Journal: bioRxiv

Article Title: PKD-1 Signaling Is Required for the Maintenance of CSCs with Epithelial-mesenchymal Plasticity in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2022.02.17.480869

Figure Lengend Snippet: Identification and distribution of CD44-positive cancer stem-like cells in human pNET tissues. A . Human pNET specimens in patient 1 were co-stained with CD44 and α-SMA antibodies followed by appropriate secondary antibodies, with DAPI staining the nuclei (blue) by immunofluorescence microscopy. CD44-positive cancer cells (red) marked by yellow arrowheads were close to the α-SMA positive (green) blood vessels. B : Human pNET specimens in patient 2 were co-stained as in A . CD44-positive cancer cells (green) were indicated by yellow arrowheads, which were close to the α-SMA positive (red) blood vessels. Note: A group of CD44-positive cells appear to distribute within the α-SMA-positive vascular niche or nearby blood vessels. Shown are representative images from different individual patients. Bar = 10 or 20 µm. C . Human pNET tissues were co-stained with CD44 antibodies and CD45 antibodies followed by appropriate secondary antibodies. Overlay images were collected by immunofluorescence microscopy. CD44-positive (green, yellow arrowheads) and both CD44-positive and CD45-positive cells (orange, blue arrowheads) were observed under a fluorescence microscope. Images were acquired by a fluorescence microscope equipped with a CCD camera. Shown are representative images from two individual patients. Bar = 10 µm. Note: Cells positive with both CD44 and CD45 are smaller and regarded as lymphatic cells (orange), a subset of cancer cells are bigger and positive for CD44 but negative for CD45, indicating their stem-like phenotype.

Article Snippet: Antibodies for immunostaining included rabbit anti-human/mouse PKD-1 (SAB4502371) and mouse anti-human/mouse α-smooth muscle actin (α-SMA, A2547) (Sigma-Aldrich), mouse anti-human/rat CD44 (5640S) and HRP-linked anti-rabbit IgG (7074) (Cell Signaling Technology), CD45 monoclonal Antibody (14-9457-80), AlexaFluor 594 conjugated donkey anti-rabbit IgG (A21207), AlexaFluor 594 conjugated donkey anti-mouse IgG(A21203), AlexaFluor 594 conjugated donkey anti-goat IgG (A11058), AlexaFluor 488 conjugated donkey anti-goat IgG(A11055), AlexaFluor 488 conjugated donkey anti-rabbit IgG (A21206), and AlexaFluor 488 conjugated goat anti-mouse IgG (A11001) (Invitrogen).

Techniques: Staining, Immunofluorescence, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: PKD-1 Signaling Is Required for the Maintenance of CSCs with Epithelial-mesenchymal Plasticity in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2022.02.17.480869

Figure Lengend Snippet: Stem-like phenotype of cancer cells in pNETs. A . A human pancreatic tissue control (top panel) and human pNET specimens from two individual patients (middle and lower panels) were co-stained with CD44 and α-SMA antibodies followed by appropriate secondary antibodies, with DAPI staining the nuclei (blue) by immunofluorescence microscopy. CD44-positive cancer stem-like cells were marked by yellow arrowheads, which were close to the α-SMA positive (green, red arrowheads) blood vessels. The fluorescence images were acquired by an immunofluorescence microscope equipped with a CCD camera, and representative images are shown, along with H & E staining for tumor tissue structures. Bar = 10 µm.

Article Snippet: Antibodies for immunostaining included rabbit anti-human/mouse PKD-1 (SAB4502371) and mouse anti-human/mouse α-smooth muscle actin (α-SMA, A2547) (Sigma-Aldrich), mouse anti-human/rat CD44 (5640S) and HRP-linked anti-rabbit IgG (7074) (Cell Signaling Technology), CD45 monoclonal Antibody (14-9457-80), AlexaFluor 594 conjugated donkey anti-rabbit IgG (A21207), AlexaFluor 594 conjugated donkey anti-mouse IgG(A21203), AlexaFluor 594 conjugated donkey anti-goat IgG (A11058), AlexaFluor 488 conjugated donkey anti-goat IgG(A11055), AlexaFluor 488 conjugated donkey anti-rabbit IgG (A21206), and AlexaFluor 488 conjugated goat anti-mouse IgG (A11001) (Invitrogen).

Techniques: Control, Staining, Immunofluorescence, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: PKD-1 Signaling Is Required for the Maintenance of CSCs with Epithelial-mesenchymal Plasticity in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2022.02.17.480869

Figure Lengend Snippet: PKD-1 signaling in the maintenance of cancer stem-like features in pNETs. A . Distribution of PKD-1 + and CD44 + CSCs within the vascular niche. Human pNET specimens were co-stained with CD44 and PKD-1 antibodies followed by appropriate secondary antibodies, with DAPI staining the nuclei (Blue). Stem-like cells with CD44-positive (green), PKD-1-positive (red) or both positive (pink) were observed under a fluorescence microscope. A few CD44-positive cancer stem-like cells tended to accumulate near the vascular lumen (red arrow heads), and PKD-1-positive CSCs might be leaving tumor nests (stars) for the vascular lumen. The fluorescence images were acquired by an immunofluorescence microscope equipped with a CCD camera. Shown are representative images. Bar = 10 µm. B . BON and QGP-1 cells were transfected with siRNA control and siPKD-1 to knock down PKD-1. Knockdown efficiency was confirmed by Western Blots (upper panel). The control and BON cells with knocked-down PKD-1 were subjected to tumorsphere formation assays. Images were acquired by the OLYMPUS CK30 microscope. Representative images are shown for tumorsphere formation (lower panel). C . Cell lysates were extracted from BON and QGP-1 cells exposed to the vehicle control, 10 μ M LPA, 2 μ M CRT0066101, or their combinations after 24 hours. The expression levels of phosphorylated PKD-1 and PKD-1 were detected by Western blots. Shown are representative images. D . BON and QGP-1 cells were exposed to 10 μ M LPA, 2 μ M CRT0066101, or their combination for 24 hours, and total RNA was extracted for the detection of mRNA levels of genes related to stemness properties by RT-qPCR. E . Effect of PKD inhibitor in tumorsphere formation. BON cells were cultured in complete MammoCult(tm) medium with the treatment of 10 μ M LPA, 2 μ M CRT0066101, or their combination for 7 days. The mammary spheres were counted under the OLYMPUS CK30 microscope. F . Control and BON cells with PKD-1 knockdown were exposed to 10 μ M LPA, 2 μ M CRT0066101, or their combination for 24 hours, and total RNA was extracted for the detection of mRNA levels of genes related to stemness properties by RT-qPCR. Note: Triplicate experiments were performed, and the results are shown as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies for immunostaining included rabbit anti-human/mouse PKD-1 (SAB4502371) and mouse anti-human/mouse α-smooth muscle actin (α-SMA, A2547) (Sigma-Aldrich), mouse anti-human/rat CD44 (5640S) and HRP-linked anti-rabbit IgG (7074) (Cell Signaling Technology), CD45 monoclonal Antibody (14-9457-80), AlexaFluor 594 conjugated donkey anti-rabbit IgG (A21207), AlexaFluor 594 conjugated donkey anti-mouse IgG(A21203), AlexaFluor 594 conjugated donkey anti-goat IgG (A11058), AlexaFluor 488 conjugated donkey anti-goat IgG(A11055), AlexaFluor 488 conjugated donkey anti-rabbit IgG (A21206), and AlexaFluor 488 conjugated goat anti-mouse IgG (A11001) (Invitrogen).

Techniques: Staining, Fluorescence, Microscopy, Immunofluorescence, Transfection, Control, Knockdown, Western Blot, Expressing, Quantitative RT-PCR, Cell Culture

Journal: bioRxiv

Article Title: PKD-1 Signaling Is Required for the Maintenance of CSCs with Epithelial-mesenchymal Plasticity in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2022.02.17.480869

Figure Lengend Snippet: Distribution of CD44-and/or PKD-1-positive cancer stem-like cells in pNET tissues. Human pNET specimens were co-stained with CD44 antibodies and PKD-1 antibodies followed by appropriate secondary antibodies, with DAPI staining the nuclei (blue). CD44-positive (green), PKD-1-positive (red) or both positive were observed under a fluorescence microscope. Cancer cells with high levels of both CD44 and PKD-1 (yellow arrowheads) likely left the tumor nests (white stars) and accumulated in the nearby vascular lumen (red arrowheads). Fluorescence images were acquired by an immunofluorescence microscope equipped with a CCD camera. Shown are representative images from two individual patients. Bar = 10 µm. Note: These are additional pictures for .

Article Snippet: Antibodies for immunostaining included rabbit anti-human/mouse PKD-1 (SAB4502371) and mouse anti-human/mouse α-smooth muscle actin (α-SMA, A2547) (Sigma-Aldrich), mouse anti-human/rat CD44 (5640S) and HRP-linked anti-rabbit IgG (7074) (Cell Signaling Technology), CD45 monoclonal Antibody (14-9457-80), AlexaFluor 594 conjugated donkey anti-rabbit IgG (A21207), AlexaFluor 594 conjugated donkey anti-mouse IgG(A21203), AlexaFluor 594 conjugated donkey anti-goat IgG (A11058), AlexaFluor 488 conjugated donkey anti-goat IgG(A11055), AlexaFluor 488 conjugated donkey anti-rabbit IgG (A21206), and AlexaFluor 488 conjugated goat anti-mouse IgG (A11001) (Invitrogen).

Techniques: Staining, Fluorescence, Microscopy, Immunofluorescence