Mocellin Simone, Provenzano Maurizio, Rossi Carlo R, Pilati Pierluigi, Nitti Donato, Lise Mario
Surgery Branch, Department of Oncological and Surgical Sciences, University of Padova, Padova, Italy.
J Immunol Methods. 2003 Sep;280(1-2):1-11. doi: 10.1016/s0022-1759(03)00274-6.
The molecular mechanisms underlying tumor responsiveness to immunotherapeutic manipulations remain elusive. Investigators are therefore searching for new technologies to study immune-related events occurring in the tumor microenvironment.
To validate the use of quantitative real-time PCR (qrt-PCR) for assessing immune cell density and cytokine (CK) gene profile in tumor biopsies obtained from patients treated with TNFalpha-based isolated limb perfusion.
We first assessed in vitro the ability of cell marker coding genes (CD4, CD8, CD14, CD56) to serve as housekeeping genes for helper and cytotoxic T-lymphocytes, macrophages and NK cells, respectively. Then, the correspondence between mRNA and protein levels of five CK (IL-2, IFNgamma, IL-4, IL-10 and TGFbeta1) expressed by stimulated PBMC was evaluated by means of qrt-PCR and ELISA, respectively. Finally, six patients affected with locally advanced soft tissue sarcomas underwent tumor biopsy before and after TNFalpha-based isolated limb perfusion. After RNA extraction and amplification, transcriptional levels of the above cell markers and CK were evaluated by qrt-PCR.
In vitro, leukocyte cell subsets constantly expressed the corresponding marker gene both under resting conditions and after cell stimulation. Cytokine mRNA levels expressed by stimulated PBMC corresponded significantly to supernatant protein concentrations. Compared to the pre-treatment gene profile, post-treatment gene expression showed higher levels of CD4 and IFNgamma and a decreased abundance of the TGFbeta1 transcript.
In vitro we found that qrt-PCR can determine accurately immune cell density and CK gene profiles in tumor biopsies. In vivo findings support the hypothesis that, after TNFalpha-based treatment, a Th1-type shift occurs in the tumor microenvironment.
肿瘤对免疫治疗操作反应的分子机制仍不清楚。因此，研究人员正在寻找新技术来研究肿瘤微环境中发生的免疫相关事件。
验证定量实时聚合酶链反应（qrt-PCR）用于评估接受基于肿瘤坏死因子α的隔离肢体灌注治疗的患者肿瘤活检中免疫细胞密度和细胞因子（CK）基因谱的用途。
我们首先在体外评估细胞标志物编码基因（CD4、CD8、CD14、CD56）分别作为辅助性和细胞毒性T淋巴细胞、巨噬细胞和自然杀伤细胞管家基因的能力。然后，分别通过qrt-PCR和酶联免疫吸附测定法评估受刺激外周血单核细胞表达的5种CK（白细胞介素-2、γ干扰素、白细胞介素-4、白细胞介素-10和转化生长因子β1）的信使核糖核酸和蛋白质水平之间的对应关系。最后，6例局部晚期软组织肉瘤患者在接受基于肿瘤坏死因子α的隔离肢体灌注治疗前后进行肿瘤活检。提取并扩增核糖核酸后，通过qrt-PCR评估上述细胞标志物和CK的转录水平。
在体外，白细胞亚群在静息条件下和细胞刺激后均持续表达相应的标志物基因。受刺激外周血单核细胞表达的细胞因子信使核糖核酸水平与上清液蛋白质浓度显著对应。与治疗前基因谱相比，治疗后基因表达显示CD4和γ干扰素水平升高，转化生长因子β1转录本丰度降低。
在体外，我们发现qrt-PCR可以准确测定肿瘤活检中的免疫细胞密度和CK基因谱。体内研究结果支持以下假设：在基于肿瘤坏死因子α的治疗后，肿瘤微环境中会发生1型辅助性T细胞偏移。
