Use of quantitative real-time PCR to determine immune cell density and cytokine gene profile in the tumor microenvironment

Background: 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. Aim: 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 TNFα-based isolated limb perfusion. Materials and methods: 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, IFNγ, IL-4, IL-10 and TGFβ1) 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 TNFα-based isolated limb perfusion. After RNA extraction and amplification, transcriptional levels of the above cell markers and CK were evaluated by qrt-PCR. Results: 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 IFNγ and a decreased abundance of the TGFβ1 transcript. Conclusion: 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 TNFα-based treatment, a Th1-type shift occurs in the tumor microenvironment.