Fig 2: Analysis of mouse pancreatic CTCs identifies Wnt2 as a candidate CTC genea, Schematic representation of strategy for DGE of RNA isolated from primary mouse pancreatic tumour, metastatic ascites, and CTC-enriched blood. b, Immunofluorescence staining of mouse pancreatic CTC, leukocyte (WBC), and CTC cluster captured on HbCTC-Chip (DAPI, blue; Cytokeratin (CK), red; CD45, green). c, Quantitation of CK+ cells captured from control mice and pancreatic cancer bearing mice (dashed line - threshold of = 6 CK+ cells/100 µL). d, DEGseq M-A plot of RNA sequence reads from mouse (MPANC-9) CTCs, comparing anti-EpCAM-captured versus IgG-coated HbCTC-Chips. (M - log2 fold change, A - averaged log2 reads, red dots – differentially expressed genes) e, RNA-ISH of CTCs and metastatic ascites cells co-expressing CK8+18 (green) and Wnt2 (red) transcripts. White arrowheads highlight Wnt2 signals. f, RNA-ISH of primary pancreatic tumours showing a small cluster of Wnt2 mRNA expressing cells near a pancreatic duct (D) (Wnt2, red dots; hematoxylin, light blue). Hematoxylin and eosin (H&E) stained serial section is shown (right). A second primary tumour imaged under fluorescence demonstrating Wnt2 expression in a subpopulation of tumour cells; high magnification shown (right, Wnt2, red; CK, green; DAPI, blue). (Scale bars=10 µm).

Fig 3: Intraurothelial macrophages phagocytose neutrophils and UPEC.a Confocal microscopy demonstrating phagocytosis of GFP+ UPEC (green) by F4/80+ macrophages (red) in the EpCAM-1+ urothelium (white) 1-day post-infection. b GFP+ UPEC in the urinary bladder determined by fluorescent image analysis (SCHNELL) after macrophage depletion by intraperitoneal application of aCSF1R antibodies. c The colony forming units of UPEC were determined in bladder homogenates three days post-infection. d Flow cytometry-based quantification of phagocytosis of neutrophils by macrophages (F4/80+Ly6G+) in bladder digests 1-day post-infection. e Confocal microscopy demonstrating phagocytosis of Ly6G+ neutrophils (green) by F4/80+ macrophages (red) in the EpCAM-1+ urothelium (white) 1-day post-infection. f Density of neutrophils in the urinary bladder after macrophage depletion by intraperitoneal application of aCSF1R antibodies 1-day post-infection. *p < 0.05, ***p < 0.001. Error bars show the mean ± SEM. lu=lumen. The scale bars in a, d indicate 5 µm and the white dashed lines distinguish the EpCAM-1+ urothelium from the lumen.

Fig 4: Macrophage relocation and the defense against UPEC depends on IL-6.Mice were infected with GFP-expressing UPEC and analyzed 1-day post-infection. IL-6 expression was topically inhibited by inoculating IL-6 antibodies (aIL-6) into the urinary bladder. Inoculating IgG antibodies served as the control. a Bladder tissue sections were stained with DAPI (blue), F4/80 (red) and EpCAM-1 (white) and the images were acquired by a fluorescence microscope. Dashed lines separate the urothelium from the connective tissue. The scale bars in the top row indicate 200 µm, bottom row 30 µm. b The density of F4/80+ macrophages in the bladder compartments was calculated by SCHNELL on immunofluorescent images. c The colony forming units of UPEC were determined in bladder homogenates in the presence and absence of IL-6. d Spider plots of the expression intensities [au] of the proteins of the GO terms “macrophage migration” (GO: 1905517) and “macrophage activation” (GO: 0042116) in the urothelium determined by MALDI-MSI. Asterisks inside the circle refer to the statistical analysis between healthy and UPEC infected (* in black), whereas the asterisks outside the spider plot (* in blue) compares UPEC versus aIL-6. *p < 0.05, **p < 0.01. Error bars show the mean ± SEM; lu, lumen.

Fig 5: TC-PTP prevents hyper-proliferation of IECs. (a) Immunostaining with the proliferation marker Ki67 using colonic tissues of WT and TC-PTPIEC-KO mice not treated with DSS. Bar, 50 µM. Representative section of n=5. (b) Statistical analysis of Ki67+ cells in 10 clearly defined colonic crypts per mouse. n=5. *P<0.05. (c) Immunoblot of lysates from IECs isolated from three WT and three TC-PTPIEC-KO mice not treated with DSS. Membranes were blotted for p-STAT1, STAT1, p-STAT3, STAT3, p-STAT5, STAT5, p-JAK1, JAK1 and TC-PTP, and actin was used as a loading control. (d, e) Live colonic stem cells (EpCam+CD45-CD44hiCD24loCD166+cKit[CD117]-) defined by flow cytometry from WT and TC-PTPIEC-KO mice not treated with DSS. (d) Gating strategy. (e) Representative plot of three independent experiments performed in triplicates, samples are shown in red and FMO controls shown in blue. (f) Quantification of the percentage of colonic stem cells (% CoSC). Ratio of colonic stem cells identified in a pool of colon epithelial cells (CoSC/colon epithelial cells). Three independent experiments performed with a group of three mice. *P<0.05. DSS, dextran sulphate sodium; FMO, fluorescence minus one; IEC, intestinal epithelial cell; KO, knockout; TC-PTP, T-cell protein tyrosine phosphatase; WT, wild type.