背景:粘膜黑素瘤(MM)是由粘膜黑素细胞引起的高度侵袭性肿瘤。目前的治疗方法为晚期MM患者提供了有限的生存益处;此外,临床前细胞系统的缺乏极大地限制了对其免疫生物学的理解.
方法:从鼻窦粘膜中产生的患者来源的MM活检中获得了五种新的细胞系,命名为SN-MM1-5。形态学,通过透射电子显微镜和免疫组织化学验证了SN-MM细胞系的超微结构和黑素细胞身份。此外,通过在NOD/SCID小鼠中皮下注射测试SN-MM1-5的体内致瘤性。SN-MM细胞系的分子表征通过质谱蛋白质组学方法进行,通过Akt激活验证了它们对PI3K化学抑制剂LY294002的敏感性,通过免疫印迹中的pAkt(Ser473)和pAkt(Thr308)测量,和MTS测定。
结果:本研究报告了五种新产生的SN-MM细胞系的验证和功能表征。与正常的对应物相比,SN-MM的蛋白质组学谱与转化的黑素细胞一致,显示出异质程度的黑素细胞分化和癌症相关途径的激活。根据aCGH分析,所有SN-MM细胞系在体内产生致瘤性并显示出复发的结构变体。相关的,相应异种移植的显微镜分析允许鉴定MITF-/CDH1-/CDH2+/ZEB1+/CD271+细胞簇,在MM中也支持黑色素瘤起始细胞的存在,在临床样本中证实。体外,SN-MM细胞系对顺铂敏感,但不是替莫唑胺.此外,SN-MM细胞系的蛋白质组学分析显示,RICTOR,mTORC2复合体的一个亚基,是最显著激活的上游调节器,提示PI3K-Akt-mTOR通路在这些肿瘤中的相关作用。始终如一,在SN-MM中观察到NDRG1的磷酸化和Akt激活,后者是构成性的,并由SN-MM2和SN-MM3中的PTEN损失维持。LY294002诱导的细胞活力损伤证实了在SN-MM细胞系中PI3K-Akt-mTOR途径的功能作用。
结论:总体而言,这些新颖而独特的细胞系统代表了相关的实验工具,可以更好地了解这些肿瘤的生物学特性,作为延伸,来自其他网站的MM。
Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology.
Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay.
This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines.
Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.