such as chemotherapy. We hypothesized that systemic doxorubicin induces tumor specific surface translocation of CALR in PDAC.
Methods Mouse 2838c3 PDAC cells were injected subcutaneously into mice. Once the tumors were palpable, the mice were treated with two intraperitoneal injections of doxorubicin (10 mg/kg) or saline, spaced 48 hours apart and then sacrificed. Tumors and major organs (heart, lung, kidney, pancreas, colon, small intestine, liver, and spleen) were harvested and prepared for either flow cytometry or immunofluorescence analyses. For flow cytometry, tumors and organs were dissociated into non-permeabilized, fixed single cell suspensions and stained with a fixable live/dead dye and an anti-CALR antibody. Immunofluorescence staining was performed under non-permeabilized conditions using an anti-CALR antibody and wheat germ agglutinin (WGA) membrane mask. Signal intensity was quantified in Fiji as integrated density/area. Unpaired t-test with Welch's correction was utilized for comparison.
Results Doxorubicin treatment significantly increased surface CALR expression in PDAC tumors compared to saline-treated controls as measured by flow cytometry (3.0-fold increase; p < 0.05) and immunofluorescence (1.3-fold increase; 9623 AU ± 522.6 vs. 7341 AU ± 485.9, mean ± SEM, p = 0.0021). Increases in surface CALR were consistent across biological replicates. Importantly, there were no significant differences in CALR expression after doxorubicin induction in non-tumor tissues such as liver, spleen, heart, lung, kidney, pancreas, colon, or small intestine via flow cytometry or immunofluorescence analyses.
利益披露 Disclosure
J. A. Watts, None..
R. Watts, None..
C. La Prairie, None..
J. B. Rose, None..
R. Guenter, None.