Garaud Soizic, Gu-Trantien Chunyan, Lodewyckx Jean-Nicolas, Boisson Anaïs, De Silva Pushpamali, Buisseret Laurence, Migliori Edoardo, Libin Myriam, Naveaux Céline, Duvillier Hugues, Willard-Gallo Karen
Molecular Immunology Unit, Université Libre de Bruxelles; Institut Jules Bordet, Université Libre de Bruxelles.
Institut d'Immunologie Médicale, Université Libre de Bruxelles.
J Vis Exp. 2014 Dec 6(94):52392. doi: 10.3791/52392.
The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45(+) cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45(+) cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.
恶性细胞逃避免疫系统的能力，其特征为肿瘤逃避先天性和适应性免疫反应，现已被公认为癌症的一个重要标志。我们对乳腺癌的研究聚焦于肿瘤浸润淋巴细胞在肿瘤进展和患者预后中所起的积极作用。为实现这一目标，我们开发了一种从正常和异常组织中快速分离完整淋巴细胞的方法，以便在接近其天然状态下对它们进行评估。与酶消化组织相比，使用机械解离器制备的匀浆显示出活力和细胞回收率均有所提高，同时保留了表面受体表达。此外，对剩余不溶性物质进行酶消化并未回收更多的CD45(+)细胞，这表明初次匀浆中的定量和定性测量可能真实反映了组织碎片中的浸润亚群。这些匀浆中的淋巴细胞可以很容易地通过免疫学（表型、增殖等）或分子（DNA、RNA和/或蛋白质）方法进行表征。CD45(+)细胞也可用于亚群纯化、体外扩增或冷冻保存。这种方法的另一个优点是，匀浆中的初次组织上清液可用于表征和比较正常组织和恶性组织中存在的细胞因子、趋化因子、免疫球蛋白和抗原。该方案对人类乳腺组织非常有效，应该适用于多种正常和异常组织。
