Figure 7. cDC1 reprogramming attenuates tumorigenicity in vitro and in vivo.

(A) Heatmap showing gene expression changes in cell cycle progression gene signatures in reprogramming. Reprogrammed (CD45⁺HLA-DR⁺; ++), partially reprogrammed (CD45^-HLA-DR⁺; +), were profiled by bulk RNA-seq at day 3 (d3), 5 (d5), 7 (d7), and 9 (d9). eGFP-transduced cells were included as day 0 (d0). cDC1s served as reference. n=4-8; biological replicates. (B) mRNA expression of cell cycle progression (CCNA2, CDK1, MCM6, CDK2, PCNA, and MKI67) and cell cycle arrest genes (TP53, RB1, and CDKN1A) (n=3-8). (C) Changes in proliferation signature imposed by cDC1 reprogramming in 17 human cancer cell lines. (D) Flow cytometry analysis of cell proliferation by dye dilution. Reprogrammed T98G cells at day 9 were labelled with CTV, recultured and analyzed by flow cytometry after 1 (day 10), 3 (day 12), 4 (day 13), 6 (day 15), 8 (day 17), and 10 (day 19) days. (E) CTV MFI in reprogrammed CD45⁺HLA-DR⁺ (red) and eGFP⁺ (black) populations from 4 cell lines (n=2). (F) Anchorage-independent growth in soft agar of purified reprogrammed and partially reprogrammed cells. Colony formation after 4 to 6 weeks of culture and quantification for three cell lines (n=3-6). Relevant mutations are indicated. (G) Anchorage-independent growth of cells reprogrammed with DOX-inducible lentiviral PIB-expressing system. Colony formation after 5 weeks of culture after removal of DOX. (n=6). (H) Assessment of tumorigenic potential of tumor-APCs in vivo (top). Survival curves of NXG mice transplanted with reprogrammed T98G-derived CD45⁺HLA-DR⁺ cells (red, n=6), eGFP-transduced controls (black, n=9) and with 3P2C PDX-derived CD45⁺HLA-DR⁺ cells (red, n=4) and eGFP-transduced cells (black, n=5). For graphs in (A-G), mean±SD is presented. n= biological replicates. P values in (H) were calculated using Mantel-Cox test.