Article Figures & Data
Figures
- Figure 1.
Expression of XCR1 in normal ovarian surface epithelium and the IOSE and borderline EOC cell lines. A, a representative image of XCR1 expression in a specimen from a normal ovary. Black arrow indicates the ovarian surface epithelium in the normal ovary. Brown – XCR1, blue – hemotoxylin. Images were generated using an Aperio ScanScope digital slide scanner. Magnification: × 10. B, expression of XCR1 in the indicated IOSE cell line T1074 was detected by flow cytometry analysis and immunofluorescent staining. Cell surface expression of XCR1 in IOSE was analyzed by flow cytometry analysis. Solid thick line – XCR1 expression, solid thin line – negative control (secondary antibody only). Images (× 40) shown are representative of at least 3 independent experiments. The fluorescent signal from XCR1 staining (red) was overlaid with DAPI staining (blue). C, Cell surface expression of XCR1 in the borderline EOC cell lines was detected with flow cytometry analysis. Solid thick line – XCR1 expression, solid thin line – negative control (secondary antibody only). Representative of at least 3 independent experiments.
- Figure 2.
Expression of XCR1 in EOC cell lines. A, cell surface expression of XCR1 in the EOC cell lines using goat anti-human PE-conjugated XCR1 antibodies. Solid thick line – XCR1 expression, solid thin line – negative control (secondary antibody only). Images shown are representative of at least 3 independent experiments. B, expression of XCR1 was tested by immunofluorescent staining of immortalized NOSE and serous EOC cell lines, as indicated. Images were generated using a Zeiss fluorescent microscope and × 40 objective lens. The fluorescent signal from XCR1 staining (red) was overlaid with 4′, 6-diamidino-2-phenylindole (DAPI) staining (blue). A magnified image of XCR1 staining in SKOV-3 converted to a black-and-white image is shown in the lower panel to show membranous immunoreactivity with XCR1 antibodies (white arrows). Intensity of XCR1 staining in cellular protrusions along the dotted line was quantified using the ImageJ (NIH) line scan feature. Bars = 20 μm and 5 μm on the top and bottom, respectively. C, expression of XCR1 was probed in total cell lysates prepared from nonimmortalized NOSE (HOSEpiC), immortalized OSE (T1074), and serous EOC cell lines (IGROV-1 and Caov-3) using goat anti-human XCR1 antibodies. β-Tubulin was used as a loading control. Images were generated using enhanced chemoluminescence application of Chemidoc (Bio-Rad). D, immunohistochemical analysis of XCR1 expression in primary and metastatic specimens of EOC. Representative images of negative and positive cases of primary EOC as well as negative and positive cases of EOC metastasis to the omentum, as indicated. Brown – XCR1, blue – hemotoxylin. Images were generated using the Aperio ScanScope digital slide scanner. Magnification: × 10.
- Figure 3.
A, expression of XCL1 and XCL2 RNA in EOC cell lines. Total RNA from IGROV-1, SKOV-3, and A2780 was extracted, cDNA was synthesized, and RT-PCR was conducted as described in the Materials and Methods section. Products of the PCR reaction were loaded in a 2% agarose gel, visualized with ethidium bromide staining, and photographed with the BIO-RAD Chemidoc software. The image has been color inverted for better visualization. M – marker, H – housekeeping gene control RPL-19, 1 – XCL1, 2 – XCL2. The position of the lowest fragment of the marker ladder (500 bp) is indicated. B, XCL1-induced proliferation. NOSE, IOSE, borderline EOC, and serous EOC cell lines, as indicated, were subjected to cell proliferation in the presence and absence of 25 nmol/L XCL1 added to the serum-starved cells (“Starved+25 nmol/L XCL1,” red bars, “Starved,” blue bars, respectively). Cell proliferation was measured using a WST-1 reagent as described in Methods and plotted. C, SKOV-3 cells were serum-starved (Starved) as well as serum starved and cultured in the presence of 25 nmol/L XCL1 (Starved+25 nmol/L XCL1) for 24 hours, photographed using a Zeiss microscope with × 5 magnification, collected, and analyzed by Western blot analysis. Expression of PCNA in total cell lysates (in duplicate) was detected by Western blot analysis using rabbit anti-human PCNA antibodies at a 1:200 dilution. β-Tubulin was used as a loading control. Images were generated using the enhanced chemoluminescence application of Chemidoc (Bio-Rad). Immunofluorescent staining of PCNA is shown in the lower panels. SKOV-3 cells were first serum starved and then cultured in the absence (Starved) and presence of 25 nmol/L XCL1 for 24 hours (Starved+XCL1). Nuclear PCNA staining (green fluorescence) was evaluated by immunofluorescent staining. Cell nuclei were stained with DAPI (blue fluorescence). Fluorescent images were generated using the Zeiss AxioObserver fluorescent microscope with × 40 magnification.
- Figure 4.
XCL1- and XCL2-mediated cell migration. A, n0on-transfected (NT), control siRNA-transfected (Ctrlsi), and XCR1 siRNA (XCR1si)-transfected SKOV-3 cells were subjected to the Transwell cell migration assay in the indicated conditions, including 0.5 and 2 nmol/L XCL2 and 5 and 10 nmol/L XCL1. B, HOSEpiC cells were subjected to the Transwell migration assay in the presence of 5 and 10 nmol/L XCL1, as indicated. C, SKOV-3 and IGROV-1 cells were transiently transfected with control and XCR1 siRNAs and subjected to the Transwell cell migration assay against human peritoneal ascitic fluid (specimens #2 and #4, respectively, Table 1). Images beneath the graph illustrate the extent of cell migration under each condition. Average of 3 experiments carried out in triplicate. *, P < 0.05; **, P > 0.05. D, expression of XCR1 in SKOV-3 and IGROV-1 cells (C) transiently transfected with either control or XCR1-specific siRNAs was detected by Western blot analysis. β-Tubulin was used as a loading control. Images were generated using the enhanced chemiluminescence application of Chemidoc (Bio-Rad).
- Figure 5.
In vivo xenograft study of the role of XCR1 in dissemination of ovarian carcinoma. A, Expression of XCR1 was probed in total cell lysates prepared from SKOV-3 and SKOV-3 stably transfected with scrambled shRNA and XCR1-specific shRNA (clone 1, clone 2, clone 3), as indicated, using goat anti-human XCR1 antibodies (Santa Cruz Biotechnology). β-Tubulin was used as a loading control. Images were generated using enhanced chemoluminescence application of Chemidoc (Bio-Rad). *, P < 0.05. B, photographs of the dissected abdomens of animals injected with parental SKOV-3 cell line (top) and SKOV-3 stably transfected with XCR1shRNA (clone 1; bottom); peritoneal metastasis from the parental SKOV-3, middle. Tumor nodules are outlined. C, average volume of ascites accumulated in each condition, as indicated. D, distribution of metastatic lesions formed on diaphragm, peritoneal wall, colon, liver, spleen, lymph, mesentery, and omentum is shown as the percentage of animals bearing metastases at the site. Statistical significance is calculated using t test. Total number of animals analyzed: 6 for the XCR1shRNA group and 5 for the parental SKOV-3 (one animal was not counted for as the tumors did not take).
Tables
- Table 1.
Concentration of XCL1 and XCL2 in samples of ovarian carcinoma and borderline gynecologic diseases determined by ELISA
Sample XCL1 XCL2 # Diagnosis pg/mL pmol/L ng/mL nmol/L 1 Borderline endometrioid cystadenoma 40 4.0 2.0 0.20 2 Serous adenocarcinoma, stage IV, Grade 3 50 5.0 1.0 0.10 3 Borderline mucinous cystadenoma 50 5.0 1.0 0.10 4 Serous adenocarcinoma, stage IV 48 4.8 1.2 0.12 5 Serous adenocarcinoma, stage IIIc, Grade 3 37 3.7 0.3 0.03 6 Serous cyst 50 5.0 1.1 0.11 7 A2780a 210 21 NDb 8 SKOV-3a 83 8.3 NDb
Supplementary Data
Files in this Data Supplement:
- Supplementary Methods - PDF file - 127K, Detailed description of some of the routine procedures used to generate the data
- Supplementary Figure 1 - PDF file - 21K, Alignment of XCL1 (NM_002995) and XCL2 (NM_003175) gene sequences using NCBI BLAST (NIH). Nucleotides that do not match are highlighted in yellow. Positions of the sequences used to construct primers for detection of XCL1 and XCL2 are indicated by blue arrows.
- Supplementary Figure 2 - PDF file - 225K, Immunohistochemical analysis of XCR1 expression in distant metastasis originating from different primary solid tumors: a) bladder to pelvic cavity, b) breast to armpit, c) cervix to pelvic cavity, d) colon to liver, e) endometrium to abdominal cavity, f) esophagus to mediastinum, g) kidney to right upper arm, h) larynx to neck, i) liver to abdominal cavity, j) lung to cerebellum, k) skin of left thigh to right groin, l) pancreas to omentum, m) prostate to costal bone, n) duodenum to mesentery, o) rectum to ovary, p) small intestine to mesentery, q) stomach to abdominal wall, r) thyroid to left frontal lobe, s) skin of penis to right groin, and t) lung to jejunum. Rabbit anti-human XCR1 antibodies (Novus Biologicals) were used at a 1:50 dilution; biotin-conjugated anti-rabbit antibodies were used at a 1:500 dilution. Brown - XCR1, blue - haemotoxylin. Images were generated using the Aperio ScanScope digital slide scanner. Bar = 50 micron.
- Supplementary Table 1 - PDF file - 58K, Sequences of primers used to detect expression of XCL1 and XCL2 by QPCR
- Supplementary Table 2 - PDF file - 65K, Expression of XCR1 RNA in samples of ovarian carcinoma and non-malignant ovary
- Supplementary Table 3 - XLS file - 69K, Expression of XCR1 in specimens of ovarian carcinoma and normal ovary
- Supplementary Table 4 - PDF file - 38K, Expression of XCR1 in specimens of metastatic ovarian carcinoma
- Supplementary Table 5 - PDF file - 66K, Expression of XCR1 in multiple organ metastatic cancer specimens
Volume 10, Issue 11
