Quantifying Tumor Immune Microenvironment

Tissue from the pathological routine diagnostics was used for histological examination. To characterize the TIME, we chose CD4 (Ventana, anti-CD4 Rabbit Monoclonal Primary Antibody, Clone SP35, dilution: 1:200) and CD8 (Ventana, anti-CD8 Rabbit Monoclonal Primary Antibody, Clone SP57, dilution: 1:200) as marker for lymphocytes, CD68 (Dako Anti-Human CD68, Clone KP1, dilution: 1:200) as a pan marker for macrophages and CD163 (Novocastra, Lyophilized Mouse Monoclonal Antibody CD163, Clone 10D6, dilution: 1:100) as a marker for M2-macrophages. First, a new hematoxylin-eosin slide was prepared, and the tumor was identified (Figure 1A,B). All immunohistochemical stains were performed on tissue sections (4 µm thickness) prepared from formalin-fixed (4% neutral buffered formalin) paraffin-embedded tissue blocks. The procedure is part of the established routine diagnostic at our institute. Briefly, immunohistochemical staining was performed using a Roche Ventana Benchmark Ultra automated slide stainer (Ventana Medical Systems, Roche, France) with the OptiView DAB IHC Detection Kit (Roche, France). The first step of the automatic staining program includes deparaffinization and rehydrating the specimens. Then antigen retrieval was completed by heat treatment lasting for 32 min with Tris-EDTA Borat Puffer (pH 8–8.5). After incubation of the diluted primary antibodies, the nuclei were counterstained with hematoxylin.
All slides were scanned (3DHISTECH Ltd. Pannoramic slide scanner 250) and evaluated using a virtual microscopy software (3DHISTECH Ltd. Case Viewer Ver.2.2). To quantify lymphocytes, ten high power fields (HPF) were identified independently by two pathologists (A.M. & C.B.). The invasion front and tumor center were identified, and the positive cells were counted. The mean values were calculated. The CD68 reaction was used to visualize all macrophages. To evaluate the quantity of the macrophages, ten HPF of the invasion tumor front and intra-tumoral area were examined and the positive cells were counted. The mean value (confidence interval 95%) of macrophages within the tumor front and the intra-tumoral area was calculated. The macrophages were then characterized concerning their subpopulations. M2-macrophages were detected using CD163-antibody. The M2-macrophages were quantified, utilizing the same previously described procedure for quantifying CD68-positive macrophages. The number of M1-macrophages per HPF was calculated through the difference between CD68-positive-macrophages and CD163-positive-M2-macrophages.