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

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 CXCR4High/CD24Low cancer stem cell population (CXCR4High) 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. CXCR4High 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 CXCR4High. 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 CXCR4High might offer a far-reaching potential as supportive chemotherapy.


Introduction
Epithelial ovarian cancer (EOC) patients who receive chemotherapy often develop drug resistance within one year. This often leads to a disease recurrence rate as high as 80%, accompanied by uncontrolled trascoelomic and hematogenous metastases. A tumor is composed of heterogeneous cell populations. Conventional chemotherapy can eradicate most cancer cells, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 but leaves small populations of innately drug-resistant cancer stem cells (CSCs) behind. These residual CSCs, with metastatic potentials, can remodel tumors to become more drug resistant.
In the past, therapeutic strategies were proposed to target the SDF-1/CXCR4 axis of different cancers [17][18][19]. Particularly in OVC, silencing CXCR4 expression by siRNA reduces tumor growth [20]. AMD3100 is a small molecule antagonist of CXCR4 that has been shown to promote the apoptosis of tumor cells and the local T-cell mediated immune responses [21]. A dimerized CXCR4-binding peptide, CTCE-9908, was reported to induce abnormal mitosis [22]. Using antibodies to neutralize SDF-1/CXCR4 could sensitize hyperthermia treatment [23]. However, all these approaches only exhibit modest anticancer effects, presumably because the drugs only block the activities of SDF-1/CXCR4 axis instead of eliminating the cancer cells. In fact, a majority of CXCR4 receptors are stored intracellularly [10,24,25]. The exposure of the cell-surface CXCR4 is a hallmark of a small distinctive cancer stem cell (CSC) that displays tumor formation, invasion, and migration capabilities accompanied with an innate resistance to chemotherapy [26]. Understanding the functional roles of this cell population would allow for the design of effective strategies to encounter drug resistance and metastasis, the common causes of mortality in OVC patients. In this study, we investigated how chemotherapy transiently enriches a small CXCR4 High /CD24 Low cancer stem cell population (CXCR4 High ) to acquire adaptive drug resistance. This CXCR4 High displayed a mesenchymal nature: enhanced migration, invasion, and colony formation properties. We used a CXCR4-targeting peptide drug (CXCR4-KLA) comprised of a CXCR4 binding sequence (KPVSLSYRC) conjugated to a mitochondrial disruption domain (KLA) to demonstrate the therapeutic merits of eradicating CXCR4 High . This peptide exhibited preferential cytotoxicity toward CXCR4 High and could be used with other cytotoxic agents to achieve synergistic OVC cell-killing effect.
Synthesis of CXCR4-KLA peptide CXCR4-KLA, (KPVSLSYRCGGKLAKLAKKLAKLAK), and its FITC-labeled analogue were synthesized on a PS3 peptide synthesizer (Protein Technologies, Tucson, AZ), employing the Fmoc strategy on rink amide [27]. The peptide was purified by reverse-phase HPLC to >98% purity and the expected molecular weights were confirmed by MALD-TOF mass spectrometer.

Quantitative real-time PCR
Quantitative real-time PCR was performed using the StepOnePlus™ Real-Time PCR System by monitoring the real-time increase in fluorescence of the SYBR Green dye (Life Technologies Corp., Grand Island, NY). Total RNAs were isolated using the TRIzol reagent (Life Technologies Corp., Grand Island, NY) and subsequently reverse transcribed to the cDNA using a SuperScript First-Strand Synthesis System (Invitrogen, Grand Island, NY). The quantitative RT-PCR was performed using a SYBR 1 Green PCR Master mix (Applied Biosystems, Grand Island, NY) according to the manufacturer's instructions. The reaction mix was subjected to real-time PCR to detect levels of the corresponding PCR primers. The specificity of each primer pair was confirmed by melting curve analysis. The employed PCR primer pairs are shown in S1 Table. Western blot The total cell extracts were obtained using a Cell Lysis Buffer (Cell Signaling Technology Inc., Danvers, MA) in the presence of protease inhibitor (Roche Applied Science, Indianapolis, IN) at 4˚C for 30 min. Briefly, cells (5x10 6 ) were washed twice with cell wash solution. The extracted proteins (40 μg) was resolved by SDS-PAGE, transferred to nitrocellulose membranes using the Novex™ Bolt™ gel electrophoresis system and iBlot 1 transfer stack system (Life Technologies Corp., Grand Island, NY), and probed with the appropriate dilution of CXCR4 antibody (1:1000, ab2074, Abcam, Cambridge, MA), β-actin antibody (1:10000, A5441, Sigma, St. Louis, MO) Na, K-ATPase antibody (1:1000, #3010, Cell Signaling Technology Inc., Danvers, MA), E-Cadherin (1:1000, #3195, Cell Signaling, Danvers, MA), and Vimentin antibody (1:1000, #3932, Cell Signaling Technology Inc., Danvers, MA). Immunoreactivity was detected using an ECL detection system (GE Healthcare Bio-Sciences Corp., Pittsburgh, PA). Films were exposed at multiple time points to ensure that images were not saturated.
Cell sorting was performed using FACSVantage (BD Bioscience, CA). Cells (2x10 7 ) were stained with CXCR4-APC (clone 12G5, BioLegend, San Diego, CA) for 1 h at 4˚C under sterile conditions. The isolated CXCR4 Low and CXCR4 High subpopulations were further characterized for purity by FACS analysis, as described above.

Wound healing assay
Cells (5x10 6 ) were cultured to >80% confluency in 12-well plates. After scratching a straight line through the cell layer with a sterile 200 μL pipette tip, cells were incubated with doxorubicin, CXCR4-KLA and combination treatment in an RPMI 1640 medium containing 10% FBS (w/v) for 48 h. The images of wound were taken using an EVOS 1 cell imaging system (Life Technologies Corp., Grand Island, NY) at 10X magnification.

Invasion assay
The in vitro migratory properties of cells were studied using Boyden chambers (NeuroProbe, Houston, TX) coated with collagen (Sigma, St Louis, MO). Bottom wells were filled with media (27 μL) containing 2% (w/v) of serum. Cells (5x10 4 /56 μL) were seeded on the upper compartment and then incubated at 37˚C for 24 h. Cells on the upper surface of the filter were then removed using a cotton swab, leaving those attached to the lower surface stained with Diff-Quik reagents (Thermo Scientific, Waltham, MA). The numbers of invaded cells were counted under a microscope with 10X magnification (5 fields/well). A representative graph of six independent experiments was performed.

Soft agar colony formation assay
Cells (5x10 4 ) were suspended in media containing 10% (w/v) FBS and 0.35% (w/v) agar and seeded in pre-solidified media containing 0.75% (w/v) agar containing 10% (w/v) FBS in on 6-well plates. The plates were then incubated at 37˚C in a humidified atmosphere of 5% CO 2 . Colonies of cells were allowed to grow for 2 weeks and any colonies larger than 0.1 mm in diameter were counted using the EVOS 1 cell imaging system (Life Technologies Corp., Grand Island, NY) at 4X magnification.

Xenograft
The protocol was approved by the Institutional Animal Care and Use Committee of Weill Cornell Medicine (Number: 2015-0014). All procedure was performed under isofluorane anesthesia, and all efforts were made to minimize suffering. Briefly, A2780/ADR and the freshly isolated CXCR4 High and CXCR4 Low cells (7 x 10 5 ) suspended in PBS (100 μL) were injected into the flank of 6-week-old female SCID (SHO) mice (Charles River Laboratories, Wilmington, MA). The resulting tumors were measured with digital calipers and tumor volumes were calculated as follows: volume = length x width 2 x 0.52. Samples of each tumor were fixed immediately in 10% (v/v) formaldehyde for further histology studies. Immunohistochemistry stainings were performed on the deparafiinized sections (6 μm), using antihuman CXCR4 monoclonal antibody (ab2074, Abcam, Cambridge, MA) and CD31 (1:100, ab125212, Abcam, Cambridge, MA).
For flow cytometry analysis, SKOV-3/GFP-Luc cells (2 x 10 6 ) suspended in PBS (100 μL) were injected into the flank of 6-week-old female SCID (SHO) mice. When tumor reached approximately 100 mm 3 (approximately 14 days following inoculation), mice were randomized into two groups (n = 4/group) for treatment with doxorubicin (5 mg/kg) in PBS (200 μL) or vehicle only via tail vein injections. After 72 h, tumor tissue was minced and digested with an enzyme cocktail (collagenase A, elastase, and DNase I, Roche Applied Science) in PBS at 37˚C for 1 hr. The cell suspension was strained through a 40 μm cell strainer (BD Biosciences). Cell were washed with PBS three times and analyzed through flow cytometry.

Statistics
All experiments were carried out three times. The results are presented as mean ± SD. For statistical comparisons, Graph Pad Prism 7.0 software was used to determine p-values using Student's t-test and two-way ANOVA. The significance levels were set at Ã p<0.05, ÃÃ p<0.01, and ÃÃÃ p<0.001.

Results
Chemotherapy transiently induces CXCR4 Low-High cell state transition in human ovarian cancer For many years, acquired drug resistance has been interpreted exclusively as a positive selection of distinct populations of innate drug-resistance cancer stem cells (CSCs) or tumor-initiating cells (TICs) after drug treatments [28]. Recently, Sengupta et al demonstrated that breast cancer cells drug resistance through an alternative route that involves a chemotherapy-induced cell state transition [29]. Here, we investigated whether such a dynamic cell state transition occurred in OVC. We first analyzed the phenotypic heterogeneity of A2780 and its doxorubicin-resistant cell lineage (A2780/ADR) by screening for the presence of live (non-fixed) cell subsets that express cell-surface CSC markers (CD44, CD133, and CXCR4) [26,[30][31][32][33][34][35]. FACS analysis showed that A2780 composed with 4.4% of CXCR4 High /CD24 Low CSC population (CXCR4 High ). On the other hand, A2780/ADR, treated weekly with doxorubicin to maintain a consistent degree of drug resistance, displayed a significantly higher percentage (10.6%) of CXCR4 High (Fig 1A). Interestingly, we could barely detect CD44 High /CD24 Low and CD133 High / CD24 Low CSC populations in both A2780 and A2780/ADR. To investigate whether other chemotherapeutic treatments could induce CXCR4 High , we incubated A2780 or SKOV-3 with suboptimal concentrations (IC 20 ) of cisplatin, doxorubicin, or paclitaxel, and then performed FACS analysis of the CSC populations. In all cases, the density of CXCR4 High was increased significantly after 72 h (Fig 1B). The results were confirmed by the increased CXCR4's protein and mRNA levels in the cell lysates (Fig 1C and 1D). Interestingly, the drug-induced CXCR4 Low-High cell transition only occurred temporarily. When the drugs were withdrawn from the cell lines, the original percentage of CXCR4 High was recovered after three passages (Fig 1B). We further investigated whether drug treatments enrich tumoral CXCR4 + -C. We treated SCID mice implanted with SK-OV3 (transfected with GFP and luciferase) with doxorubicin. After 72 h, we isolated the live OVC cells (GFP+) from the tumors, and determined the percentage of CXCR4 + -C. The CXCR4 + -C population in the tumors of the drug-treated mice were significantly higher (7.9 vs. 26%) than in those animals received no treatment (Fig 1E), thus, suggesting that drug treatment could enrich the tumoral CXCR4 + -C population in addition to cell cultures. The chemotherapy-induced CXCR4 High may represent a unique mechanism of acquiring drug resistance. To test this hypothesis, we first isolated CXCR4 High and CXCR4 Low /CD24 Low (CXCR4 Low ) from A2780/ADR in high purity by flow cytometry (Fig 2A). The difference in CXCR4 expression between the two isolated cell clones was confirmed using western blot and real-time PCR (Fig 2B and 2C). Both cell clones lost purity after 20 days of continuous culturing (three passages in the absence of doxorubicin), as shown by the CXCR4 High clone's dramatically reduction in the CXCR4-positive cell density (from 93% to 25%) and whereas the CXCR4 Low clone showed an increase of the CXCR4 High population (from 1% to 5%).
To investigate whether this small CXCR4 High played a significant role in drug resistance, we freshly isolated CXCR4 High and CXCR4 Low from A2780/ADR and compared their susceptibility to drug treatments. MTS assay showed that CXCR4 High was intrinsically resistant to cisplatin, doxorubicin, and paclitaxel ( Fig 2D). The IC 50 values of each drug toward CXCR4 High and its parent A2780/ADR were similar ( Table 1). The drug-induced CXCR4 High contributed drug-resistance, as removing CXCR4 High from A2780/ADR recovered the sensitivity of the cell lines to drug treatments.
Increasing studies have shown that chemotherapy induced drug resistance as well as tumorigenesis via epithelial-mesenchymal transition (EMT) in OVC [26,36]. During our studies, we observed significant differences in the morphology between the isolated CXCR4 High and CXCR4 Low clones (Fig 2E), suggesting that chemotherapy might also induce the EMT of a small population of OVC cells. Despite the two cell clones showed a similar proliferation rate (Fig 2F), CXCR4 High expressed lower E-cadherin (epithelial marker) and higher Vimentin (mesenchymal marker) levels than CXCR4 Low (Fig 2G). The real-time PCR analysis also confirmed that CXCR4 High expressed lower levels of epithelial markers, including E-cadherin and Occludin, and higher levels of mesenchymal markers, including Fibronectin, Snail, N-cadherin, Twist, Zeb1, Zeb2, and Vimentin ( Fig 2H). Moreover, CXCR4 High expressed higher level of mRNA stem cell markers including ABCB1, ABCG2, ALDH1, ALDH2, Lin28, and MyD88 ( Fig 2I). Our data supported that the drug-resistant CXCR4 High population of A2780/ ADR displayed mesenchymal CSC-like characteristics.

CXCR4 High displays enhanced invasion, migration, and tumor formation properties
The mesenchymal nature of CXCR4 High may play significant roles in enhancing the tumor invasion, migration, and formation properties of OVC [26,33]. This prompted us to investigate the migration properties of CXCR4 High . A wound healing assay showed that the cells closed scratched wounds within 48 h, but CXCR4 Low was unable to complete the wound healing at this time frame (Fig 3A and 3B). We also performed Boyden chamber assay to confirm the enhanced migration of CXCR4 High , and showed that three times more cells migrated through the chamber. When compared to CXCR4 Low , CXCR4 High also produced larger and more colonies in a soft agar assay. In terms of tumor formation, the subcutaneously implanted CXCR4 High initiated a much faster tumor growth rate in SCID mice and after 21 days, the average size of tumors was four times larger than the implanted A2780/ADR and CXCR4 Low tumors (Fig 3C). Further immunohistology study showed that the excised CXCR4 High tumors consisted of a higher blood vessel density (Fig 3D). This was expected since the SDF-1/CXCR4 axis has been shown to participate in angiogenesis [10]. Taken together, our data strongly suggested that drug-induced CXCR4 High plays significant roles in tumor progression. The enhanced drug resistance, metastasis, and tumor formation indicated that CXCR4 High could be a therapeutic target for OVC treatment. Currently, there are several clinical trials of AMD 3100, CTCE-9908, and neutralizing antibody that target SDF-1/CXCR4 signaling. However, simply truncating the SDF-1/CXCR4 signaling by CXCR4 antagonists would only lead to moderate anticancer effects [21,37]. An approach that can eliminate rather than antagonize CXCR4 High may increase the therapeutic efficacy. Here, we synthesized a bifunctional peptide (CXCR4-KLA) composed with a CXCR4-binding sequence (KPVSLSYRC) [38] conjugated to a mitochondrial disruption domain (KLA). Once the peptide is delivered inside a cancer cell via receptor-mediated endocytosis, the intracellular KLA domain can physically disrupt the mitochondrial membrane to induce apoptosis by releasing cytochrome c [27]. We first determined the specificity of the peptide. Initial FACS studies demonstrated that CXCR4-KLA selectively bound to CXCR4 High (Fig 4A). MTS assay showed that the peptide was 5-fold more cytotoxic toward CXCR4 High than CXCR4 Low (Fig 4B). The calculated IC 50 values were 2.6 μM and 11.3 μM, respectively. Such a preferential cell-killing effect was further confirmed by CXCR4-KLA selectively reducing the CXCR4-positive cell density in CXCR4 High as well as in its parent A2780/ADR over time, as shown in FACS analysis (Fig 4C). The peptide was more potent than AMD3100 or CTCE-9908 (Fig 4D and S1 Fig). When used together with doxorubicin or cisplatin at the suboptimal concentrations (IC 20 ), the drug-peptide combinations executed synergistic cell killing to both CXCR4 High and CXCR4 Low (Fig 4E).
To demonstrate the therapeutic merits of targeting CXCR4 High , we evaluated the cytotoxicity of CXCR4-KLA alone or in combination with other cytotoxic agents toward A2780, A2780/ADR, and SKOV3. Since the potency of a drug is cell-line specific, we first determined the cytotoxicity of individual drugs or peptide toward each cell line (Table 1), and then formulated a drug-peptide combination with the ratio of the drug (doxorubicin or cisplatin) and peptide (CXCR4-KLA) selected according to the calculated IC 50 values (e.g., Drug A: Drug B = IC 50 of drug A: IC 50 of drug B). We also compared the therapeutic efficacy of the drug-peptide combinations with the drug-AMD3100 combinations. Given to the AMD3100 displayed no cytotoxic effect on OVC cell lines (S2 Fig), we used the same dose of CXCR4-KLA and AMD3100 for the comparative study. As expected, drug-peptide combinations (at suboptimal IC 20 or IC 50 of drug and peptide dosages) were more effective than doxorubicin, cisplatin, AMD3100, or peptide alone, or any of the drug-AMD3100 combinations for eradicating A2780, A2780/ADR, and SKOV-3 (Fig 4F and S3 Fig). This is because using a cytotoxic agent and CXCR4-KLA together could simultaneously eliminate both chemo-sensitive and drug-resistant cancer cells. We also performed Chou-Talalay Plots to confirm that both the doxorubicin-CXCR4-KLA and cisplatin-CXCR4-KLA combinations were synergistic [39], with calculated combination index values of < 1 (S4 Fig). Finally, using a combination of doxorubicin and CXCR4-KLA, we further demonstrated that it could synergistically inhibit the invasion, migration, and colony formation of A2780/ADR and the isolated CXCR4 High (Fig 5). Overall, our results strongly suggested that CXCR4-KLA  The therapeutic merits of targeting CXCR4 cancer stem cells in ovarian cancer

Discussion
Most OVC patients respond well to chemotherapy but quickly develop drug resistance, leading to treatment failure. Currently, there is no effective approach to prevent drug resistance in OVC. The five-year survival rate of patients is low, 30-40% [40]. To design new and more 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, ***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 IC 50 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).
doi:10.1371/journal.pone.0171044.g004 The therapeutic merits of targeting CXCR4 cancer stem cells in ovarian cancer effective treatments, it is essential to understand how chemotherapy induces drug resistance. CXCR4 is a cancer stem cell (CSC) marker [26,41]. In the present studies, we demonstrated that drug treatments caused a dynamic shift of the cell-state equilibrium in OVC cells, a transient increase in CXCR4 High cell population. Such a drug-induced transition event only occurred temporarily. Withdrawing the drug treatments returned to the original equilibrium status overtime. Interestingly, CXCR4 High and CXCR4 Low that were physically isolated from A2780/ADR by flow cytometry was shown to spontaneously re-establish cell-state equilibrium. After three cell passages, the isolated CXCR4 High population lost its purity (with 68% of the cells underwent CXCR4High)Low transition). On the other hand, a small percentage (4%) of the pure CXCR4 Low population transit to CXCR4 High over time. Our results were in agreement with the Markov model, which states that cancer cells can stochastically transit between states [42]. We also found the isolated CXCR4 Low and CXCR4 High were phenotypically different. CXCR4 High was not only more resistance to doxorubicin, paclitaxel, or cisplatin treatment but also played a dominant role in controlling acquired and possibly innate drug resistance. A selective removal of CXCR4 High from doxorubicin-resistant A2780/ADR could recover the sensitivity to drug treatments. The CXCR4 High isolated from A2780/ADR was resistance to paclitaxel and cisplatin in addition to doxorubicin, most likely because of a relative higher expression of ATP-binding cassette sub-family B member 1 (ABCB1), a common cell membrane protein that involves in acquiring multidrug resistance, compared to CXCR4 Low . Other studies also supported ABCB1 is associated with paclitaxel-resistant A2780 cell line, leading to cross-drug resistance [43]. However, further studies are required to investigate the role of CXCR4 High or its associated SDF-1/CXCR4 axis to regulate ABCB1 or vice versa.
Our studies also showed that SDF-1/CXCR4 axis is involved in EMT of various cancers including lung, pancreatic, hepatic, and ovarian cancers [44][45][46]. Here, it was not a surprise to find CXCR4 High displaying mesenchymal-like characteristics, a lower epithelial marker and higher mesenchymal marker levels with enhanced invasion, migration and tumor formation properties compared to CXCR4 Low . Given to the dominant role of CXCR4 High in acquiring drug resistance and metastatic potential, we suggested that a means to truncate CXCR4 High would improve chemotherapeutic treatment outcomes. We designed and synthesized CXCR4-KLA to investigate the therapeutic merits of targeting CXCR4 High . Unlike the conventional antagonists that only block the activities of SDF-1/CXCR4 axis, CXCR4-KLA can eliminate CXCR4 High . This peptide was shown to be more potent than AMD3100 and CTCE-9908. When used in combination with other cytotoxic agents including doxorubicin and cisplatin, it displays synergistic cytotoxicity and the inhibition of cell invasion, migration, and colony formation in OVC. Currently, there are very few reports of using CXCR4 antagonists in combination therapy of OVC [22,37]. Overall, we demonstrated that therapeutic strategies designed to eradicate rather than antagonize CXCR4 High /CD24 Low might offer far-reaching potential as a supportive therapy. One advantage of CXCR4-KLA is that it can be easily modified to target other cancer cells [47]. Given to a tumor consists of heterogeneous cell populations, we could easily replace the binding domain of CXCR4-KLA with other ligands to target other CSCs that express different cell-surface biomarkers such as CD133, CD44, CD117, and MyD88 of OVC in the future [48][49][50][51]. This can be a useful approach to study the functional roles and targeting benefits of a particular or multiple CSC populations. The data were analyzed using the CalcuSyn software as previously described [39]. Data are averages of triplicate determinations ± SEM. (TIF) S1 Table. A list of the primers used for RT-PCR analysis.