Figure 2. Tumor mtDNA is selectively increased inside the cytosol of DCs in response to CD47 blockade.

(A) BMDCs were co-cultured with MC38 in the presence of rat Ig or anti-CD47 mAb for four hours. CD11c⁺ DCs were purified and cytosolic DNA was extracted and quantitated via qPCR using the primer specific for genomic (Polg1) or mitochondrial DNA(nd1). (B) C57BL/6 mice were injected s.c. with 1 × 10⁶ MC38 cells and treated i.t. with 50 µg of anti-CD47 mAb or isotype control rat Ig on day 12. 24 to 72 hours after anti-CD47 mAb or rat Ig treatment, DCs were sorted from tumors. Cytosolic amount of mtDNA and gDNA were quantitated by real-time PCR assay. (C) MC38 cells were injected s.c. into C57BL/6 mice. Tumor-bearing mice were treated i.t. with 50 µg of anti-CD47 or rat Ig on day 12. Two days after treatment, the single-cell suspensions from tumors were sorted into monocytes, macrophages and DCs. (D) NSG mice were injected s.c. with 1 × 10⁶ HCT116 cells and treated i.t. with 50 µg of anti-human CD47 mAb (clone B6H12) or isotype control mouse Ig on day 14. 24 hours after anti-human CD47 or mouse Ig treatment, DCs were sorted from tumors. Cytosolic amount of mtDNA and gDNA that originated from host (mouse DNA) or tumor cells (human DNA) were quantitated and normalized to mouse gDNA and mtDNA respectively obtained from the whole-cell extract. Data are represented as mean ± SEM after normalizing to the copy number of gDNA obtained from the whole-cell extract. * p< 0.05, ** p< 0.01, *** p< 0.001, ns, not significant (Two-tailed student’s t test). Data are representative of two (C–D) or three (A–B) independent experiments. See also Figure S2.