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Clinical Trial
. 2016 Jun;26(6):713-27.
doi: 10.1038/cr.2016.53. Epub 2016 May 10.

Reversing Drug Resistance of Soft Tumor-Repopulating Cells by Tumor Cell-Derived Chemotherapeutic Microparticles

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Free PMC article
Clinical Trial

Reversing Drug Resistance of Soft Tumor-Repopulating Cells by Tumor Cell-Derived Chemotherapeutic Microparticles

Jingwei Ma et al. Cell Res. .
Free PMC article

Abstract

Developing novel approaches to reverse the drug resistance of tumor-repopulating cells (TRCs) or stem cell-like cancer cells is an urgent clinical need to improve outcomes of cancer patients. Here we show an innovative approach that reverses drug resistance of TRCs using tumor cell-derived microparticles (T-MPs) containing anti-tumor drugs. TRCs, by virtue of being more deformable than differentiated cancer cells, preferentially take up T-MPs that release anti-tumor drugs after entering cells, which in turn lead to death of TRCs. The underlying mechanisms include interfering with drug efflux and promoting nuclear entry of the drugs. Our findings demonstrate the importance of tumor cell softness in uptake of T-MPs and effectiveness of a novel approach in reversing drug resistance of TRCs with promising clinical applications.

Figures

Figure 1
Figure 1
Drug-packaging MPs target TRCs in cancer patients with malignant pleural effusion. (A) Malignant pleural effusion fluids from three end-stage lung cancer patients were collected before and after one-week treatment with intrathoracic injection of Cis-MP. A part of cells harvested from the fluids were smeared on glass slides and observed under microscope (magnification = 200×). A part of cells were stained with FITC-labeled anti-CD45 antibody and analyzed with flow cytometry. (B) Cytologic analysis of pleural effusion. The cells in pleural effusion fluids were collected and smeared on glass slides. HE staining showed abundant aggregates of neoplastic cells with conspicuous nucleoli and scanty cytoplasm (magnification = 200×) before treatment. After one-week treatment, most tumor cells in the malignant fluids disappeared and abundant small immune cells were left. (C) Malignant pleural effusion fluids from three end-stage lung cancer patients were collected before and after treatment with intrathoracic injection of free Cis for one week. The pleural effusion cells were smeared on glass slides and observed under microscope (magnification = 200×). (D) Formation of spheroids of primary tumor cells cultured in 3D soft fibrin gels. CD45 tumor cells were collected from the pleural effusion fluids and seeded in soft 3D fibrin gels (1 mg/ml, gel stiffness = 90 Pa). Five days later, the spheroids were observed under microscope. Scale bar, 50 μm. (E) Treatment with conventional chemotherapeutic drugs induces TRC drug resistance; however, treatment with chemotherapeutic drug-packaging MPs reverses TRC drug resistance in cancer patients with malignant pleural effusions. See also Supplementary information, Figures S1-S4 and Table S1.
Figure 2
Figure 2
Drug-packaging MPs could reverse H22 TRC drug resistance in vitro. (A) H22 hepatocarcinoma tumor cells cultured on conventional rigid plates (control cells) and H22 TRCs (5 × 104) were treated with different concentrations of Cis and Cis-MP for 36 h, MTX and MTX-MP for 24 h or DOX and DOX-MP for 36 h. Drug-packaging MPs used in the drug-MP group contained a similar dose of drugs compared with the corresponding free drug group. Then the cells were collected and stained with APC-conjugated Annexin-V for apoptotic detection by flow cytometry. (B) The reversal of TRC drug resistance was estimated by the IC50 assay. H22 tumor cells cultured on conventional rigid plates and H22 TRCs (5 × 103) were seeded into 96-well plates. Different concentrations of free drug or drug-MPs were added to the experimental group. 24 or 36 h later, the cells were collected and subjected to the MTT assay. (C) Drug-packaging MP-induced cell death is dependent on the caspase pathway. H22 TRCs were pre-treated with the pan-caspase inhibitor z-VAD-FMK for 30 min. All groups were then treated with MTX-MP for 20 h and the cells were collected and stained with APC-conjugated Annexin-V for apoptotic detection by flow cytometry. (D-E) Analysis of multicellular tumor spheroid (round colony) number and colony size in soft 3D fibrin gels. H22 tumor cells were pre-treated with free drugs (Cis or MTX) or drug-MPs (Cis-MP or MTX-MP) for 4 h. The cells (n = 2 500) from each group were seeded into soft 3D fibrin gels. Five days later, tumor spheroid number (D) and colony size (E) were calculated. Scale bar, 50 μm. For all graphs, data represent mean ± SEM; n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student's t-test). See also Supplementary information, Figure S5.
Figure 3
Figure 3
Soft TRCs efficiently take up drug-packaging MPs. (A) Comparison of the abilities of H22 cells and their TRCs to take up T-MPs. PKH26-labeled H22-MPs were incubated with TRCs or control tumor cells (MP/cell = 5 : 1) for 4 h and PKH26-positive cells were detected by flow cytometry (left). The uptake efficiency of different cancer cell lines (H22, Lewis, MCF-7 and A549) and their TRCs was indicated (right). (B) MCF-7 and A549 TRCs were more deformable than their differentiated counterparts. Cells were bound with magnetic microbeads and their deformability was measured under an oscillatory magnetic field. n = 3 independent experiments (at least 150 cells per experiment). (C) Blebbistatin treatment increased the uptake of MPs. MCF-7 or A549 cells cultured on conventional rigid plates were treated with different concentrations of blebbistatin for 6 h and incubated with PKH26-MPs for 4 h. The cells were then collected and analyzed by flow cytometry. (D) Jasplakinolide treatment decreased the uptake of MPs. MCF-7 or A549 TRCs were treated with different concentrations of jasplakinolide for 12 h and incubated with PKH26-MPs for 4 h. The cells were then collected and analyzed by flow cytometry. For all graphs, data represent mean ± SEM; n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student's t-test). See also Supplementary information, Figure S6.
Figure 4
Figure 4
MPs inhibit drug efflux and increase drug retention in TRCs. (A) DOX-MP treatment resulted in enhanced DOX retention in TRCs compared with DOX treatment. H22, MCF-7 TRCs or their control counterparts were incubated with free DOX (1.2 μg/ml) or 1.5 × 106 DOX-MPs (with 1.2 μg/ml DOX) for 4 h and then were incubated in fresh culture medium for additional 6 h. The drug retention was measured by flow cytometric analysis of mean fluorescent intensity (MFI) of DOX. (B-D) MP pretreatment inhibited the drug efflux in TRCs. H22 (B, C) or MCF (D) TRCs or control cells were pretreated with drug-free MPs for 12 h, and then treated with 1 μg/ml DOX for 4 h. After refreshing the culture medium, cells were subjected to various assays as listed in B-D. In B, cells were further cultured and analyzed by flow cytometry at different time points (0, 3, 6, 18 and 24 h). Differences between MFI measured at the end of the 4-h DOX treatment versus MFI measured at different time points were plotted as the efflux rate. In C, cells were further incubated in fresh culture medium for 24 h and supernatants were collected to measure the concentrations of free drugs by HPLC. In D, MCF-7 TRCs were observed under two-photon confocal microscope. Scale bar, 20 μm. (E, F) MP treatment downregulates ABCB1 expression in ADR/MCF-7 cells. ADR/MCF-7 cells were treated with MPs for 12 h (E) or 48 h (F). The expression of ABCB1 was analyzed by real-time PCR (E) and western blot (F). (G) MP treatment downregulates ABCB1 expression in MCF-7 TRCs. MCF-7 TRCs were treated with or without MPs for 12 h. The expression of ABCB1 was analyzed by real-time PCR. For all graphs, data represent mean ± SEM; n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test). See also Supplementary information, Figure S7.
Figure 5
Figure 5
Drug-packaging MPs facilitate the entry of DOX into the nucleus. (A) MP membranes were not present in mitochondria, ER or Golgi apparatus, but showed enhanced co-localization with lysosomes in MCF-7 TRCs. MCF-7 TRCs were incubated with PKH26-labeled MPs for 12 h, and then analyzed with mitochondria, ER, Golgi and lysosome Green Trackers under two-photon confocal microscope. Scale bar, 20 μm. (B) MCF-7 cells, MCF-7 TRCs or ADR/MCF-7 cells were treated with DOX (1.2 μg/ml) or 1.5 × 106 DOX-MP (with 1.2 μg/ml DOX) for 12 h, stained with LAMP-2 antibody (green; a lysosomal marker) and DAPI (blue), and observed under two-photon confocal microscope. Scale bar, 20 μm. See also Supplementary information, Figures S8-S10.
Figure 6
Figure 6
Microtubules are involved in MP-mediated entry of drugs into the nucleus of TRCs. (A) PTX can enhance MP-induced drug entry into nuclei. MCF-7 cells with or without the 12-h MP pretreatment were treated with PTX (6 μg/ml) for 6 h. The cells were then treated with 1 μg/ml DOX for 4 h and observed under two-photon confocal microscope. (B) MPs co-localize with β -tubulin in MCF-7 TRCs. PKH26-labeled MPs were incubated with MCF-7 TRCs for 12 h. Then the cells were stained with FITC-labeled anti-β -tubulin antibody (green). DAPI was used to stain the cell nuclei (blue). (C) Much enhanced co-localization between LAMP-2 and β -tubulin in MP-treated MCF-7 TRCs. MCF-7 TRCs were incubated with MPs for 12 h. Then the cells were stained with fluorescence-labeled anti-LAMP-2 (green) and anti-β-tubulin (red) antibodies. DAPI was used to stain the cell nuclei (blue). (D) Efficiency of Dynein knockdown by siRNAs. Dynein siRNAs or control siRNA (NC) were transfected into MCF-7 TRCs. Twenty-four hours later, the expression of dynein was analyzed by real-time PCR. Error bars indicate mean ± SEM; n = 3 independent experiments. ***P < 0.001 (Student's t-test). (E) Dynein knockdown effectively blocked the entry of DOX into the nucleus. Dynein siRNA#1 or control siRNA were transfected into MCF-7 TRCs. Twenty-four hours later, MCF-7 TRCs were incubated with DOX-MPs for 12 h and then observed under two-photon confocal microscope. For all graphs, scale bars indicate 20 μm. See also Supplementary information, Figure S11.
Figure 7
Figure 7
Drug-packaging MPs effectively target TRCs in vivo. (A) Drug-packaging MPs effectively kill TRCs in mice bearing H22 malignant ascites. 5 × 104 H22 tumor cells were intraperitoneally (i.p.) injected into BABL/c mice. The next day, 2 × 106 Cis-MPs (containing ∼2 μg Cis) or MTX-MPs (containing ∼2 μg MTX) were i.p. injected into the mice once every day for 10 days. On day 11, mice were sacrificed and ascites were collected to count the number of CD45 tumor cells by flow cytometry. (B) 5 × 104 H22 tumor cells were i.p. injected into BABL/c mice. The next day, different concentrations of MTX (2, 10, 50 μg) or 2 × 106 MTX-MPs (containing ∼2 μg MTX) were i.p. injected into the tumor-bearing mice once every day for 10 days . The long-term survival of tumor-bearing mice was assessed (n = 10). (C) Primary tumor cells (n = 2 500) from each group were seeded in soft 3D fibrin gels and incubated for 5 days. The number of tumor spheroids was counted. (D-F) Drug-packaging MPs were capable of killing TRCs in the Lewis lung carcinoma-bearing C57BL/6 mouse model. 8 × 105 Lewis tumor cells were intravenously (i.v.) injected into C57BL/6 mice. Twenty-four hours later, MTX (2 μg) or 2 × 106 MTX-MPs (containing ∼2 μg MTX) were i.v. injected into the mice once every day for 10 days. On day 11, mice were sacrificed and the tumors were indicated by the white arrow (D) and confirmed by HE staining (E). The isolated tumor cells were seeded in soft 3D fibrin gels. The tumor spheroid number was counted (F). For all graphs, data represent mean ± SEM; n = 3 independent experiments. **P < 0.01, ****P < 0.0001 (Student's t-test). (G) 8 × 105 Lewis tumor cells were i.v. injected into C57BL/6 mice. Twenty-four hours later, different concentrations of MTX (2, 10, 50 μg) and 2 × 106 MTX-MPs (with ∼2 μg MTX) were i.v. injected into the tumor-bearing mice once every day for 10 days. The long-term survival of tumor-bearing mice was assessed by the Kaplan-Meier method (n = 12). Data are representative of three independent experiments. P < 0.0001; MTX-MP group vs PBS control group. See also Supplementary information, Figures S12 and S13.

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