title: Use of quantitative real-time PCR to determine immune cell density and cytokine gene profile in the tumor microenvironment.
abstract: 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.
  
  
  OBJECTIVE
  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.
  
  
  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, 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.
  
  
  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 IFNgamma and a decreased abundance of the TGFbeta1 transcript.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Surgery Branch, Department of Oncological and Surgical Sciences, University of Padova, Padova, Italy.
authors.forename: Simone
authors.initials: S
authors.lastname: Mocellin
authors.forename: Maurizio
authors.initials: M
authors.lastname: Provenzano
authors.forename: Carlo R
authors.initials: CR
authors.lastname: Rossi
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Pilati
authors.forename: Donato
authors.initials: D
authors.lastname: Nitti
authors.forename: Mario
authors.initials: M
authors.lastname: Lise
mesh_terms: D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000473:pathology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000473:pathology; D002452:Cell Count; D010478:Chemotherapy, Cancer, Regional Perfusion; D016207:Cytokines / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D066298:In Vitro Techniques; D007694:Killer Cells, Natural / Q000276:immunology / Q000473:pathology; D009000:Monocytes / Q000276:immunology / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D012509:Sarcoma / Q000235:genetics* / Q000276:immunology* / Q000473:pathology / Q000628:therapy; D012983:Soft Tissue Neoplasms / Q000235:genetics* / Q000276:immunology* / Q000473:pathology / Q000628:therapy; D014409:Tumor Necrosis Factor-alpha / Q000008:administration & dosage / Q000627:therapeutic use
chemical_list: D016207:Cytokines; D017931:DNA Primers; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D014409:Tumor Necrosis Factor-alpha