Fig 1.
Chemotherapy transiently induced the CXCR4High cell state transition in human OVC.
(A) Representative FACS analysis showed that A2780/ADR displays a higher percentage of CXCR4High than A2780. Cells were stained with fluorescently labeled anti-CD44, anti-CD133, or anti-CXCR4, and also with anti-CD24. The numbers in the quadrants indicate the average percentage of CXCR4High. (B) Chemotherapy reversibly induced CXCR4High in A2780 and SKOV-3. A comparison of the changes in the cell population 72 h after treatment with suboptimal concentrations of cisplatin (1 μM), doxorubicin (10 nM), and paclitaxel (100 pM). At 72 h after withdrawing the drugs, the percentage of CXCR4High returned to the basal level. (C and D) The drug-induced CXCR4High was confirmed using western blot and real-time PCR analysis of the CXCR4 expression in the cell lysates. All the results represent means ± SD of three independent experiments (t-test, *p<0.05, ***p<0.001). (E) Doxorubicin induces tumoral CXCR4+-C. Mice (n = 3/group) were implanted with SKOV3/GFP-Luc (tagged with GFP and luciferase) and then treated with PBS (control) or doxorubicin (5mg/kg) via IV injection. After 72 h, the tumors were dissociated and analyzed for the percentage of CXCR4+-C by FACS analysis.
Fig 2.
CXCR4High displayed drug-resistant and mesenchymal properties.
(A) FACS analysis showed that the freshly isolated CXCR4High and CXCR4Low eventually lost their purity over time. The cells were isolated by flow cytometry using APC-labeled CXCR4 antibody. (B and C) The CXCR4 expressions in A2780/ADR, CXCR4High, and CXCR4Low were determined using western blot and real-time PCR analysis. Results of western blot and real-time PCR were normalized to β-actin expression and the CXCR4 mRNA expression of CXCR4 in A2780/ADR cells were set to 1. (D) Plots of relative cell viability versus different drug treatments reveal that CXCR4High cells were intrinsically drug-resistant. The cell viability was determined by MTS assay. (E) Microscopic images (10x magnification) of CXCR4High and CXCR4Low. The two freshly isolated cell lineages from A2780/ADR display significantly different morphologies. After continuous culturing for 21 days, their morphology became similar, presumably because OVC prefers to maintain an equilibrium state between the two cell populations. (F) Comparison of the cell proliferation among A2780/ADR and its isolated CXCR4High and CXCR4Low lineages. CXCR4High exhibits the similar cell proliferation rate. (G and H) CXCR4High displayed mesenchymal phenotypes, as shown by the higher and lower expressions of the epithelial and mesenchymal markers, respectively, compared to CXCR4Low using western blot and real-time PCR. (I) CXCR4High also displayed higher expressions of cancer stem cell markers. Results of real-time PCR was normalized to β-actin expression and the mRNA gene expression of epithelial, mesenchymal and stem cell markers in CXCR4 High cells were set to 1. All our data represent means ± SD of three independent experiments (t-test, *p<0.05, **p<0.01 and ***p<0.001).
Table 1.
Comparison of the IC50 values among different drugs against various OVC cell lines or cell clones.
Fig 3.
The freshly isolated CXCR4High showed enhanced invasion, migration, and tumor formation properties compared to the CXCR4Low.
(A) Microscopic images of wound healing, Boyden-chamber, and soft agar colony formation assays. (B) The results were also presented quantitatively to reflect the percentage and number of cells that invaded the wounds and migrated through the chambers, as well as the number of colonies formed. All results are presented as means ± SD of three independent experiments (t-test, **p<0.01 and ***p<0.001). (C) A comparison of the A2780/ADR, CXCR4High and CXCR4Low tumors’ growth rate in SCID mice (n = 5/group). The tumors were subcutaneously implanted on each side of the back of the animals. Data are presented as mean tumor volumes (mm3) ± SD versus time (two-way ANOVA, ***p<0.001). (D) Representative microscope images of the H&E staining and immunohistochemistry of the tumor sections show that CXCR4High tumor consist of higher expressions of CXCR4 and CD31 (blood vessel). Arrows indicate the vascular core areas.
Fig 4.
CXCR4High can be a useful target for OVC treatment.
(A) CXCR4-KLA (labeled with FITC) specifically bound to CXCR4High. Cells were incubated with the peptide (5 μM) in 1.5% (w/v) FBS containing medium for 2h at 37°C prior to performing FACS analysis. (B) CXCR4-KLA displays the preferential cell killing of CXCR4High compared to CXCR4Low. MTS assays were performed on the cells 72 h after incubation with different concentrations (0–100 μM) of the peptide. (C) The specificity of CXCR4-KLA was confirmed by a reduction of the CXCR4High fraction of both CXCR4High and A2780/ADR after treatment with a suboptimal concentration (2 μM) of CXCR4-KLA for 72 h. (D) CXCR4-KLA is more potent than the conventional CXCR4 antagonists, including AMD3100 and CTCE9908, toward A2780, A2780/ADR, and SKOV3. Cells were incubated with the peptide (10 μM) for 72 h prior to determine the cell viability using MTS assay. (E) Using a combination of drugs (doxorubicin or cisplatin) and CXCR4-KLA (at suboptimal concentrations) showed synergistic cell-killing effects on CXCR4High and CXCR4Low (two way ANOVA, ***p<0.001). (F) The drug-peptide combinations also show synergistic cytotoxicity toward A2780, A2780/ADR, and SKOV-3. The drug dosages were selected according to the IC50 values of the drug or peptide alone against individual cell lines. All the experiments were performed in triplicate and the results were presented as means ± SD of three independent experiments (t-test, *p<0.05, **p<0.01, ***p<0.001).
Fig 5.
Using a combination of doxorubicin and CXCR4-KLA could synergistically inhibit invasion, migration, and colonization of OVC.
(A-C) Representative images showing the wound healing, migration, and colony formation assays on A2780/ADR, CXCR4High, and CXCR4Low. The cells were treated with vehicle alone, doxorubicin alone, CXCR4-KLA alone and a combination of the drug and peptide. (D-F) The results were also presented quantitatively to reflect the percentage and number of cells that invaded the wounds and migrated through the chambers, as well as the number of colonies formed. The results expressed on the graph represent the means ± SD of three independent experiments (t-test, *p<0.05, **p<0.01, ***p<0.001).