Fig 1.
Immunostaining of Cultured MTFs.
Representative confocal images of cultured MTFs. Nuclei were stained with DAPI (Blue); individual cells are shown in Panels [A,E,I,M, and Q]). The same cells were also stained with pairs of various fluorescent markers specific for melanocyte, macrophage, or epithelial differentiation (shown in each row). Panel [B,C]: Melanocytic marker ALCAM (Green) and M2- polarization macrophage marker CD204 (Red). Panel [F,G]: Pan-Cytokeratin (Green) and M2- polarization macrophage marker CD204 (Red). Panel [J,K]: M2- polarization Macrophage marker CD206 (Green) and epithelial cell adhesion molecule EpCAM (Red). Panel [N,O]: Melanocyte marker MLANA (Green) and M2- polarization macrophage marker CD204 (Red). Panel [R,S]: Melanocyte marker MLANA (Green) and M2- polarization macrophage marker CD206 (Red). Composite images are shown in Panels [D, H, L, P, and T].
Fig 2.
Immunostaining of human melanoma cell lines SK-MEL-28 and SK-MEL-31.
Representative confocal images of human melanoma cell lines SK- MEL-28 and SK-MEL-31 are shown. Nuclei were stained with DAPI (Blue) shown in Panels [A, E, I, and M]. The same cells were also stained with various fluorescent markers specific for melanocyte, macrophage, or epithelial differentiation. Panel [B,C]: SK-MEL-28 stained with M2- polarization macrophage marker CD206 (Green) and melanocyte marker Melan-A. Panel [F,G]: SK-MEL-31 stained with M2-polarization macrophage marker CD206 (Green) and melanocyte marker Melan-A. Panel [J,K]: SK-MEL-31 stained with pro-carcinogenic cytokine MIF (Green) and pan-macrophage marker CD68 (Red, no signal on the majority of cells). Panel [N,O]: SK-MEL-31 stained with M2- polarization macrophage marker CD204 (Green) and epithelial cell adhesion molecule EpCAM (Red). Composite images are shown in Panels [D,H,L and P].
Fig 3.
Comparative Immunostaining of human Pancreatic Carcinoma (Panc-1) Cells.
Representative confocal images of Human Pancreatic Carcinoma (PANC-1) cells are shown in rows. Nuclei were stained with DAPI (Blue) are shown in Panels [A, E, I, M, and Q]. The same cells were also stained with various fluorescent markers specific for melanocyte, macrophage, or epithelial differentiation. Panel [B,C]: pan-Macrophage marker CD68 (Green, No signal) and M2-polarization macrophage marker CD204 (Red). Panel [F,G]: Pan-Cytokeratin (Green) and M2- polarization macrophage marker CD204 (Red). Panel [J,K]: M2- polarization macrophage marker CD206 (Green) and epithelial cell adhesion molecule EpCAM (Red). Panel [N,O]: Melanocyte marker MLANA (Green, No Signal) and M2- polarization macrophage marker CD204 (Red). Panel [R,S]: Melanocyte marker MLANA (Green, No Signal) and M2- polarization macrophage marker CD206 (Red). Composite images are shown in Panels [D, H, L, P, and T].
Fig 4.
MIF Expression in Cultured MTFs.
Shown are representative 3D deconvolved confocal images of Cultured MTFs. Nuclei were stained for nuclei with DAPI (Blue), and immunostained for the pro-carcinogenic cytokine MIF (Green) and the pan-macrophage marker CD68 (Red). Nuclei appeared to have “holes” or “tunnels” through them, and these holes/tunnels stained strongly for MIF. Panels [A,B]: 3D projections of DAPI and composite immunostaining. Panels [C-F]: Sectional (XY views) views, with XZ views shown beneath them, localizing the accumulated MIF to the holes visible in the nuclei.
Fig 5.
CXCR4 and CD44 Expression in Cultured MTFs.
Representative images are shown. Nuclei were stained with DAPI (upper left), and cells were immunostained for CXCR4 (green, upper right) and CD44 (red, lower left). Composite images are shown in the lower right panel.
Fig 6.
Transmission Electron Microscopy of Cultured MTFs.
Cultured MTFs were transferred to coverslips, and grown for ~ 3 days, and then processed and stained as described. Panels [A-F] show representative photomicrographs. Essentially all cells appear large (50 μm diameter or larger), and show extensive pseudopod formation. Mitochondria are prominent, as are lysosomes, indicative of active phagocytosis. Heterogeneously-sized autophagic vacuoles and autolysomes at various stages of maturation are readily apparent in most all cells (Panel E shows a higher power view of autophagosomes containing micronuclei and chromatin, as well as melanosomes). Cross-sections of “tunnels” through nuclei are evident in many cells (Panel A, and Panel F shows a higher power view), where subcellular organelles and membranous structures can be seen within the cytoplasmic confines of a tunnel. Melanosomes are prominent in many cells (Panels B, G, and F, for examples).
Fig 7.
3D Confocal Rendering of DNA in Cultured MTFs.
Representative 3D surface rendered confocal images of normal diploid HPDE Cells [A], Panc-1 cells [B], and cultured MTFs [C-E] stained for nuclei with DAPI (Blue). DNA ploidy measurements were performed as described in Methods. HPDE cells serve as a standard diploid control. The cultured MTF populations were very heterogeneous with respect to DNA content (similar results were observed with DAPI and TO-PRO-3 staining). Occasional large cells were bi-nucleate or tri-nucleate, containing 2 or 3 physically attached para-diploid nuclei, and there was also considerable heterogeneity in the size of individual nuclei.
Fig 8.
DNA Ploidy Distributions of Cultured MTFs.
Graphs [A-B]: Populations of Cultured MTFs from 2 representative patients, showed cells with DNA distribution peaks corresponding to “para-diploid” and “para-tetraploid”, but with many aneuploidy cells distributed throughout the range, including some with DNA contents ranging up to 8n or 10n. Also shown on Graphs A-B are control ploidy measurements for normal diploid human HPDE cells. P values show the probabilities that the cultured MTF populations are diploid. Graph [C]: Human Panc-1 cells also showed very heterogeneous DNA contents. The distribution of DNA content within the Panc-1 cell populations resembled the DNA distribution observed in the CTC populations.
Fig 9.
DNA Ploidy Distributions of Melanomas in situ and SK-MEL Cell Lines.
DNA content analysis of dual-staining MTFs in primary melanomas in situ was assessed as described, and DNA content distributions were also assessed for the various SK-MEL human melanoma cell lines. DNA contents of all samples differed significantly from that of the normal diploid HDPE cell line. The DNA content of the SK-MEL-24 cells (derived from a metastatic lesion) most closely resembled that observed in melanomas in situ.
Fig 10.
Extrusion of Chromatin in Cultured MTFs.
Representative 3D surface rendered confocal images of cultured MTFs stained for nuclei with DAPI (Blue), showing extrusion of DNA from nuclei. There was apparent “Shedding” of DNA from the nuclei into the cytoplasm, which was evident in essentially all of the cultured MTFs. In many cases, this appeared as tubes of chromatin being extruded, in other cases as sheets. Panc-1 cells showed a reduced extrusion of DNA, which appeared much finer than that seen in CTCs.
Fig 11.
Chromatin Texture Analysis of Cultured MTFs.
Representative 3D confocal images of normal human HPDE cells [A-B] and MTFs [C-F] stained for nuclei with DAPI (Blue). Chromatin texture analysis was performed as described, and the images are color-coded to demonstrate the intensity level (red is most condensed; intensity scale is shown in Panel D).
Fig 12.
Hematoxylin & Eosin stained tissue sections from cutaneous human melanomas.
Formalin-fixed paraffin-embedded tissues from melanoma cases were sectioned and stained with H&E, and examined microscopically. The 4 panels show a range of lesions, including early lesions, which were subsequently examined for “dual-staining” MTFs (macrophage and epithelial markers) using confocal microscopy. Bar in upper left panel represents ~ 100 μm.
Fig 13.
Representative confocal images of MTFs in primary human melanomas.
Cells were stained for nuclei with DAPI (Blue), shown in Panels [A, E, I, M, Q, U, and Y]. The same cells were also stained with various fluorescent markers specific for melanocyte, macrophage, or epithelial differentiation, and images are shown in rows. Panels [B,C]: Melanocyte marker MLANA (Red) and M2- polarization macrophage marker CD204 (Green). Panels [F,G]: Melanocyte marker MLANA (Red) and M2- polarization macrophage marker CD206 (Green). Panels [J,K]: Melanocyte marker MLANA (Red) and M2- polarization macrophage marker CD163 (Green). Panels [N,O]: Melanocytic marker ALCAM (Green) and M2- polarization macrophage marker CD206 (Red). Panels [R,S]: M2- polarization Macrophage marker CD206 (Red) and epithelial marker pKRT (Green). Panels [V,W,Z, and A1]: M2- polarization Macrophage marker CD163 (Green) and epithelial cell adhesion molecule EpCAM (Red). As is evident, there are distinct populations of cells (in each of 6 primary melanoma specimens examined, as well as the metastatic lesions) which dual-stain for macrophage-melanocyte markers, which are often seen surrounding nests of melanoma cells. These cells also stain for epithelial markers. Composite images are shown in Panels [D, H, L, P, T, X, and B1]. Panels underneath Panels [A-D] represent XZ views of the panels above.
Fig 14.
Metastatic Foci after subcutaneous implantation of Cultured MTFs in Athymic Nude Mice.
MTFs from 2 separate patient samples were grown in culture for ~ 4 weeks, and 5 x 105 cells were subcutaneously implanted in hind limbs of nude mice. Mice were sacrificed and necropsied 47 days later. Sections were taken from multiple locations and examined for human MTFs. Shown are two distinct metastatic foci in mouse pancreas, stained with antibodies specific for human CD204 (Upper row), MLANA (Middle row), and CD206 (Lower row). Left column shows a low power (20X) view, and Right column shows a higher power (40X) view of 2 distinct foci. Inset in the Left Middle Panel shows a blown-up view of a single binucleate human cell in mouse pancreas, stained for human MLANA. Many of the cells in the foci also contained pigment (melanin), which was visible on standard H&E-stained sections.