Figure 6.

Assessment of the Ability of EnAd to Mediate Release of Pro-inflammatory Cytokines from Clinical Tumor Biopsies

(A–C) Quantification by ELISA of intracellular (A) and extracellular (B) HSP70 protein in supernatants of cells infected with different viruses, as shown. (C) shows temperature-induced positive controls. Significance was assessed by one-way ANOVA, and Tukey’s multiple-comparison test, *p ≤ 0.05, **p ≤ 0.01. Error bars represent SEM; n = 3. (D) Dot blot for presence of HMGB1 protein in the supernatants of cells infected with different viruses. (E) Expression of calreticulin in A549 cells following treatment with viruses (100 ppc). Cells were harvested at 24, 48, 72, 96, and 120 hr post-infection (HPI), fixed in formalin, and exposed to an anti-calreticulin antibody, and then analyzed using flow cytometry. The y axis represents the mean expression of calreticulin (FL1). (F and G) Freshly resected human colorectal cancer biopsies were cultured ex vivo and treated immediately with virus particles (2 × 10⁹ virus particles/well) or drugs (50 μM cisplatin or 1 mM H₂O₂). Levels of TNF-α and IL-6 were measured in the supernatant after 72 hr, with significance determined using one-way ANOVA, with Tukey’s multiple-comparison test. **p ≤ 0.01. Error bars represent SEM; n = 3.