Fig 2: cDC1 Function Is Inhibited by PGE2, Related to Figure 5(A) Quantification of intratumoral cDC1 4 days after s.c. transplantation of 2x106 COX-sufficient control BRAFV600E melanoma cells EMPTY or expressing CCL5 or XCL1.(B) Tumor growth of COX-sufficient control BRAFV600E melanoma cells EMPTY or expressing CCL5 or XCL1 after s.c. transplantation of 2x105 cells into WT mice.(C and D) Same as (A-B) but using CT26 cancer cells.(E–I) CD103+ cDC1 from in vitro DC cultures were sorted by FACS and incubated for 16h with PGE2 (100ng/ml) or conditioned medium (CM) from COX-sufficient control BRAFV600E melanoma cells before testing in vitro migration toward (E) recombinant CCL5 or (F) recombinant XCL1. (G-H) Analysis of Xcr1 (G) or Ccr5 (H) mRNA by RT-PCR or XCR1 surface protein by flow cytometry after the incubation period .(J) Flow cytometric analysis of XCR1 surface expression on intratumoral cDC1 isolated from control or Ptgs1/Ptgs2-/- BRAFV600E tumors.Pooled data from at least two independent experiments are shown in A-D and G, H and depicted as mean of all mice per group ± SEM. Representative data from one of at least three independent experiments are shown in E, F, I and J and depicted as mean per duplicate wells or mice per group from one experiment ± SD.

Fig 3: Intratumoral cDC1 Accumulation Depends on NK Cells(A) Quantification of tumor-infiltrating lymphocytes in control or Ptgs1/Ptgs2-/- BRAFV600E tumors (day 4).(B) Image of a Ptgs1/Ptgs2-/- BRAFV600E tumor. Insets show colocalization of CD103+ cDC1 and NK1.1+ cells in multicellular clusters. Scale bar, 50 µm. The dashed line indicates the tumor margin. Data are representative of 6 tumors from two experiments.(C) Distance analysis based on (B). Data represent quantification across 6 images from 6 tumors.(D) Image of a tumor from a MMTV-PyMT mouse. Insets show colocalization of CD103+ cDC1 and NK1.1+ cells. Scale bar, 100 µm. Data are representative of 4 independent experiments.(E) Distance analysis based on (D). Data represent quantification across 8 images from 4 tumors.(F and G) Quantification of cDC1 in control or Ptgs1/Ptgs2-/- BRAFV600E tumors 4 days after s.c. inoculation of 2 × 106 tumor cells into WT mice, Rag1-/- mice, or Rag2-/-Il2rg-/- mice with or without NK cell depletion.(H) Quantification of NK cells in control or Ptgs1/Ptgs2-/- BRAFV600E tumors 4 days after inoculation of WT mice or Batf3-/- mice.(I) Effect of NK cell depletion on the growth of Ptgs1/Ptgs2-/- BRAFV600E tumors in WT or Batf3-/- mice.Data shown in (A) and (F)–(I) are pooled from at least two independent experiments with 4–6 mice per group and represented as mean ± SEM; (A), (C), and (E–I): n.s., non-significant, *p < 0.05, **p < 0.01, ***p < 0.001.See also Figure S2.

Fig 4: cDC1 Accumulate in the TME of PGE2-Deficient Tumors(A and B) Wild-type (WT) mice were injected s.c. with 2 × 106Ptgs1/Ptgs2-/- BRAFV600E cells and tumor-infiltrating immune cells were analyzed 4 days later by flow cytometry. Plots representative of 2-3 experiments are shown.(A) Gating strategy to identify intratumoral cDC1. Numbers represent % cells within depicted gate.(B) Analysis of intratumoral cDC1 and CD103-CD11c+MHCII+ cells for cDC1 markers.(C) Quantification of tumor mass and intratumoral immune cells 4 days after inoculation of WT mice with 2 × 106 control or Ptgs1/Ptgs2-/- BRAFV600E melanoma cells. Data are pooled from 4 independent experiments with 4–6 mice per group and depicted as mean ± SEM; each circle represents an individual tumor.(D) Representative sections of control and Ptgs1/Ptgs2-/- BRAFV600E tumors. Arrows indicate multicellular clusters of cDC1. Scale bar, 50 µm.(E) Surface reconstruction of images from (D) showing the localization of cDC1 versus CD103-MHCII+ cells.(F) Distance analyses based on (E). Data represent quantification across 8 images from 6 tumors.(G) WT or Batf3-/- mice were inoculated with 2 × 105 control or Ptgs1/Ptgs2-/- BRAFV600E cells and tumor growth was analyzed over time. Data are represented as mean ± SEM and are from one of two independent experiments with 3–5 mice per group.(H and I) WT and Batf3-/- mice were injected s.c. with 2 × 106 control or Ptgs1/Ptgs2-/- BRAFV600E cells. 12 days later, T cells were quantified (H) and stained for intracellular GzmB (I).In (C) and (F)–(I): n.s., non-significant, *p < 0.05, **p < 0.0, ***p < 0.001.See also Figure S1.

Fig 5: Accumulation and Positioning of cDC1 in COX-Deficient Tumors, Related to Figure 1WT mice were injected s.c. with 2x106 4T1 breast cancer cells, CT26 colorectal cancer cells or BRAFV600E melanoma cells. Tumors were excised 4 days later.(A and B) Immunofluorescence images of parental 4T1 or Ptgs1/Ptgs2-/- 4T1 tumors (A) or WT CT26 or Ptgs2-/- CT26 tumors (B). Upper panels show original images, lower panels show visualization of CD103+ cDC1 localization by surface reconstruction. Scale bar 100µm. Images are representative of individual tumors from 5-6 mice in two independent experiments. The dashed lines indicate the tumor margin, arrows indicate multicellular clusters of cDC1.(C and D) Quantification of intratumoral cDC1 in immunofluorescent images of 4T1 tumors (C) or CT26 tumors (D). Each circle represents data from one individual tumor. Data are mean ± SEM and were pooled from two independent experiments.(E) Distance analysis based on (A).(F) Distance analysis based on (B). Line indicates mean value, **p < 0.01, ***p < 0.001.