The case series consisted of a subset of the 19 patients enrolled onto a phase I/II vaccination trial for advanced melanoma and renal cell carcinoma in which the first 9 patients were treated with iDC and the remaining 10 received mDC, both pulsed with autologous tumor lysate and keyhole limpet hemocyanin (Biosyn, Fellbach, Germany). In the present study 8 patients were analyzed (7 melanoma, 1 renal carcinoma) for a total of 11 treatments. In vivo migration was assessed using a part of the DC obtained for one of the therapy cycles. Three of the 8 patients were evaluated twice.
Two melanoma patients were treated with iDC (one of whom twice), while 4 other patients with melanoma and 1 with renal cell carcinoma (treated twice) received mDC. The remaining melanoma patient was treated with iDC and subsequently with mDC.
The clinical trial was approved by the Italian Ministry of Health and by the Ethical Committee of Forlì Health and Social Services (Azienda USL – Forlì, Italy). All patients gave written informed consent.
Tumor samples surgically removed from the patients were immediately placed in PBS. Adjacent non malignant tissue was removed by scalpel and tumor cells were dispersed to create a single-cell suspension. Cells were lysed by incubation in sterile distilled water. Lysis was monitored by light microscope. Larger particles were removed by centrifugation (10 min at 600 g) and the supernatant was passed through a 0.2-μm filter. Protein contents were determined and aliquots were stored at -80°C until use.
Patients were generally vaccinated intradermally with DC (4–6 inoculations at the base of the thigh, about 10 cm from the groin, in the absence of visible disease). From days 2–6, IL-2 (Chiron, Milan, Italy) was administered subcutaneously at a dose of 3 million IU/die. This procedure was repeated after two weeks and once a month until progression occurred.
DC were prepared from peripheral blood monocytes (PBMC) obtained by leukapheresis without previous mobilization. 5–9 liters of blood were processed in each collection. PBMC were purified on Ficoll-Paque. An aliquot of PBMC was utilized immediately for DC generation and the rest was frozen in bags for use at a later date (4–5 bags/1 collection).
PBMC were incubated in tissue culture flasks with CellGro DC Medium (Cell Genix, Freiburg, Germany) at 10 × 106 cells/ml for 2 h. The non-adherent cells were discarded and the adherent cells were incubated in CellGro DC Medium containing 1000 IU/ml rhIL-4 (Cell Genix) and 1000 IU/ml rhGM-CSF (Shering Plough, Milan, Italy) for 7 days to generate a DC-enriched cell population. On day 6 DC were pulsed with autologous tumor lysate (100 mg/ml) and with KLH (50 mg/ml) and incubated overnight. On day 7, they were defined as iDC. After eliminating the previous culture medium, pulsed iDC were cultured for a further 2 days with a cocktail of cytokines (TNFα, IL-1β, IL-6, Endogen, Pierce Biotechnology, Rockford, USA; PGE2, Cayman Chemical, Ann Arbor, MI, USA). On day 9 they were defined as mDC. iDC or mDc were removed, washed and suspended in sterile saline for therapeutic infusion into the patient.
DC labelling and migration evaluation
Labelling of DC was performed according to the methods described for leucocyte radiolabelling [24–26]. A part of both iDC and mDC destined for vaccination (about 9.106) were resuspended in platelet-poor autologous plasma (CFP1) and incubated for 15 min at room temperature with 99mTc-HMPAO (20 mCi) (Nycomed Amersham plc, Little Chalfont, UK) 111In-Oxine (1 mCi) (Altana Pharma, Milan, Italy). After two washes to eliminate the unbound isotope, the cells were resuspended in a total volume of 1.5 ml of CFP1. Radiolabelling of the DC and of the culture supernatant was evaluated with a gamma counter, after which DC were inoculated intradermally into the patient near healthy lymph nodes and in the contralateral zone not used for therapeutic vaccination (3 inoculations at 10 cm from inguinal or axillary lymph nodes). The patient then underwent serial acquisitions with gamma-camera positioned at the site of inoculation, with a field of view that included all the lymphatic regions of interest. The first acquisition was performed with a dynamic study of 20 min, followed by 10-min static acquisitions carried out every 30 min for the first 4–6 h and from 18 to 28 h. Other static determinations were made at 36, 48 and 72 h. The maximum duration of observation of DC migratory activity, which depended on the half-life of the radioisotope used, was 72 h for 111In-Oxine and 36 h for 99mTc-HMPAO.
The identification of lymph node stations involved in the migratory activity was initially visual, after which we carried out a semiquantitative evaluation of the percentage of DC that migrated to lymph nodes from the inoculation site and an assessment of the speed of DC migration, expressed by activity/time curves obtained through the compartmental mathematical model.
Evaluation of labelling stability
DC obtained from the culture of frozen PBMC were divided into two parts: one was labelled with 99mTc-HMPAO and the other was labelled with 111In-Oxine. The labelled cells were then suspended in CellGro DC Medium, divided into 4–5 culture flasks for each labelling molecule and incubated for 0 h, 4 h, 21 h, 24 h (99mTc-HMPAO) and 0 h, 4 h, 21 h, 24 h, 48 h (111In-Oxine). The DC from one flask were removed and centrifuged. The supernatant containing the free molecule, and the pellet containing the labelled cells, were then measured with a gamma counter.
iDC and mDC phenotypes were determined by single or two-color fluorescence analysis. 3–5·105 cells were suspended in 100 μl of buffer (PBS, 2% FCS, 1% sodium azide) and incubated for 30 min at 4°C with 10 μl of appropriate fluorescein isothiocyanate or phycoerythrin-labelled monoclonal antibodies (mAbs). The cells were then washed twice and resuspended in 500 μl of assay buffer. The fluorescence was analyzed by a FACS Vantage flow cytometer (Becton Dickinson, Milan, Italy). mAbs specific for human CD1a, CD14, CD80, CD86, (Becton Dickinson) CD83 (Immunotech, Marseille, France) and CCR7 (BD Pharmingen, Milan, Italy) were used.
At each pre-set time the supernatant was collected and stored at -80°C until analysis was carried out using commercially available ELISA Kit (Biosource, Nivelles, Belgium) to measure the production of IL-12 + p40 (bioactive heterodimer of IL-12) and IL-10 by DC.
Single cell-based measurement of endocytosis was carried out as described (27). Dendritic cells were incubated for 30 min at 37°C with 0.5 mg/ml FITC-Dextran (40S DX-FITC Sigma, Milan, Italy). DX-FITC (average MW 42,000) was centrifuged before use to remove aggregates. As negative control, cells were incubated with DX-FITC at 4°C. The cells were washed with cold PBS containing 2% FCS and 2 nM sodium azide to exclude dead cells and were then analyzed on a FACS Vantage flow cytometer (Becton Dickinson) .