Research ArticleBioengineering

Active targeting of chemotherapy to disseminated tumors using nanoparticle-carrying T cells

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Science Translational Medicine  10 Jun 2015:
Vol. 7, Issue 291, pp. 291ra94
DOI: 10.1126/scitranslmed.aaa5447
  • Fig. 1. SN-38 is a potent cytotoxic agent against Eμ-myc lymphoma cells but fails to access sites of lymphoma dissemination in vivo.

    C57BL/6J recipients were injected intravenously with 1 × 106 luciferase- and GFP-expressing Eμ-myc cells. (A) On day 21 after inoculation, lymphoma biodistribution was imaged by IVIS in intact animals, followed by flow cytometry to detect GFP+ tumor cells (green gates). Images are representative ventral and side views of the same animal. LN, lymph node; PE, phycoerythrin. (B) On day 17 after inoculation, free SN-38 (10 mg/kg) was injected intravenously into tumor-bearing mice, and tissue drug concentrations were measured by high-performance liquid chromatography (HPLC) over time. Data are means ± SEM (n = 3 per group). (C) On day 17 after inoculation, tumor-bearing mice were intravenously injected with empty fluorescent liposomes (36.3 mg/kg lipid). Tissues were collected 24 hours after injection for histology. (D) On day 17 after inoculation, tumor-bearing mice were intravenously injected with SN-38–containing liposomes (1 mg/kg SN-38). Tissues were collected 24 hours after injection for HPLC analysis. All data are representative of one of two independent experiments (n = 3 animals per group). Dashed lines in (B) and (D) denote limit of detection (0.5 ng/g tissue) for SN-38 by HPLC.

  • Fig. 2. IL-2/rapamycin–expanded T cells express homing receptors to traffic to lymphoma sites and are resistant to SN-38 toxicity.

    (A) Schematic of T cell functionalization and cell-mediated delivery of SN-38 NCs into tumors. (B and C) Polyclonal T cells from C57BL/6J mice were primed with concanavalin A and IL-7 for 2 days, then expanded in IL-2 with or without rapamycin for 2 days, and analyzed for expression of tissue-homing receptors by flow cytometry. Shown are representative staining histograms (B) and quantification markers (C) of n = 7 replicate cultures from two independent experiments. Data are means ± SEM. P value was determined by t test. MFI, median fluorescence intensity. (D) Eμ-myc cells or IL-2/rapamycin–expanded T cells were cultured in vitro with SN-38 at indicated doses, and viability was assessed by flow cytometry after 24 hours. Data are means ± SEM of pooled four to eight replicate cultures from four independent experiments. (E) IL-2– or IL-2/rapamycin–cultured T cells were taken directly from culture or incubated with SN-38 (20 ng/ml) for 12 hours, then stained for intracellular Bcl-2 expression, and analyzed by flow cytometry. Data are representative plots for n = 3 cultures per condition from one of three independent experiments. P values were determined by two-way analysis of variance (ANOVA) with Bonferroni post-test. (F) IL-2– or IL-2/rapamycin–cultured Thy1.1+ T cells (40 × 106) were adoptively transferred into C57BL/6J mice (n = 6 to 7 per group). The number of Thy1.1+ T cells in blood, spleen, and lymph nodes was enumerated by flow cytometry 2 days after transfer. Data are means ± SEM from one of two independent experiments. P values were determined by t test.

  • Fig. 3. T cells conjugated with SN-38 NCs kill bystander lymphoma cells but not the T cells themselves.

    (A) Cryo–electron microscopy image of SN-38 NCs. (B) Kinetics of SN-38 release from NCs at 37 °C in 10% serum. Data are means ± SEM (n = 8). (C) T cells stained with carboxyfluorescein diacetate succinimidyl ester (blue) and conjugated to fluorescently labeled NCs (pink) were imaged by confocal microscopy. (D) T cells were conjugated to fluorescent DiD NCs either lacking maleimide-lipid (control NCs) or containing maleimide-headgroup lipids (maleimide NCs), washed, and then analyzed by flow cytometry. (E) T cells were conjugated with SN-38 NCs over a range of NC/cell ratios and then lysed to measure the final conjugated amount of SN-38. (F and G) Eμ-myc cells were cocultured with unmodified T cells, empty (“blank”) NC–conjugated T cells, or SN-38 NC–conjugated T cells at indicated T cell/lymphoma cell ratios. Viability of Eμ-myc cells (F) and T cells (G) was measured by flow cytometry 24 hours later. Data are means ± SEM (n = 3 to 8 samples per group). ns, not significant, two-way ANOVA with Bonferroni post-test. All data are representative of two to three independent experiments.

  • Fig. 4. SN-38 NC–conjugated T cells traffic into lymphoma-bearing lymph nodes in vivo and sustain elevated intranodal SN-38 levels over time.

    C57BL/6J mice (n = 3 to 5 per group) were injected intravenously with 1 × 106 Eμ-myc cells and, 17 days later, received intravenous injection of 2 × 108 luciferase+ SN-38–carrying NC-T cells (1 mg/kg equivalent SN-38), control unmodified T cells, or an equivalent dose of free NCs. (A) T cell biodistribution was assessed by whole-animal bioluminescence 38 hours after transfer. Images are ventral views of three different animals and side view of one representative animal. (B) Kinetics of T cell and SN-38 NC-T cell accumulation in tissues assessed by flow cytometry. Data are means ± SEM (n = 4 to 12 animals per group per time point), pooled from five independent experiments. **P < 0.01 versus T cells alone, by two-way ANOVA with Bonferroni post-test. (C) Mice (n = 4 per group) were injected with SN-38 NC–conjugated fluorescently labeled T cells, and tumor (Eμ-myc)–bearing lymph nodes were harvested at 24 hours after injection for histological analysis. Representative image from one of two experiments. (D) Retention of fluorescently labeled SN-38 NCs by T cells (Thy1.1+) in tumor-bearing lymph nodes was measured by flow cytometry 15 hours after transfer. Plot is from one of two representative experiments. (E) Animals were treated with free SN-38 NCs, SN-38 NC-T cells, or free SN-38 at a dose 10-fold greater than that in the NCs. Tumor-bearing lymph nodes were harvested at various times after treatment for HPLC analysis. Data are means ± SEM (n = 3 to 5 animals per group per time point, pooled from four independent experiments). Dashed line denotes limit of SN-38 detection (0.5 ng/g tissue). *P < 0.05, ****P < 0.0001 versus SN-38 NCs, by two-way ANOVA with Bonferroni post-test.

  • Fig. 5. T cell–mediated delivery of NCs improves the therapeutic efficacy of SN-38 without toxicity.

    (A and B) Albino C57BL/6J mice (n = 5 animals per group) were inoculated with 1 × 106 Eμ-myc cells on day 0 and then received four treatments of free SN-38, free SN-38 NCs, or 2 × 108 NC-T cells (1 mg/kg SN-38 in each group), as indicated [arrows on (B)]. Shown are representative results from one of two independent experiments. (A) Bioluminescence images of tumor burden on day 16. (B) Tumor burden as assessed by Eμ-myc bioluminescence (normalized to signal at the start of therapy) over time. *P < 0.01 by two-way ANOVA with Bonferroni post-test on day 16. (C and D) Albino C57BL/6J mice (n = 5 to 6 animals per group) were inoculated as in (A) and then received free SN-38 at 1 or 10 mg/kg, free SN-38 NCs at 1 mg/kg, or 2 × 108 or 5 × 107 NC-T cells (1 or 0.25 mg/kg SN-38, respectively) every 3 days, starting on day 5, for a total of seven treatments. Shown are representative results from one of two independent experiments. (C) Normalized total body bioluminescence measured on day 15. **P < 0.01, by t test. (D) Overall survival. ***P < 0.001 by log-rank test. (E and F) Animals were inoculated and treated as in (C). (E) Weights of animals normalized individually to the pretherapy weight. (F) On day 28, serum was collected for alanine aminotransferase (ALT) and blood urea nitrogen (BUN) measurement; dashed lines indicate reference healthy ranges. Data shown are means ± SEM (n = 4 to 6 animals per group). Shown are representative results from one of three independent experiments.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/291/291ra94/DC1

    Materials and Methods

    Fig. S1. Eμ-myc lymphoma cells express homing markers for peripheral lymph nodes, gut, and bone marrow.

    Fig. S2. T cells expanded in IL-2 with or without rapamycin express similar activation and subset markers.

    Fig. S3. NCs are synthesized by fusion and covalent stabilization of SN-38–containing liposomes.

    Fig. S4. T cell transfer alone has no impact on lymphoma progression.

    Fig. S5. Animals treated with SN-38 therapies show no systemic toxicity.

    Reference (47)

  • Supplementary Material for:

    Active targeting of chemotherapy to disseminated tumors using nanoparticle-carrying T cells

    Bonnie Huang, Wuhbet D. Abraham, Yiran Zheng, Sandra C. Bustamante López, Samantha S. Luo, Darrell J. Irvine*

    *Corresponding author. E-mail: djirvine{at}mit.edu

    Published 10 June 2015, Sci. Transl. Med. 7, 291ra94 (2015)
    DOI: 10.1126/scitranslmed.aaa5447

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Em-myc lymphoma cells express homing markers for peripheral lymph nodes, gut, and bone marrow.
    • Fig. S2. T cells expanded in IL-2 with or without rapamycin express similar activation and subset markers.
    • Fig. S3. NCs are synthesized by fusion and covalent stabilization of SN-38–containing liposomes.
    • Fig. S4. T cell transfer alone has no impact on lymphoma progression.
    • Fig. S5. Animals treated with SN-38 therapies show no systemic toxicity.
    • Reference (47)

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