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Nanoparticle interactions with immune cells dominate tumor retention and induce T cell–mediated tumor suppression in models of breast cancer

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Science Advances  25 Mar 2020:
Vol. 6, no. 13, eaay1601
DOI: 10.1126/sciadv.aay1601
  • Fig. 1 Particle design and in vitro demonstration of targeting for studies in mice.

    (A) Schematic of particle chemistry showing amine functionalization of BP nanoparticles using maleimide precursors for conjugation with thiol moieties of the antibody (trastuzumab). (B) Western blot analysis showing HER2 protein expression by human breast cancer cell lines used in the study. (C) Immunofluorescence results showing HER2 protein surface expression in six human breast cancer cell lines. MDA-MB-231 is a triple-negative ER/PR/HER2- cell line. MCF7/neo and MCF7/HER2 are an isogenic pair with HER2-expressing (MCF7/HER2) variant having a single copy of HER2 gene and HER2- (MCF7/neo), which received a scrambled gene. Other cell lines are wild type and express varying amounts of HER2 protein. (D) In vitro iron content analysis (ferene-s assay) after exposure of cells to BP and BH nanoparticles shows a positive correlation with HER2 protein level and iron uptake in the breast cancer cells. For the assay, cells were incubated at 37°C for 3 hours with BP or BH nanoparticles (0.5 mg/ml) and evaluated for total iron content after washing unbound particles. Untreated cells, Herceptin alone, and BNF-IgG were used as controls. The average of three independent experiments is shown. Statistical differences among BP, BH, and BNF-IgG were obtained by two-tailed Student’s t test (*P < 0.05 and **P < 0.01). (E) Schematic of the overall study design using mouse models of human breast cancers. See text for details.

  • Fig. 2 Iron content and histopathology analyses demonstrate higher accumulation of antibody-labeled nanoparticles in tumors.

    (A) Gross morphology of tumors following intravenous injection with BP or BH nanoparticles shows different tissue color. Darker (black) color indicates greater particle uptake. Tumors from NOD/scid γ (NSG) mice show more BH than BP. Photo credit: Preethi Korangath, Johns Hopkins University. (B) Representative images of HER2 immunohistochemistry (IHC) from breast xenografts showing that expression correlates with in vitro expression. (C and D) Inductively coupled plasma mass spectrometry (ICP-MS) of Fe recovered from tumors excised from mice injected with BH nanoparticles demonstrates consistently higher Fe content than tumors from mice injected with BP nanoparticles regardless of HER2 status of the tumor. Recovered iron was higher in tumors excised from NSG mice (D) than that from athymic nude mice (C) (*P < 0.05, **P < 0.01, and ***P < 0.001). (E and F) Prussian blue–stained tissue slides recovered from the same tumors as in (C) and (D) and digitally analyzed for percent positive area that showed a similar trend as observed with ICP-MS. (G and H) ICP-MS analysis of Fe from the livers showed higher iron content in mice injected with BP nanoparticles than mice injected with BH nanoparticles, mirroring the results of Fe recovered from tumors (**P ≤ 0.01 and ***P < 0.0001).

  • Fig. 3 Histopathology analysis of tumors reveals antibody-labeled nanoparticles localize to regions rich with immune cells.

    (A) Analysis of Prussian blue–positive (nanoparticle-rich) areas of tumors from nude mice injected with BH nanoparticles reveals only weak correlation with HER2 expression. (B) Conversely, this correlation is stronger in tumors from NSG mice. (C and D) Weak or no correlation was observed between BH nanoparticle presence and CD31+ (vascular endothelium) regions. (E) Representative histology images of sequential sections showing IBA-1+ cells associated with Prussian blue–positive areas in HCC1954 (HER2+) tumors grown in NSG mice and treated with BH (a) hematoxylin and eosin (H&E), (b) Prussian blue, (c) HER2 IHC, (d) IBA-1 IHC, (e) CD-31 IHC, (f) H&E of another area from same tumor, (g) sequential section stained for Prussian blue shows positive staining for iron nanoparticles, and (h) immunofluorescence (IF) staining for IBA-1 shows positivity in the nanoparticle accumulated region. (F and G) Iron recovery from HER2+ (HCC1954) or HER2 (MDA-MB-231) tumors is similar whether BNF nanoparticles have trastuzumab (anti-HER2) or human IgG (polyclonal), suggesting that antibody-antigen binding does not drive intratumor nanoparticle accumulation. ns, not statistically significant.

  • Fig. 4 Comparison of nanoparticle accumulation in syngeneic transgenic (HER2+) tumors reveals that strongest correlation between tumor uptake of antibody-labeled nanoparticles (BH) occurs in immune competent mice.

    (A) Schema of transgenic huHER2 tumor allograft development and IHC confirmation of HER2 protein expression on cancer cells in tumors. (B) IHC analysis demonstrates that HER2 protein expression in syngeneic huHER2 allografts is comparable among the range of immune strains of mice tested: FVB/N, athymic nude, and NSG mice. (C) Gross appearance of huHER2 allograft tumors grown to 150 to 200 mm3 in FVB/N, athymic nude, or NSG mice 24 hours after they were injected via tail vein with BP or BH nanoparticles shows that BH accumulation is greatest in tumors growing in immune competent host(s). Photo credit: Preethi Korangath, Johns Hopkins University. (D) ICP-MS results showing absolute iron recovery from tumors grown in all mice reveals highest accumulation of BH nanoparticles in FVB/N mice (*P < 0.05, **P < 0.005, and ***P ≤ 0.0001). (E) Histology analysis revealed that Prussian blue–positive area was seen in stromal area and colocalized more with IBA-1+ cells than HER2+ tumor cells.

  • Fig. 5 In vitro and in vivo analysis shows preferential uptake of nanoparticles in immune cells.

    (A) Undifferentiated RAW 264.7 (M0) or differentiated M1 or M2 (LPS + IFN-γ or IL-4, respectively) macrophages were incubated for 24 hours with BP or BH, and ferene-s assay was conducted to measure the total amount of iron uptake per cell. As a control, BP and Her, added together, were also used. As shown in the figure, BH nanoparticles were taken up more significantly than BP by macrophages irrespective of their phenotype. The uptake was significantly higher in M1 macrophages than either M0 or M2, which indicates that proinflammatory macrophages take up more BP and BH nanoparticles with preference toward BH. (B) Likewise, LPS-activated neutrophils (induced) preferentially sequestered BH over BP, whereas no difference in uptake was observed with naïve bone marrow neutrophils (uninduced). (C) Total cell count obtained from magnetically separated BP- or BH-injected tumors shows significant difference. Immune competent FVB/N mice (n = 3 per group, two tumors each) bearing huHER2 tumors were intravenously injected with BP or BH. After 24 hours, tumors were harvested and digested to isolate single cells and were magnetically separated to collect nanoparticle-associated cells to determine the total cell count. (D) Analysis of magnetically sorted cells obtained from in vivo tumors showed that nanoparticles were associated with immune cells, not tumor cells. Immune competent FVB/N mice (n = 5 to 8 per group) bearing huHER2 tumors were intravenously injected with PBS, BP, or BH. After 24 hours, tumors were harvested and digested to isolate single cells and were magnetically separated to collect nanoparticle-associated cells for analysis by flow cytometry. Gating strategy is provided in fig. S8. Cell numbers measured from BP- and BH-injected mice are shown as change in ratio relative to PBS-injected mice (PBS ratio = 1). (a) Populations of cancer cells were not changed in nanoparticle-associated cancer cells. Ratios of NK cells (b), monocytes (c), TAMs (d), neutrophils (e), and dendritic cells (f) are increased in nanoparticle fractions, suggesting uptake of nanoparticles by immune cells rather than tumor cells. (*P ≤ 0.05, **P ≤ 0.01, and ***P < 0.001).

  • Fig. 6 Flow cytometry analysis demonstrates impact of nanoparticles on TME in response to intravenous nanoparticle delivery.

    Immune competent FVB/N mice (n = 5 to 8 per group) bearing huHER2 tumors were intravenously injected with PBS, BP, BH, or Herceptin (Her). After 24 hours, tumors were harvested and digested to isolate single cells and evaluated by polychromatic fluorescence-activated cell sorter (FACS). Gating strategy is provided in fig. S8. (A) Relative decreases in T cell (a) and B cell (b) populations were observed following injection of nanoparticles. By contrast, relative increases were measured in many innate immune cell populations within the TME: NK cells (c), neutrophils (d), TAMs (e), and monocytes (f) 24 hours after nanoparticle exposure. Except for TAMs, no significant increase was seen in any other immune cell population after Her injection. (*P ≤ 0.05 and **P ≤ 0.01). (B) Graphic representation of distributions of nanoparticle-associated CD45+ immune cells among the cohorts.

  • Fig. 7 Nanoparticles stimulate T cell infiltration into the TME, inhibiting tumor growth.

    (A) Female FVB/N mice bearing huHER2 allograft tumors (n = 7 to 18 per group) were intravenously injected with either PBS, BP, BH (5 mg per mouse), or Herceptin (175 μg per mouse) 5 days after tumor implantation (day 0). Growth of the tumors was monitored by caliper measurements twice per week for 4 weeks (means ± SEM). Final tumor weight is given in inset (**P < 0.005 and &P ≤ 0.0001). (B) On day 28, all mice were euthanized, and representative images of tumors are shown. Photo credit: Preethi Korangath, Johns Hopkins University. [C (a and b)] Female athymic nude mice bearing huHER2 allograft tumors (n = 6 to 7 per group) were similarly treated as above, and 3 weeks of tumor growth and tumor weight is reported (means ± SEM, *P < 0.05). [D (a and b) and E (a and b)] Flow analysis of tumors: As in (A), mice (n = 5 per group) were intravenously injected with either PBS, BP, BH (5 mg per mouse), or Herceptin (175 μg per mouse) on the 10th day after tumor implantation. Seven days after injection, mice were euthanized; tumors were harvested, and single cells were isolated and evaluated by FACS. Infiltration of CD3+ T cells with increases in CD8+ T cells was measured following nanoparticle exposure, likely leading to growth inhibition observed in (A) (*P < 0.05). FITC, fluorescein isothiocyanate.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/13/eaay1601/DC1

    Supplementary Materials and Methods

    Table S1. Summary of analytical data of (80 nm) BP nanoparticles.

    Table S2. Summary of analytical data of all BNF-HER nanoparticles prepared.

    Table S3. Characteristics of breast cancer cell lines used in the study.

    Table S4. Summary of analytical data of BNF-IgG nanoparticles.

    Table S5. Summary of analytical data of BNF-HER nanoparticles that passed in vitro qualification testing.

    Table S6. Summary of immune modifications in mouse strains used for study.

    Table S7. Summary of Aperio imaging settings used for digital analysis of tissue sections.

    Table S8. Definitions of parameters used for Aperio imaging settings.

    Table S9. Antibodies used for flow cytometry and their dilutions.

    Table S10. Summary of numbers and strains of mice used in the study.

    Table S11. Summary of one-factor model statistical analysis of iron measurements in xenograft models.

    Table S12. Summary of two-factor model statistical analysis of iron measurements in xenograft models.

    Table S13. Summary of three-factor model statistical analysis of iron measurements in xenograft models.

    Table S14. Summary of one-factor model statistical analysis of Prussian blue histopathology analyses in xenograft models.

    Table S15. Summary of two-factor model statistical analysis of Prussian blue histopathology analyses in xenograft models.

    Table S16. Summary of three-factor model statistical analysis of Prussian blue histopathology analyses in xenograft models.

    Table S17. Summary of statistical analysis of whole tumor digests flow cytometry in huHER2 allograft model.

    Table S18. Summary of statistical analysis of nanoparticle-associated fractions (magnetic-sorted sediment) from flow cytometry in huHER2 allograft model.

    Table S19. Summary of statistical analysis of nanoparticle-depleted fractions (magnetic-sorted supernatant) from flow cytometry in huHER2 allograft model.

    Table S20. Summary of statistical analysis of iron measurements (ICP-MS) obtained from the livers of xenograft models.

    Table S21. Ratio of Fe level between groups (treatment).

    Table S22. Ratio of Fe level between groups (strains).

    Table S23. Statistical analysis of ICP-MS huHER2-FVB/N lymph node data.

    Table S24. Statistical analysis of ICP-MS huHER2-FVB/N spleen data.

    Table S25. Statistical analysis of ICP-MS huHER2-FVB/N liver data.

    Table S26. Ratio of percent positive between groups.

    Table S27. Statistical analysis of tumor weight in huHER2-FVB/N.

    Table S28. Statistical analysis of tumor growth in huHER2-FVB/N.

    Table S29. Statistical analysis of whole tumor flow data third day.

    Table S30. Statistical analysis of whole tumor flow data seventh day.

    Table S31. Statistical analysis of whole tumor flow data 14th day.

    Table S32. Statistical analysis of tumor weight–huHER2 allograft in nude mice.

    Table S33. Statistical analysis of tumor growth–huHER2 allograft in nude mice (from initial day to 21st day).

    Fig. S1. Representative images showing immunofluorescence staining of BH particles.

    Fig. S2. Subtracting endogenous iron using PBS controls reveals little tumor retention of plain nanoparticles, and retention of BH nanoparticles is independent of tumor expression of the target antigen HER2.

    Fig. S3. Retention of Herceptin-labeled BNF nanoparticles by xenograft tumors depends on immune strain of host.

    Fig. S4. Weak correlations were found between deposits of plain nanoparticles and HER2, CD31+, or IBA-1+ regions in tumors of mice injected with BP nanoparticles.

    Fig. S5. BNF nanoparticles labeled with a nonspecific IgG polyclonal human antibody were retained by tumors.

    Fig. S6. Histopathology data support ICP-MS results for tumor retention of nanoparticles, and ICP-MS data show nanoparticles accumulated in lymph nodes, spleens, and livers of injected mice.

    Fig. S7. Within tumors, nanoparticles localized in stromal regions rather than in cancer cell–rich regions.

    Fig. S8. Gating for flow cytometry was conducted to ascertain immune cell populations residing in tumors.

    Fig. S9. Flow cytometry analysis of huHER2 tumors harvested from immune competent mice reveals tumor immune microenvironment changes, and magnetically sorted tumor immune cell populations demonstrates impact of nanoparticles on tumor immune cells in response to intravenous nanoparticle delivery.

    Fig. S10. Pan-leukocyte inhibition abrogates BH nanoparticle retention in tumors.

    Fig. S11. Systemic exposure to BNF nanoparticles resulted in tumor growth inhibition but only if the host has an intact (adaptive) immune system (i.e., T cells).

    Fig. S12. Following systemic exposure to nanoparticles, intratumor T cell populations decline through the third day and then increase by day 7 relative to PBS controls.

    Fig. S13. Exposure to nanoparticles induces changes in adaptive immune signaling in tumors of nanoparticle-treated mice.

    Fig. S14. Changes in innate cell population in tumors of nanoparticle-treated mice.

    Fig. S15. Data suggest that systemically delivered BNF nanoparticles are preferentially sequestered by inflammatory immune cells within the TME, resulting in immune recognition of the tumor.

  • Supplementary Materials

    This PDF file includes:

    • Materials and Methods
    • Table S1. Summary of analytical data of (80 nm) BP nanoparticles.
    • Table S2. Summary of analytical data of all BNF-HER nanoparticles prepared.
    • Table S3. Characteristics of breast cancer cell lines used in the study.
    • Table S4. Summary of analytical data of BNF-IgG nanoparticles.
    • Table S5. Summary of analytical data of BNF-HER nanoparticles that passed in vitro qualification testing.
    • Table S6. Summary of immune modifications in mouse strains used for study.
    • Table S7. Summary of Aperio imaging settings used for digital analysis of tissue sections.
    • Table S8. Definitions of parameters used for Aperio imaging settings.
    • Table S9. Antibodies used for flow cytometry and their dilutions.
    • Table S10. Summary of numbers and strains of mice used in the study.
    • Table S11. Summary of one-factor model statistical analysis of iron measurements in xenograft models.
    • Table S12. Summary of two-factor model statistical analysis of iron measurements in xenograft models.
    • Table S13. Summary of three-factor model statistical analysis of iron measurements in xenograft models.
    • Table S14. Summary of one-factor model statistical analysis of Prussian blue histopathology analyses in xenograft models.
    • Table S15. Summary of two-factor model statistical analysis of Prussian blue histopathology analyses in xenograft models.
    • Table S16. Summary of three-factor model statistical analysis of Prussian blue histopathology analyses in xenograft models.
    • Table S17. Summary of statistical analysis of whole tumor digests flow cytometry in huHER2 allograft model.
    • Table S18. Summary of statistical analysis of nanoparticle-associated fractions (magnetic-sorted sediment) from flow cytometry in huHER2 allograft model.
    • Table S19. Summary of statistical analysis of nanoparticle-depleted fractions (magnetic-sorted supernatant) from flow cytometry in huHER2 allograft model.
    • Table S20. Summary of statistical analysis of iron measurements (ICP-MS) obtained from the livers of xenograft models.
    • Table S21. Ratio of Fe level between groups (treatment).
    • Table S22. Ratio of Fe level between groups (strains).
    • Table S23. Statistical analysis of ICP-MS huHER2-FVB/N lymph node data.
    • Table S24. Statistical analysis of ICP-MS huHER2-FVB/N spleen data.
    • Table S25. Statistical analysis of ICP-MS huHER2-FVB/N liver data.
    • Table S26. Ratio of percent positive between groups.
    • Table S27. Statistical analysis of tumor weight in huHER2-FVB/N.
    • Table S28. Statistical analysis of tumor growth in huHER2-FVB/N.
    • Table S29. Statistical analysis of whole tumor flow data third day.
    • Table S30. Statistical analysis of whole tumor flow data seventh day.
    • Table S31. Statistical analysis of whole tumor flow data 14th day.
    • Table S32. Statistical analysis of tumor weight–huHER2 allograft in nude mice.
    • Table S33. Statistical analysis of tumor growth–huHER2 allograft in nude mice (from initial day to 21st day).
    • Fig. S1. Representative images showing immunofluorescence staining of BH particles.
    • Fig. S2. Subtracting endogenous iron using PBS controls reveals little tumor retention of plain nanoparticles, and retention of BH nanoparticles is independent of tumor expression of the target antigen HER2.
    • Fig. S3. Retention of Herceptin-labeled BNF nanoparticles by xenograft tumors depends on immune strain of host.
    • Fig. S4. Weak correlations were found between deposits of plain nanoparticles and HER2, CD31+, or IBA-1+ regions in tumors of mice injected with BP nanoparticles.
    • Fig. S5. BNF nanoparticles labeled with a nonspecific IgG polyclonal human antibody were retained by tumors.
    • Fig. S6. Histopathology data support ICP-MS results for tumor retention of nanoparticles, and ICP-MS data show nanoparticles accumulated in lymph nodes, spleens, and livers of injected mice.
    • Fig. S7. Within tumors, nanoparticles localized in stromal regions rather than in cancer cell–rich regions.
    • Fig. S8. Gating for flow cytometry was conducted to ascertain immune cell populations residing in tumors.
    • Fig. S9. Flow cytometry analysis of huHER2 tumors harvested from immune competent mice reveals tumor immune microenvironment changes, and magnetically sorted tumor immune cell populations demonstrates impact of nanoparticles on tumor immune cells in response to intravenous nanoparticle delivery.
    • Fig. S10. Pan-leukocyte inhibition abrogates BH nanoparticle retention in tumors.
    • Fig. S11. Systemic exposure to BNF nanoparticles resulted in tumor growth inhibition but only if the host has an intact (adaptive) immune system (i.e., T cells).
    • Fig. S12. Following systemic exposure to nanoparticles, intratumor T cell populations decline through the third day and then increase by day 7 relative to PBS controls.
    • Fig. S13. Exposure to nanoparticles induces changes in adaptive immune signaling in tumors of nanoparticle-treated mice.
    • Fig. S14. Changes in innate cell population in tumors of nanoparticle-treated mice.
    • Fig. S15. Data suggest that systemically delivered BNF nanoparticles are preferentially sequestered by inflammatory immune cells within the TME, resulting in immune recognition of the tumor.

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