Chapter 3 │ Page 79 2.9. Gold Standard Vaccination Assay in Syngeneic HNSCC Mouse Model To validate bona fide ICD induction, the gold standard in vivo vaccination assay[39, 40] was performed in a syngeneic mouse model for HNSCC. 56 six-week-old female C3H/HeN mice were purchased from Janvier-Labs and maintained in a pathogenfree environment at the Animal Core Facilities of the University of Antwerp. All animal procedures were approved by the University of Antwerp Animal Research Ethical Committee (ECD reference number 2023-67). For the syngeneic setting of this experiment, vaccines were prepared with SCC7 mouse HNSCC tumor cells. SCC7 cells were seeded in 24-well plates and treated with one of the five experimental conditions: i) 1000 Hz NTP for 10 sec, ii) 1.71 µM of CDDP (CDDPlow), iii) 5.85 µM of CDDP (CDDPhigh), the combination of NTP with iv) the lowest CDDP concentration (COMBOlow), or v) highest CDDP concentration (COMBOhigh). Cultures treated with PBS or mitoxantrone (2 µg/ml) were used as negative and positive ICD control, respectively. Cells were harvested 24 hours after therapy, washed twice with PBS and resuspended in PBS at a concentration of 1x106 cells/ml. The cell suspensions were incubated overnight at 37°C and 5% CO2 to reduce cell viability to below 10%, thereby minimizing the risk of tumor growth at the vaccination site. At the day of vaccination, eight mice were randomly assigned to each of the seven experimental conditions, whereafter mice were injected subcutaneously in the left flank with 1x 105 treated SCC7 cells in 100 μL sterile PBS (Life Technologies). One week later, on day 7, all mice were rechallenged with 5x104-1x105 live SCC7 cells in the opposite (right) flank. Tumor progression in the rechallenged flank was monitored by measuring two orthogonal tumor diameters, and volumes were calculated using the formula Vtumor= (1/2 x π) x (short side tumor) 2 x (long side tumor). Simultaneously, the vaccination site was routinely examined for signs of tumor development. Humane endpoints for the study were set prior to the start of
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