Pd l1 clone e1l3n

Slides created from cell line control (CLC)-spiked HD samples or bladder cancer patient samples were subjected to automated IF staining for cytokeratin (CK), CD45 (hematopoietic marker) and PD-L1 (clone E1L3N, Cell Signaling Technology). Stained slides were analyzed with fluorescent scanners and morphology algorithms for the identification of traditional CK⁺ CTCs, CTC clusters, apoptotic CTCs and CK⁻CTCs morphologically distinct from hematological cells [26 ]. Trained classifiers conducted final classification of CTC subpopulations based on morphological parameters and biomarker expression. CLC slides were stained in parallel with patient samples. Threshold for PD-L1 cell positivity in patient samples was set to 95 % specificity of negative control CLC signal (i.e., 95 % negative control cell line cells lie below threshold).

To investigate potential differences in the tumor microenvironment (TME) between adjacent lymph nodes with disparate treatment effects, surgical specimens were stained using antibodies to PD-L1 (clone E1L3N 1:50, Cell Signaling, MA, USA), CD8 (clone SP57 prediluted, Roche, Switzerland) CD163 (clone MRQ-26 prediluted, Roche) or FoxP3 (clone SP97 1:100, Spring Bioscience, CA, USA) to determine the number of positive cells per high-powered field. For each tumor assessed, image analysis was performed at three different regions of interest (ROI) (tumor-stromal interface, tumor, and stroma). For each ROI, three separate areas representing the highest concentrations of target cell population were analyzed per slide. Quantitative analysis of CD8, CD163, and FoxP3 was performed using Visiopharm (Visiopharm, Denmark), utilizing a linear type Bayesian classification to determine cell positivity. PD-L1 expression was performed semi-quantitatively, with cell counts determined by pathologist interpretation (<1, 1–20, and >20% TPS score). Importantly, patients receiving tadalafil were excluded from all TME analysis, in order to avoid potential confounding effects of tadalafil on TME.

Immunohistochemistry was performed to detect the presence of PD-L1, CD8, FoxP3, and 4-1BB/CD137 in the whole tumor and invasive margin (IM) of pretreatment tumor biopsies. Immunohistochemistry testing of PD-L1 (clone E1L3N; Cell Signaling, Danvers, MA), CD8 (clone C8/144B; Dako, Carpinteria, CA), FoxP3 (clone 236A/E7; Cell Signaling), and 4-1BB/CD137 (BBK-2; ThermoFisher, Rockford, IL) was performed by Mosaic Laboratories, LLC (Lake Forest, CA).

Total cellular proteins were extracted using lysis buffer (5 mM EDTA, 300 mM NaCl, 0.1% NP-40, 0.5 mM NaF, 0.5 mM Na3VO4, 0.5 mM PMSF, and 10 μg/mL each of aprotinin, pepstatin, and leupeptin; Sigma-Aldrich). A total of 30–50 μg protein was separated using 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Then immunoblotting was performed using antibodies against PD-L1 (clone E1L3N), GLUT1, HK2, PKM2, P-Akt, Akt (Cell Signaling Technology, Danvers, MA, USA), P-Erk, Erk, P-p38MAPK, p38MAPK, and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). Most images of western blots were from parallel gels and actin images were obtained from the stripped and re-probed blots. The immunoblots were visualized using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Uppsala, Sweden).

The data of CD4+, CD8+, and FOXP3+ TILs and PD-L1+ ICs were adopted from our previous studies [25 (link), 26 (link)] for all of the cases of pre-invasive carcinoma and 307 cases of invasive carcinoma. Immunohistochemical staining had been carried out using the following antibodies: CD4 (clone SP35; ready to use; Dako), CD8 (clone C8/144B; ready to use; Dako), FOXP3 (clone 236A/E7; 1:100; Abcam) and PD-L1 (clone E1L3N; 1:100; Cell Signaling, Danvers, MA, USA). CD4+, CD8+, and FOXP3+ T cells had been counted in intratumoral and stromal areas as absolute numbers per high-power field. Detailed information on the counting method of TILs is described in the previous studies [25 (link), 26 (link)]. For this study, CD4+, CD8+, and FOXP3+ TILs were dichotomized into high- and low-infiltration groups using cutoff values obtained by ROC curve analyses. PD-L1+ ICs were considered to be present when at least 1% of the tumor stromal area was occupied by PD-L1+ ICs.