DCs are collected and washed 3 times with PBS.

Advanced melanoma patients are vaccinated with the DC vaccine.

Vaccination with a vaccine composed by autologous DCs pulsed with apoptotic autologous ovarian carcinoma cells. Apoptosis is induced by UV.

Patients with advanced prostate carcinoma are vaccinated with the DC vaccine.

The pellets are washed with 70% Ethanol two times.

Hybridized slides are washed in a solution of water, 20× SSC, and 10% SDS, rinsed in water and 20× SSC, and dried via centrifugation for 2 min at 1000 rpm.

The patients will be vaccined with the DC vaccine that consists of monocyte derived DC loaded with tumor lysate generated from the excised lesion.

The frozen MoDC are subsequently used for tumor peptide specific stimulation of autologous T lymphocytes at a ratio of 1 DC per 10 T cells. Co-cultures led to antigen specific, IFN-? secreting T cells after 1 restimulation at a DC:T cell ratio of 1:20, as evaluated by IFN- ? Elispot, showing that the generated DC have good antigen presenting and co-stimulatory capacity.

Before injection all cell products will be thoroughly tested to exclude contamination and presence of viable tumor cells.

DCs are treated with zymolyase.

Transfection of immature DCs with different constructs encoding for the oncogene Her-2/neu and as control PSA using the technology of Amaxa biosystems or an adeno virus Her-2 full length construct.

Transfection of CD40L-encoding RNA along with the tumor RNA payload.

Patients with stage III or stage IV melanoma will undergo surgical resection of a melanotic lesion.

The supernatants are transferred to a new tube and 2 ml of acid phenol is added to repeat the extraction.

Day 2: transfection

Trypsinize cells and fix in 4 % PFA (or formalin)

After thawing cells showed good viability (>90%) and the recovery was approximately 80%.

Patients will undergo surgical removal of a metastatic lesion.

Take supernatant and freeze at -80°C for analysis of DC-stimulation

Staining of GM-DC/Raw with anti-CD11c antibodies