Fig 2: The interactions among CXCL12, KRT19, and TGM2. (A) Biotinylated human CXCL8 or CXCL12 was immobilized on SA beads, which were incubated with KRT19. The bead-bound proteins were eluted with SDS and subjected to SDS-PAGE and immunoblotting with SA-HRP or anti-KRT19 antibody. (B) SA beads bearing CXCL12–biotin were incubated with increasing concentrations of KRT19. Bound KRT19 was detected by SDS-PAGE and immunoblotting with anti-KRT19 antibody, and intensities of the KRT19 bands were measured. (C) CXCL12–biotin that was immobilized on SA beads was incubated at 4 °C with TGM2 in the absence or presence of KRT19 and in the absence or presence of 10 mM CaCl2. The bound proteins were eluted and subjected to SDS-PAGE and immunoblotting with SA-HRP and antibodies to KRT19 and TGM2. (D) SA beads bearing preformed complexes of CXCL12–biotin and KRT19 were incubated at 20 °C for 15 min with TGM2 in the absence or presence of the TGM2 inhibitor ERW1041E. The proteins were eluted from the beads and detected by SDS-PAGE and immunoblotting with antibodies to CXCL12 and KRT19.

Fig 3: Responses of hepatic metastases formed with control, Krt19-edited, or Tgm2-edited PDA cells to treatment with anti–PD-1 antibody. (A and B) Sections of hepatic metastases formed with luciferase-expressing sgScramble control PDA cells, sgKRT19-edited (A), or sgTGM2-edited (B) PDA cells were stained with fluorochrome-conjugated antibodies to KRT20 or KRT19 to reveal cancer cells and with CD3 to reveal T cells. The distance of each cancer cell from the nearest T cell was calculated for entire cross-sections, and the distance distribution of the sgScramble control tumors and their matching sgKRT19 or sgTGM2 tumors were plotted. (Scale bars, 100 µm.) More than 4,000 cancer cells from two to seven tumor sections of each group were analyzed; ****P < 0.0001, Kolmogorov–Smirnov test. (C) Mice bearing hepatic metastases formed with these luciferase-expressing PDA cells were treated with nonimmune IgG or anti–PD-1 IgG (arrows), and growth of the metastases was measured by bioluminescent imaging.

Fig 4: The CXCL12–KRT19 coating of cancer cells in human adenocarcinomas. (A) Sections of freshly resected human PDA, CRC, and breast cancers, respectively, were stained with fluorochrome-conjugated antibodies to KRT19 to reveal cancer cells and to CXCL12. (Scale bar, 100 µm.) (B) SDS eluates of the six tumors were subjected to SDS-PAGE and immunoblotting with anti-CXCL12 or anti-KRT19 antibodies. (C) The proteins that were immunoprecipitated by control IgG or anti-CXCL12 antibody from the SDS eluates of the human PDA tumors were subjected to SDS-PAGE and immunoblotting with anti-CXCL12 or anti-KRT19 antibodies. (D) The human breast cancer and CRC cell lines SKBR3 and Caco2, respectively, were stained with fluorochrome-conjugated antibodies to KRT19, KRT8, and CXCL12 prior to fixation and permeabilization. Confocal images of representative cells are shown. (Scale bar, 10 µm.) (E) SDS eluates of SKBR3 and Caco2 cells were subjected to SDS-PAGE and immunoblotting with antibodies to CXCL12 or KRT19. (F) The detergent lysates of SKBR3 and Caco2 cells were immunoprecipitated by control IgG or anti-KRT19 antibody and subjected to SDS-PAGE and immunoblotting with anti-CXCL12 and anti-KRT19 antibodies. Ab, antibody.

Fig 5: The immunological responses of tumors formed with Krt19-edited mouse PDA cells. (A) Sections of s/c tumors formed with sgScramble control PDA cells and sgKRT19-edited PDA cells were stained with fluorochrome-conjugated antibodies to KRT20, KRT19, CXCL12, and CD3. (Scale bars, 100 µm.) (B) Growth curves are shown of s/c tumors formed with sgScramble control PDA cells and sgKRT19-edited PDA cells in mice that were untreated or treated with either nonimmune IgG or T cell–depleting antibodies to CD4 and CD8; n = 5. (C) The mRNA levels of immune genes in tumors formed with sgKRT19-edited PDA cells were compared to those of tumors formed with sgScramble control PDA cells; n = 8. (D) Sections of s/c tumors formed with sgScramble control PDA cells without or with Dox-induced icOVA expression were stained with fluorochrome-conjugated antibodies to KRT19 and CD3. (Scale bar, 100 µm.) (E) Growth curves are shown of s/c tumors formed with sgScramble control PDA cells or sgKRT19-edited PDA cells without or with Dox-induced icOVA expression; n = 5; mean ± SEM; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test. (F) The heat map depicts the relative expression of immune genes as assessed by RNA-seq assay of individual s/c tumors formed with paired sgScramble control PDA cells with icOVA-expressing sgScramble control PDA cells and paired sgScramble control PDA cells with sgKRT19-edited PDA cells.