Research ArticleCancer

Autologous tumor cell–derived microparticle-based targeted chemotherapy in lung cancer patients with malignant pleural effusion

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Science Translational Medicine  09 Jan 2019:
Vol. 11, Issue 474, eaat5690
DOI: 10.1126/scitranslmed.aat5690
  • Fig. 1 Characterization of TMPs and TMPs-MTX.

    (A) TEM of (left) multiple TMPs, (middle) a single TMP of rounded shape, and (right) a single TMP of cup shape. Scale bar, 200 nm. (B) Representative size and particle distribution plots of A549-derived TMPs-MTX. (C) Flow cytometry analysis of A549-derived TMPs labeled with annexin V–fluorescein isothiocyanate (FITC), EpCAM–Brilliant Violet 421, and CD147–Alexa Fluor 647 (negative control shown as pale gray histograms). (D) Western blot of EpCAM, CD63, CD9, and TSG101 expression in A549 whole-cell lysates (positive control) and TMP pellets. (E) Standard curve of MTX (left) and concentration of MTX loaded in LLC-derived MPs. LLC cells (4 × 107) were exposed to a concentration gradient of MTX (0, 1, 10, 100, and 500 μg/ml) under UV irradiation for the preparation of TMPs (L0, L1, L10, L100, and L500, respectively). The concentration of MTX loaded in LLC-derived MPs suspended in 500 μl of filtered PBS was measured by HPLC. (F to I) C57BL/6 mice received intrapleural injection of LLC cells (day 0), followed by intrapleural treatment 5 days later with phosphate-buffered saline (PBS), TMPs induced by UV irradiation (TMP0), and TMPs secreted by tumor cells naturally without UV exposure (TMPauto) every other day for 5 days. Mice were euthanized at day 14 (n = 8 per group). (F) Representative photographs of pleural tumors on the parietal and visceral pleura. (G) Pleural tumor number. (H) MPE volume. (I) Life-span analysis in the three groups (n = 7 per group). *P < 0.05 and ***P < 0.0001.

  • Fig. 2 Accumulation and cytotoxicity effect of TMPs-MTX in tumor cells and immune cells in vitro.

    (A and B) Representative confocal microscopy images of A549 cells incubated with A549-derived TMPs for 24 hours. QD-labeled TMPs (red) were added into the culture medium of parental cells overnight at 37°C. A549 cells were stained by dye carboxyfluorescein diacetate succinimidyl ester (CFSE) (green), and the nucleus was counterstained with dye 4′,6-diamidino-2-phenylindole (DAPI) (blue). (A) Most A549 cells showed an accumulation of TMPs. Scale bars, 100 μm. (B) Zoomed image of a single A549 cell showed the TMPs’ internalization in the cytoplasm. Scale bars, 10 μm. (C) Flow cytometric analysis of TMPs’ internalization into A549 cells at multiple time points (n = 3). (D) Detection of apoptosis in LLC cells treated with TMPs-MTX for 48 hours. TMPs-MTX (1 × 106) packaging MTX at a total dose of ~1 μg were used for the cytotoxicity assay, compared to the naive empty TMPs group (TMP0) and to the free MTX group at concentrations of 10 μg/ml (MTX10) and 100 μg/ml (MTX100), with PBS as control. Treated cancer cells were stained with an annexin V–FITC apoptosis detection kit, and apoptotic cells were defined as annexin V–positive cells as measured by flow cytometry (n = 3). (E and F) Detection of apoptosis in MPE-derived malignant cells (E) and other target cancer cell lines such as A549/MCF-7/B16-F10 cells (F) treated with TMPs-MTX for 48 hours (n = 3). (G) Representative confocal microscopy images of CFSE-labeled RAW264.7 macrophages (green) incubated with red fluorescent TMPs overnight at 37°C (scale bars, 10 μm) and the incidence of RAW264.7 apoptosis were analyzed by flow cytometry after 48 hours of incubation (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 In vivo tissue distribution and cell targeting of TMPs and TMPs-MTX.

    (A) In vivo imaging of C57 MPE models induced by LLC-Luc cells at 1, 3, 24, and 48 hours after intrapleural (IP) injection with DiR-labeled LLC-derived TMPs-MTX. A scale of the radiance efficiency is presented to the right of the images. (B and C) MPE mice were treated by intravenous (IV) or IP injections with DiR-labeled LLC-derived TMPs. Major organs were excised and weighed at 24 hours after injection. Representative ex vivo fluorescence images (B) and fluorescence intensity (C) of major organs and tumors are shown (n = 4). (D and E) Mice with MPE induced by LLC cells were intrapleurally injected with PKH67-labeled LLC-derived TMPs. Twenty-four hours later, mice were euthanized. Representative confocal images of immunofluorescent staining of frozen tumor sections with CD11b (scale bars, 20 μm) (D) and F4/80 (scale bars, 20 μm) (E) primary antibodies are shown. Insets are magnified. (F) Representative flow cytometry dot plots of PKH67-labeled TMP accumulation in lymphocytes and macrophages in tumor tissues. SSA, side scatter. (G) Quantification of PKH67-labeled TMP accumulation in various cell types in tumors and MPE (means ± SEM, n = 4 mice). *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 4 Impact of TMP-MTX treatment on murine MPE models.

    (A) Graphical outline of in vivo TMP-MTX treatment experiments. C57BL/6 mice received intrapleural injection of LLC cells or MC38 cells (white arrow) followed by intrapleural treatment with TMPs-MTX packaging 3 μg of MTX, TMP0, MTX (2.5 mg/kg), or PBS three times (on days 5, 7, and 9 after tumor inoculation) (black arrows). Mice were euthanized after 14 days for LLC models and 12 days for MC38 models (gray arrow). (B to E) In vivo bioluminescence images (B) and quantitation of luciferase activity (C) (n = 6 per group), pleural tumor number (D), and pleural fluid volume (E) of mice with LLC-induced MPE treated with TMPs-MTX (n = 14 per group). (F and G) Pleural tumor number (F) and MPE volume (G) of mice with MC38-induced MPE after TMP-MTX treatment (n = 12 per group). The graphs depict data pooled from two independent experiments for MPE volume and pleural tumor number for both LLC and MC38 models. (H and I) Comparisons of CD31-positive cells (H) and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining among cells (I) in the four groups (each n = 6). Microvessel density (MVD) defined as the mean number of microvessels determined by CD31 immunostaining. Scale bar, 100 μm. (J and K) Life-span analysis in four groups of LLC and MC38 models. Kaplan-Meier survival analysis shows a comparison of TMP-MTX treatment with the three control groups in LLC models (J, n = 12) and in MC38 models (K, n = 9). *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 5 Infiltrating immune cell proportion alternation in pleural tumor and MPE after TMP-MTX treatment.

    (A to D) Flow cytometry analysis of MPE samples in MC38 models after different treatments. (A) CD45 cells within the live gate. (B) Lymphocyte infiltrates including CD45+CD3+ T cells, CD45+CD3+CD4+ T cells, CD45+CD3+CD8+ T cells, CD4/CD8 T cell ratio, and CD45+CD3+CD4+Foxp3+ Tregs. The graphs depict data pooled from two independent experiments. (C and D) Myeloid cell infiltrates including PMNs (C) and TAMs (D) within the CD45+ gate. (E to J) Flow cytometry analysis of tumor samples in MC38 models receiving different treatments. (E) Total infiltrates (CD45+) within the live gate. (F) Lymphocyte infiltrates including CD45+CD3+ T cells, CD45+CD3+CD4+ T cells, and CD45+CD3+CD8+ T cells. (G) CD4/CD8 T cell ratio. (H) CD45+CD3+CD4+Foxp3+ Treg infiltrates. (I and J) Myeloid cell infiltrates including PMNs (I) and TAMs (J). Data (shown as means ± SEM) were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis. (K to N) Mice bearing LLC tumors and MPE were treated intrapleurally with TMPs-MTX, clodronate liposomes, or their combination. Fourteen days later, MPEs were analyzed by flow cytometry. Representative dot plots of CD11b+F4/80+ cells of MPE samples for each treatment (K) and quantification of TAMs (L), MPE volume (M), and survival rates (N) of mice receiving different treatments are shown (P < 0.001 for combination therapy compared to the PBS group and P = 0.045 compared to the TMP-MTX group by log-rank test). *P < 0.05 and **P < 0.01; ns, no significance.

  • Fig. 6 Therapeutic effect of ATMP-MTX in lung cancer patients with MPE.

    (A) ATMP-MTX treatment and biomarker assessment schedule. Patients were intrapleurally infused with ATMPs-MTX on days 1, 3, 5, 7, 9, and 11 (blue arrows), accompanied by the routine examination of pleural fluid at baseline and on treatment after each infusion (white triangles), as well as ultrasound and CT scans at baseline, at the end of the treatment (day 12) and at 1-month follow-up (black squares). In addition, blood sampling was performed for assessment of possible adverse effects and immune responses (red triangles). (B) Top: CT images of the thorax of a 56-year-old patient with MPE. The red dashed line indicates the pleural effusion (left, before treatment; right, 28 days after treatment). Bottom: Pleural effusion volume assessment of the same patient by CT analysis (left, before treatment; right, 28 days after treatment). (C) Kaplan-Meier curve of pleurodesis success in all 11 patients. Most participants achieved the predefined pleurodesis criteria at a median of 7 days. Day 1 was defined as the first day of intrapleural treatment with ATMPs-MTX. (D) Top: Light microscope examination of MPE cells from a patient treated with ATMPs-MTX (left, before treatment; right, after treatment). Bottom: Wright-Giemsa stain of MPE cell smear from a patient treated with ATMPs-MTX under a light microscope (left, before treatment; right, after treatment). Scale bars, 100 μm. (E) The proportions of CD45 cells within the live gate in MPE samples from clinical subjects before and after ATMP-MTX treatment. Data are represented as means ± SEM. ***P < 0.001.

  • Fig. 7 Alteration of immune infiltrates in MPE after ATMP-MTX treatment.

    MPE samples from 10 patients (MPE of 1 patient resolved after one injection) were collected to investigate the immune infiltrates by flow cytometry at the indicated time as shown [baseline, before treatment (D0); 1st, 48 hours after the first intrapleural infusion (D3); 2nd, 48 hours after the second infusion (D5)]. (A) The proportions of lymphocytes within the CD45+ gate, CD4+ cells within the CD45+CD3+ T cell gate, and CD8+ T cells within the CD45+CD3+ T cell gate. (B to D) Quantification of cytokines produced by MPE-derived tumor-infiltrating T lymphocytes. TILs were isolated from MPE and analyzed for the expression of IL-2 and IFN-γ after phorbol 12-myristate 13-acetate/ionomycin restimulation by intracellular cytokine staining. (B) IFN-γ+ T cells within the CD45+CD3+ T cell gate. (C) IFN-γ+ cells and IL-2+ cells (n = 9) within CD45+CD3+CD4+ cells. (D) IFN-γ+ cells and IL-2+ cells (n = 9) within CD45+CD3+CD8+ cells. (E) The proportions of regulatory T lymphocytes. (F) The proportions of myeloid cells (CD45+CD3CD11b+). (G) The change in the numbers of tumor-associated neutrophils (TANs) (CD45+CD11b+CD15+CD66b+) of three patients during ATMP-MTX infusions. MPE of the subjects resolved quickly after ATMP-MTX treatment, and MPE samples at 28 days were only available in three patients. Black arrows indicate the days of ATMP-MTX intrapleural infusion therapy. (H and I) (H) The proportions of monocytes (CD45+CD3CD14+) in patients’ MPE samples over time. (I) Representative flow cytometry plots of MPE samples stained for CD14 and CD163, gated on CD45+ cells from a patient with lung adenocarcinoma before and after therapy (left). Quantification of TAMs (CD45+CD14+CD163+) (middle). Percentages of TAMs in three patients at different time points during ATMP-MTX infusions (right). Black arrows indicate the days of ATMP-MTX intrapleural infusion therapy. Data (shown as median ± interquartile range) were analyzed using a repeated-measures model. *P < 0.05, **P < 0.01, and ***P < 0.001. (J) The concentrations of pleural fluid IFN-γ, IL-17, TNF-α (tumor necrosis factor–α), and MCP-1 (monocyte chemoattractant protein 1) (n = 8; the concentration of MCP-1 was below the detection range in two patients’ MPE) were measured. Red squares represent data from the only nonresponder (patient 13).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/474/eaat5690/DC1

    Materials and Methods

    Fig. S1. Characterization of TMPs and TMPs-MTX.

    Fig. S2. Effect of TMPs on tumor cell viability and invasion in vitro.

    Fig. S3. Cellular uptake and cytotoxicity of TMPs-MTX and the interaction of TMPs-MTX with immune cells.

    Fig. S4. Biodistribution and internalization of TMPs-MTX in vivo.

    Fig. S5. Analysis of immune infiltrates in pleural tumor and MPE of LLC cell–induced murine MPE models after ATMP-MTX treatment.

    Fig. S6. Immune cell components in peripheral blood after ATMP-MTX treatment and macrophage depletion by clodronate liposome treatment.

    Fig. S7. ATMP-MTX treatment in advanced lung cancer patients with MPE.

    Fig. S8. Immune cell components in peripheral blood and cytokine concentrations in MPE of lung cancer patients treated with ATMPs-MTX.

    Table S1. Patient demographics.

    Table S2. Patient adverse events related to ATMP-MTX treatment.

    References (5760)

  • This PDF file includes:

    • Materials and Methods
    • Fig. S1. Characterization of TMPs and TMPs-MTX.
    • Fig. S2. Effect of TMPs on tumor cell viability and invasion in vitro.
    • Fig. S3. Cellular uptake and cytotoxicity of TMPs-MTX and the interaction of TMPs-MTX with immune cells.
    • Fig. S4. Biodistribution and internalization of TMPs-MTX in vivo.
    • Fig. S5. Analysis of immune infiltrates in pleural tumor and MPE of LLC cell–induced murine MPE models after ATMP-MTX treatment.
    • Fig. S6. Immune cell components in peripheral blood after ATMP-MTX treatment and macrophage depletion by clodronate liposome treatment.
    • Fig. S7. ATMP-MTX treatment in advanced lung cancer patients with MPE.
    • Fig. S8. Immune cell components in peripheral blood and cytokine concentrations in MPE of lung cancer patients treated with ATMPs-MTX.
    • Table S1. Patient demographics.
    • Table S2. Patient adverse events related to ATMP-MTX treatment.
    • References (5760)

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