Fig. 4: Regional AP1 heterogeneity determines macrophage recruitment.

a NMRI-Foxn1^nu/nu mice were orthotopically transplanted with CAPAN2 cells with stable HA-tagged cJUN overexpression (HA-cJUN-OE) or empty vector (EV) control. b H&E staining of CAPAN2 HA-cJUN-OE and EV tumors. Immune infiltrates are indicated. Scale bar 100 μm. n = 8 animals. c-n, QuPath-based analysis of HA-cJUN-OE tumors for HA-cJUN IHC, DAPI-JUNB IF, CD68 IHC, CD163 IHC, CD86 IHC, and DAPI-CCL2 IF. IHC for the HA tag of cJUN (c) or IF for JUNB (d) in serial sections. Nuclear-positive cell detections (red/green) are indicated. Density maps of positive cells were created and thresholded to derive hotspot regions for HA-cJUN⁺ and JUNB⁺ cells, respectively, in the same tumors. Whole tumor overviews as well as a HA-cJUN (mid) and a JUNB (bottom) hotspot ROIs are shown. Scale bar: tumor overview, 1 mm; large ROI, 100 µm; ROI insert, 20 µm. e Quantification of (c, d) for HA-cJUN⁺ and JUNB⁺ cells relative to the total number of detected cells in cJUN (red) and JUNB (blue) hotspot regions, with mean ± s.d. shown. One outlier is indicated (red circle), which was excluded for mean and s.d. n = 5 tumors. f IHC for CD68 in HA-cJUN-OE tumors, with CD68⁺ cell detection (purple), as well as JUNB and HA-cJUN hotspots. Exemplary 2D distance measurement strategy which was used in (g-n) is shown. Scale bar 100 µm. Distance analysis of CD68⁺ (g), CD163⁺ (i), CD86⁺ (k), or CCL2⁺ (m) cells to JUNB or HA-cJUN hotspots. Scatter plots show each individual cell, with mean ± s.d. Two-tailed Student’s t-test with Welch’s correction. g, n = 14774 CD68⁺ cells from n = 5 tumors, with a total of n = 1,003,639 cells analyzed. i, n = 7691 CD163⁺ cells from n = 4 tumors, with a total of n = 654,297 cells analyzed. k n = 8384 CD86⁺ cells from n = 4 tumors, with a total of n = 632,792 cells analyzed. m n = 14,925 CCL2⁺ cells from n = 5 tumors, with a total of n = 745,208 cells analyzed. h, j, l, n As in (g, i, k, m) showing the shortest distances of each cell towards both the HA-cJUN and JUNB hotspots. Source data are provided as a Source Data file.