Fig 1.
Immunostaining of cultured MTFs from PDAC patients.
Representative confocal images of cultured MTFs from different PDAC patients. The cell populations showed uniform staining, and subcellular localization, of the various pairs of markers. Since the plates were sparsely populated, low power panoramic photomicrographs did not allow adequate visualization of the subcellular localizations, so photomicropraphs of individual cells were generally taken. Nuclei were stained with DAPI (Blue) shown in Panels [A, E, I, M, Q and U]. The same cells were also stained for various markers specific for pancreas, PDAC stem cell, or macrophage markers. Panels [B, C, F and G]: PDAC stem cell marker ALDH1A1 (Green) and pancreas marker ZG16B (red). Panels [J, K, N and O]: PDAC stem cell marker ALDH1A1 (Green) and pan-macrophage marker CD68 (Red). Panels [R, S]: Pancreas marker S100PBP (Green) and PDAC stem cell marker CD44 (Red). Panels [V, W]: M2-polarization macrophage marker CD206 (Green) and PDAC stem cell marker CD44 (Red). Composite images are shown in Panels [D, H, L, P, T and X].
Fig 2.
Immunostaining of cultured MTFs from PDAC patients.
Representative confocal images of cultured MTFs from different PDAC patients. Nuclei were stained with DAPI (Blue) shown in Panels [A1, E1, I1, M1 and Q1]. The same cells were also stained with various fluorescent markers specific for pancreas, macrophage, the pre-carcinogenic cytokine MIF and its receptor CXCR4. Panel [B1, C1]: MIF (Green) and pancreas-specific marker ZG16B (red). Nuclei had “tunnels” which were stained strongly for MIF. Panels [F1, G1, J1 and K1]: CXCR4 (Green) and pancreas-specific marker ZG16B (red). Panels [N1, O1, R1 and S1]: CXCR4 (Green) and M2-polarization macrophage marker CD204 (Red). Composite images are shown in Panels [D1, H1, L1, P1 and T1].
Fig 3.
Representative confocal images of MTFs in PDAC tissues from different patients.
Nuclei were stained with DAPI (Blue), shown in Panels [A, E, I, M, Q and U]. The same cells were also stained with various markers specific for pancreas, macrophage, or epithelial differentiation. Panels [B, C, F and G]: Epithelial marker pan-cytokeratin (Green) and M2-polarization macrophage marker CD206 (Red). Panels [J, K]: Epithelial marker pan-cytokeratin (Green) and M2-polarization macrophage marker CD163 (Red). Panels [N, O]: Epithelial marker pan-cytokeratin (Green) and M2-polarization macrophage marker CD204 (Red). Panels [R, S]: M2-polarization macrophage marker CD206 (Green) and epithelial marker EpCAM (Red). Panels [V, W]: M2-polarization macrophage marker CD206 (Green) and pancreas-specific marker ZG16B (Red). Composite images are shown in Panels [D, H, L, P, T and X].
Fig 4.
Representative 3D confocal images of MTFs in PDAC tissues from different patients which show various nuclear geometries (Yellow) of dual-stained cells.
[Panels A—I]: Dual stained for pancreatic tumor marker ZG16B (Red) and macrophage marker CD 206 (Green); [Panels J & K]: Dual stained for pancreatic tumor marker ZG16B (Red) and macrophage marker CD 204 (Green); [Panel L]: Dual stained for pancreatic tumor marker S100BPB (Green) and macrophage marker CD 204 (Red).
Fig 5.
Ploidy analysis of dual-staining MTFs in PDACs.
DNA content analysis of dual-staining cells was performed as described in PDACs in archival FFPE tissues. DNA content was also analyzed in adjacent “normal” pancreas (gray bars). Populations of MTFs from 2 patients, showed cells with DNA distribution peaks corresponding to “para-diploid” but with many aneuploidy cells distributed throughout the range, including some with DNA contents ranging up to 5n (black bars).
Fig 6.
Transmission electron microscopy of cultured MTFs.
Panels A-H show representative cells with distinctive features, including cytoplasmic “tunnels” through nuclei (Panel G). Focal hyper-dense regions of chromatin (Panel A, E, and F), autophagic bodies (Panel D), etc. are evident within the individual cells.
Table 1.
Cancer-related genes of interest.
Fig 7.
Immunohistochemical staining of mouse tissues after orthotopic transplantation of cultured MTFs.
Human MTFs were cultured from blood as described, and orthoptically transplanted into mouse pancreas. After various periods of time, tissues were harvested and stained for an epithelial marker (cytokeratin, KRT), human macrophage M2 polarization markers CD204 and CD206, and human pancreatic stem cell marker ALDH1A1. Tissues (pancreas, spleen, and liver) are as indicated. Panel A shows samples harvested at 4 weeks after implantation, B shows samples after 8 weeks, and C shows samples after 12 weeks.
Fig 8.
Immunostaining of human MTFs after orthotopic transplantation into mice.
Cultured human MTFs were orthotopically transplanted into mouse pancreas and tissues were harvested at various times. Representative confocal images are shown from pancreas after 4, 8 and 12 weeks (PANC-4W, PANC-8W, PANC-12W) after orthotopic implantation. Nuclei were stained with DAPI (Blue). The same cells were also dual stained with various fluorescent markers specific for the epithelial marker cytokeratin (KRT), human macrophage M2 markers (CD204/206) or the human pancreatic stem cell marker (ALDH1A1).
Fig 9.
Immunostaining of human MTFs after orthotopic transplantation into mice.
Cultured human MTFs were orthotopically transplanted into mouse pancreas. At 8 weeks following implantation, tissues were harvested and examined by confocal microscopy. Representative confocal images of liver or spleen (LIVER-8W, SPLEEN-8W) are shown. Nuclei were stained with DAPI (Blue). The same cells were also dual stained with various fluorescent markers specific for epithelial marker cytokeratin (KRT) or macrophage (CD204/206). Note that the CD204-expressing cell is no longer expressing KRT.
Table 2.
Antibodies used for immunophenotypic characterizations.