Chapter 3 │ Page 95 facilitating immune activation. However, these factors serve primarily as indicators of ICD rather than definitive proof of immunogenicity and immune engagement. True confirmation that cancer cells died in an immunogenic way requires validation in a complete biological system, as individual markers alone nor grouped evaluation fully capture the complexity of the immune response. The gold standard for ICD validation remains the in vivo vaccination assay[39, 40], which assesses whether dying tumor cells can elicit a protective immune response in the mouse, which highlights functional immunogenicity. Hence, to test our NTP-CDDP combination in vivo, female C3H/HeN mice were injected with one of our seven pre-treated vaccines (1x106 cells/ml, 100 µL, viability < 10%) in the left flank (“UT” = PBS; “NTP” = 1000 Hz – 10 sec; “CDDPlow” = 1.71 µM; “CDDPhigh” = 5.85 µM; or their respective combinations “COMBOlow” and “COMBOhigh”, or positive control for ICD “Mito”). All mice were rechallenged with viable SCC7 cells one week later (1x105 cells/ml, 100 µL) in the opposite flank (Figure 11A). None of the injected mice developed tumors at the vaccination site, confirming the safety of our vaccine preparation. Tumor volumes at the rechallenge site were measured three times per week using digital calipers, and volumes were calculated accordingly (Figure 11B). The study follow-up was set at 100 days, but was terminated early if tumors exceeded 1500 mm³ or showed signs of ulceration. In general, tumor kinetics demonstrated a delayed onset and slower growth in mice that received a treated vaccine compared to untreated controls, reaching significance for the strongest combination (Figure 11B-C). Notably, we could appreciate that the addition of NTP to CDDP treatment successfully delayed tumor development and significantly suppressed tumor progression in both cases. Analysis of survival data showed a strong benefit in incorporating NTP into CDDP administration (Figure 11D-F). The plasma-based vaccine groups consistently outperformed their CDDP counterparts, displaying a notable increase in mean survival. Mice receiving COMBOlow survived significantly
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