Given the fact that activated fibroblasts play an important role in the progression of malignant disease as well as in various non-malignant diseases of the lung, we aimed to AG-013736 investigate the prevalence of Hsp27 positive tumor stroma in CRC lung metastases and corresponding Gefitinib primary tumors. Furthermore, we sought to describe the implications on the clinical outcome after metastasectomy and the presence of cellular and secreted Hsp27 in these patients. If patients had undergone pulmonary metastasectomy before, specimen of the first metastasectomy was also examined and the date of the first metastasectomy was used for outcome calculation. Lung metastasis free survival was defined as the time between diagnosis of the primary tumor and diagnosis of the metastatic spreading to the lung. R0 resection was achieved in all patients. Of 33 patients, specimens of the corresponding primary tumor could be obtained. Follow- up examinations were carried out in 3 to 6 months intervals. Recurrence-free survival was defined as the period from the first pulmonary metastasectomy to evidence of recurrent disease at any site verified by CT scans. In 10 consecutive cases, serum samples obtained before metastasectomy and during follow-up were available. Follow-up serum samples were collected 3 to 6 months after surgery. Additionally, serum samples from age-, gender- and smoking status- matched healthy volunteers were collected. All patients gave their written informed consent prior to blood collection and participation. Immunohistochemical staining was performed according to a standard protocol. Shortly, formalin- fixed, paraffin-embedded tissue specimens were cut in 4-��m thick sections and deparaffinized. Heat-mediated antigen retrieval was performed. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide. Sections were incubated with the appropriate primary antibody for 1h at room temperature. For immunohistochemistry, as secondary step, the polymer- based ImmPRESS kit was used according to the manufacturer��s protocol. 3,3��-Diaminobenzidine was used as substrate. Finally, the sections were counterstained with hematoxylin. Vimentin staining was performed in a Ventana ES Immunostainer System. For immunofluorescence, appropriate fluorescent secondary antibodies were used and cell nuclei were counterstained with DAPI. As negative controls, the primary antibody was omitted. Positive controls were tissue samples with known presence of the respective target protein. Immunohistochemistry staining score was calculated as described previously. The percentage of positive tumor cells could reach values between 0 and 100%, and were multiplied by the staining intensity. Thus, IHC scores could range from 0 to 300. Two blinded observers rated the staining. In case that the two ratings differed, the slide was discussed and re-evaluated. The continuous IHC score was dichotomized by applying the median score of metastases or primary tumors as cut-off value. Distant metastases are the main cause of cancer-related mortality. Today, the tumor microenvironment and especially fibroblasts become of growing interest, providing additional information on tumor behavior and potential therapeutic targets.