Chemotherapy induces adaptive drug resistance and metastatic potentials via phenotypic CXCR4-expressing cell state transition in ovarian cancer

<div><p>Ovarian cancer (OVC) patients who receive chemotherapy often acquire drug resistance within one year. This can lead to tumor reoccurrence and metastasis, the major causes of mortality. We report a transient increase of a small distinctive CXCR4^High/CD24^Low cancer stem cell population (CXCR4^High) in A2780 and SKOV-3 OVC cell lines in response to cisplatin, doxorubicin, and paclitaxel, treatments. The withdrawal of the drug challenges reversed this cell-state transition. CXCR4^High exhibits dormancy in drug resistance and mesenchymal-like invasion, migration, colonization, and tumor formation properties. The removal of this cell population from a doxorubicin-resistant A2780 lineage (A2780/ADR) recovered the sensitivity to drug treatments. A cytotoxic peptide (CXCR4-KLA) that can selectively target cell-surface CXCR4 receptor was further synthesized to investigate the therapeutic merits of targeting CXCR4^High. This peptide was more potent than the conventional CXCR4 antagonists (AMD3100 and CTCE-9908) in eradicating the cancer stem cells. When used together with cytotoxic agents such as doxorubicin and cisplatin, the combined drug-peptide regimens exhibited a synergistic cell-killing effect on A2780, A2780/ADR, and SKOV-3. Our data suggested that chemotherapy could establish drug-resistant and tumor-initiating properties of OVC via reversible CXCR4 cell state transition. Therapeutic strategies designed to eradicate rather than antagonize CXCR4^High might offer a far-reaching potential as supportive chemotherapy.</p></div

CXCR4^High displayed drug-resistant and mesenchymal properties.

<p>(A) FACS analysis showed that the freshly isolated CXCR4^High and CXCR4^Low 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, CXCR4^High, and CXCR4^Low 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 CXCR4^High cells were intrinsically drug-resistant. The cell viability was determined by MTS assay. (E) Microscopic images (10x magnification) of CXCR4^High and CXCR4^Low. 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 CXCR4^High and CXCR4^Low lineages. CXCR4^High exhibits the similar cell proliferation rate. (G and H) CXCR4^High displayed mesenchymal phenotypes, as shown by the higher and lower expressions of the epithelial and mesenchymal markers, respectively, compared to CXCR4^Low using western blot and real-time PCR. (I) CXCR4^High 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, *<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001).</p

The freshly isolated CXCR4^High showed enhanced invasion, migration, and tumor formation properties compared to the CXCR4^Low.

<p>(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, **<i>p</i><0.01 and ***<i>p</i><0.001). (C) A comparison of the A2780/ADR, CXCR4^High and CXCR4^Low 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, ***<i>p</i><0.001). (D) Representative microscope images of the H&E staining and immunohistochemistry of the tumor sections show that CXCR4^High tumor consist of higher expressions of CXCR4 and CD31 (blood vessel). Arrows indicate the vascular core areas.</p

CXCR4^High can be a useful target for OVC treatment.

<p><b>(A) CXCR4-KLA (labeled with FITC) specifically bound to CXCR4</b>^<b>High</b><b>.</b> 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 CXCR4^High compared to CXCR4^Low. 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 CXCR4^High fraction of both CXCR4^High 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 CXCR4^High and CXCR4^Low (two way ANOVA, ***<i>p</i><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, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001).</p

Comparison of the IC₅₀ values among different drugs against various OVC cell lines or cell clones.

<p>Comparison of the IC₅₀ values among different drugs against various OVC cell lines or cell clones.</p

Using a combination of doxorubicin and CXCR4-KLA could synergistically inhibit invasion, migration, and colonization of OVC.

<p>(A-C) Representative images showing the wound healing, migration, and colony formation assays on A2780/ADR, CXCR4^High, and CXCR4^Low. 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, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001).</p

Smart Nanotransformers with Unique Enzyme-Inducible Structural Changes and Drug Release Properties

We previously reported a high aspect ratio peptide nanofiber that could be effectively delivered to tumors with minimal nonspecific uptake by other organs. The peptidic nature offers the design flexibility of smart formulation with unique responsiveness. Two new formulations that behave congruously as nanotransformers (NTFs) are reported herein. NTF1 and NTF2 could biomechanically remodel upon enzyme activation to generate a degradable and an aggregable effect, respectively, within the lysosomal compartment. These NTFs were further evaluated as carriers of mertansine (DM1), a microtubule inhibitor. DM1-loaded NTF1 could be degraded by cathepsin B (CathB) to release the same active metabolite, as previously described in the lysosomal degradation of antibody-DM1 conjugate. In contrast, CathB only partially digested DM1-loaded NTF2 and induced aggregate formation to become a storage reservoir with slow payload release property. The DM1-loaded NTF1 exhibited a comparable cytotoxicity to the free drug and was more effective than the NTF2 formulation in eradicating triple negative breast cancer. Our data suggested that biological transformers with distinct enzyme-induced structural changes and payload release profiles could be designed for the intracellular delivery of cytotoxic and imaging agents

Immunofluorescence study to demonstrate the presence of S100A8/A9-positive cells in the kidney from an acute kidney injury model.

<p>Immunofluorescence study for neutrophils (CD15), monocytes/macrophages (CD68), and S100A8/A9-staining cells in an acute kidney injury model demonstrating that S100A8/A9, CD15, and CD69 staining were found frequently in the CDDP group which might reflect the inflammatory damage in this group. S100A8/A9 staining appeared to correlate with areas of CD15 (a) and CD68 staining (b). These findings also suggest that increased S100A8/A9 expression reflects the infiltration of inflammatory cells in intrinsic AKI. Magnification, × 400 in (a) and (b)</p

Serum and urinary Klotho, S100A8/A9, and Neutrophil gelatinase-associated lipocalin (NGAL) concentrations in an acute kidney injury model.

<p>(a) Urinary Klotho and urinary Klotho/creatinine (Cr) decreased significantly in the volume depletion (VD) group. (b) Serum and urinary S100A8/A9 and urinary S100A8/A9/Cr increased significantly in the cisplatin (CDDP) group. (c) Serum and urinary NGAL showed no significant difference between the VD and CDDP groups. Data are given as means±SD. (<i>n</i> = 6 for each group). *, p < 0.05, vs. the control group; †, p < 0.05, vs. the VD group.</p

Semiquantitative immunoblotting and immunohistochemistry of the kidney from the acute kidney injury model.

<p>(a) Renal Klotho abundance was reduced in the volume depletion (VD) group (48%) versus the control (100%) and cisplatin (CDDP) groups (74%). Immunohistochemical analysis also showed decreased renal Klotho labeling in kidneys from VD group. (b) S100A8/A9 abundance was increased in the CDDP group (220%) versus the control (100%) and VD groups (83%). Immunohistochemical analysis also showed increased renal S100A8/A9 labeling in kidneys from CDDP group. (c) Neutrophil gelatinase-associated lipocalin (NGAL) abundance and labeling showed no significant difference between the VD and CDDP groups. Data are given as means±SD. *, p < 0.05, vs. the control group; †, p < 0.05, vs. the VD group; magnification, × 200.</p