Human serum albumin (hsa)

Peripheral blood, uninvolved LNs, metastatic LNs, and tumor samples were obtained from individuals with HNSCC, melanoma, colon cancer, rectal cancer, lung cancer, CRLM, and ovarian cancer. All subjects signed written informed consent approved by the Providence Portland Medical Center Institutional Review Board (IRB protocol no. 06-108A). At the time of sample collection, patients were not undergoing therapy. Previously, patients had undergone a wide range of therapies, including chemotherapy, radiotherapy, surgery and immunotherapy, or none of the above. Peripheral blood mononuclear cells (PBMCs) were purified from whole blood over Ficoll-Paque PLUS (GE Healthcare) gradient and cryopreserved prior to analysis. Tumor specimens were prepared as follows: under sterile conditions, tumors were cut into small pieces and digested in RPMI-1640 supplemented with hyaluronidase at 0.5 mg/ml, collagenase at 1 mg/ml (both Sigma-Aldrich), DNase at 30 U/ml (Roche) as well as human serum albumin (MP Biomedicals) at 1.5% final concentration. Cells were digested for 1 h at room temperature under agitation with a magnetic stir bar. Cell suspensions were filtered through a 70 µm filter. Tumor-infiltrating lymphocytes were enriched as described above by Ficoll-Paque PLUS density centrifugation. Tumor single-cell suspensions were cryopreserved until further analysis.

Peripheral blood, uninvolved LNs, metastatic LNs, and tumor samples were obtained from all HNSCC patients. All subjects signed written informed consent approved by the Providence Portland Medical Center Institutional Review Board (PH&S IRB # 14-042). At the time of enrollment, patients were not undergoing therapy. PBMC were purified from whole blood over Ficoll-Paque PLUS (GE Healthcare) gradient and cryopreserved prior to analysis. Tumor specimens were prepared as follows: Under sterile conditions, tumors were cut into small pieces and digested in RPMI-1640 supplemented with hyaluronidase at 0.5 mg/ml, collagenase at 1 mg/ml (both Sigma-Aldrich), DNase at 30 U/ml (Roche) as well as human serum albumin (MP Biomedicals) at 1.5% final concentration. Cells were digested for 1 h at room temperature under agitation with a magnetic stir bar. Cell suspensions were filtered through a 70-μm filter. TIL were enriched as described above by Ficoll-Paque PLUS density centrifugation. Tumor single-cell suspensions were cryopreserved in LN₂ until further analysis.

Human serum albumin (MP biomedicals, Irvine, CA, USA) was dissolved in PBS as 22 mg/mL concentration. CDDP (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in PBS as a 1 mg/mL concentration. HSA and CDDP solutions were added to a new bottle at a molar ratio of 1:10 (1:1 volume ratio) and stirred for 24 h at 37 °C. To remove unconjugated CDDP and concentrate HSA–CDDP, centrifugal filtration was conducted using an Amicon Ultra centrifugal filter unit (nominal molecular weight limit 30 kDa; Millipore, Burlington, MA, USA). The HSA–CDDP concentration was measured with a bicinchoninic acid (BCA) protein assay kit (Pierce Endogen, Rockford, IL, USA), and the molecular weight was analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) using the TOF-TOF 5800 System (AB SCIEX, Framingham, MA, USA) to check the amount of CDDP per HSA in HSA–CDDP.