Figure 6.

Release of Innate Immune-Stimulatory DAMPs and Calreticulin from Human Cancer Cells after Treatments with Immunogenic Cell Death-Inducing Agents

(A) WM266-4 cells (2 × 10⁵ cells/well) were transfected with ICR2 or ICR4 (0.5 μg/mL each) in a 24-well plate and harvested 24 hr after transfection. Surface expression of Calreticulin was determined by flow cytometry. (B) Uptake of dead/dying WM266-4 cells treated with ICR2, ICR4, or Dox by DCs was determined by phagocytosis assay. (C) Secretion of nuclear protein HMGB1 from cells treated with ICR2, ICR4, or Dox was determined by ELISA. (D–G) DAMPs were isolated from WM266-4 cells treated with transfection agent alone (Mock DAMP), ICR2 (ICR2 DAMP), ICR4 (ICR4 DAMP), poly(I:C) (pIC DAMP), or Dox (Dox DAMP) as described in Materials and Methods. HEK-TLR2, HEK-TLR3, HEK-TLR4, and HEK-TLR9 reporter cells (5 × 10⁴ cells/well) were incubated with DAMPs (25% v/v). Activation of TLRs was determined by colorimetric assay. Pam3CSK4, non-transfected poly(I:C), LPS, and CpG 2006 were used as positive controls in TLR reporter assays. In (A) and (B), the data represent two individual experiments. In (C)–(G), the data are the mean of three experiments. Error bars show SD. *p < 0.05.