Harshman Sean W, Canella Alessandro, Ciarlariello Paul D, Agarwal Kitty, Branson Owen E, Rocci Alberto, Cordero Hector, Phelps Mitch A, Hade Erinn M, Dubovsky Jason A, Palumbo Antonio, Rosko Ashley, Byrd John C, Hofmeister Craig C, Benson Don M, Paulaitis Michael E, Freitas Michael A, Pichiorri Flavia
Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH, USA.
Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.
J Proteomics. 2016 Mar 16;136:89-98. doi: 10.1016/j.jprot.2015.12.016. Epub 2016 Jan 13.
Multiple myeloma (MM) is a hematological malignancy of clonal plasma cells in the bone marrow (BM). The microenvironment plays a key role in MM cell survival and drug resistance through release of soluble factors, expression of adhesion molecules and release of extracellular vesicles (EVs). The aim of this manuscript is to use proteomic profiling of EVs as a tool to identify circulating tumor associated markers in MM patients. First, we characterized the EV protein content obtained from different MM cell lines. Then, we established differences in protein abundance among EVs isolated from MM patient serum and BM and the serum of healthy donors. These data show that the Major Histocompatibility Complex Class I is highly enriched in EVs of MM cell lines and MM patient's serum. Next, we show that CD44 is highly expressed in the EVs isolated from the corticosteroid resistant MM cell line, MM.1R. Furthermore, CD44 was found to be differentially expressed in EVs isolated from newly diagnosed MM patients. Finally through ELISA analysis, we establish the potential of serum CD44 as a predictive biomarker of overall survival. These results support the analysis of EVs as an easily accessible source for MM biomarkers.
Extracellular vesicles are becoming a research focus due to their roles in cancer cell biology such as immune evasion, therapeutic resistance, proliferation and metastases. While numerous studies of vesicle characterization and biology have been conducted in many cancer models, the role of EV in MM remains relatively unstudied. Here we found that EVs isolated from MM cells are enriched in MHC-1 antigen presenting complex and its binding protein β2-MG, this observation is compatible with the enhanced proteasome activity of MM cells compared to other cancers and the ability of functional MHC-1 to bind and present peptides, generated from protein degradation by the proteasome. Additionally, our experiments show that CD44 is particularly enriched in the EV fraction of corticosteroid resistant MM.1R cells and is differentially expressed in the EV fraction of MM patients. This is of high significance due to the established role of CD44 in adhesion of MM cells to BMSC and induction of IL-6, the primary cytokine for MM cell survival, secretion by the BMSC. Furthermore, ELISA assays for CD44 content from the serum of 254 newly diagnosed MM patients enrolled in a Phase 3 randomized trial show highly variable CD44 levels and those patients with >280 ng/mL serum CD44 showing a reduced overall survival time. These results suggest the potential use of CD44 as a prognostic biomarker in MM.
多发性骨髓瘤（MM）是一种骨髓中克隆性浆细胞的血液系统恶性肿瘤。微环境通过释放可溶性因子、表达黏附分子以及释放细胞外囊泡（EVs）在MM细胞存活和耐药性中起关键作用。本论文的目的是利用EVs的蛋白质组学分析作为一种工具，来识别MM患者循环中的肿瘤相关标志物。首先，我们对从不同MM细胞系获得的EV蛋白含量进行了表征。然后，我们确定了从MM患者血清和骨髓以及健康供体血清中分离出的EVs之间蛋白质丰度的差异。这些数据表明，主要组织相容性复合体I类在MM细胞系和MM患者血清的EVs中高度富集。接下来，我们表明CD44在从皮质类固醇耐药的MM细胞系MM.1R中分离出的EVs中高表达。此外，发现CD44在从新诊断的MM患者中分离出的EVs中差异表达。最后通过ELISA分析，我们确定了血清CD44作为总生存期预测生物标志物的潜力。这些结果支持将EVs分析作为MM生物标志物的一个易于获取的来源。
细胞外囊泡因其在癌细胞生物学中的作用，如免疫逃逸、治疗耐药性、增殖和转移，正成为一个研究热点。虽然在许多癌症模型中已经对囊泡的表征和生物学进行了大量研究，但EV在MM中的作用仍相对未被研究。在这里我们发现，从MM细胞中分离出的EVs富含MHC-1抗原呈递复合体及其结合蛋白β2-MG，这一观察结果与MM细胞相比其他癌症增强的蛋白酶体活性以及功能性MHC-1结合和呈递由蛋白酶体蛋白降解产生的肽的能力相一致。此外，我们的实验表明，CD44在皮质类固醇耐药的MM.1R细胞的EV部分中特别富集，并且在MM患者的EV部分中差异表达。这具有高度重要性，因为CD44在MM细胞与骨髓间充质干细胞（BMSC）的黏附中以及诱导IL-6（MM细胞存活的主要细胞因子，由BMSC分泌）方面已确立的作用。此外，对参与一项3期随机试验的254名新诊断MM患者血清中CD44含量的ELISA检测显示，CD44水平高度可变，血清CD44>280 ng/mL的患者总生存时间缩短。这些结果表明CD44在MM中作为预后生物标志物的潜在用途。
