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Cancer Prevention Research
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Research Article

Analysis of Immune Cells from Human Mammary Ductal Epithelial Organoids Reveals Vδ2+ T Cells That Efficiently Target Breast Carcinoma Cells in the Presence of Bisphosphonate

Nicholas A. Zumwalde, Jill D. Haag, Deepak Sharma, Jennifer A. Mirrielees, Lee G. Wilke, Michael N. Gould and Jenny E. Gumperz
Nicholas A. Zumwalde
1Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
2McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
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Jill D. Haag
2McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
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Deepak Sharma
2McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
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Jennifer A. Mirrielees
3Department of Surgery, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
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Lee G. Wilke
3Department of Surgery, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
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Michael N. Gould
2McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
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  • For correspondence: jegumperz@wisc.edu gould@oncology.wisc.edu
Jenny E. Gumperz
1Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
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  • For correspondence: jegumperz@wisc.edu gould@oncology.wisc.edu
DOI: 10.1158/1940-6207.CAPR-15-0370-T Published April 2016
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    Figure 1.

    Purified organoid fragments demonstrate epithelial enrichment compared with stromal fraction. A, light microscopic image of representative organoid fragments purified from human breast reduction tissue. B, flow cytometry analysis of the CD31− (non-endothelial) and CD45− (non-hematopoietic) cells shows that a higher percentage of cells express EpCAM (an epithelial marker) in the organoid fraction compared with the stromal fraction. C, quantification of ≥10 different patient reduction samples. **, P = 0.0003 (Mann–Whitney). D, quantification of the CD31+ or CD45+ cells from the organoids from ≥13 different patient reductions. Each symbol represents a different donor's tissue sample.

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    Figure 2.

    Characterization of immune cells from breast-derived organoids yields unique lymphocyte percentages compared with blood. A, gating strategy to delineate CD3+CD45+ T lymphocytes. B, quantification of CD3+CD45+ T lymphocytes. C, expression of the coreceptors CD8α+ and CD4+ by CD3+ T cells. D, quantification of CD8α+ expression by the CD3+ T cells. E, CD45RO−/+ and CD27−/+ (markers of T cell activation and differentiation) staining of CD8α+ T cells. F, quantification of CD8α+ T effector memory cells (CD45RO+CD27−). G, expression of CD103+ (an intraepithelial cell associated integrin) by cells of the CD8α+ gate. H, quantification of CD103+ CD8α+ T cells. ***, P < 0.0001 (Student t test).

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    Figure 3.

    Breast organoids contain specifically targetable lymphocytes that respond to an FDA-approved BP drug. A, flow cytometry analysis of an organoid preparation from tissue sample L1 (see Table 1) for γδ T cells and MAIT cells (Vα7.2 T cell receptor). B, quantification of T cell subsets from organoid preparations from the indicated tissue samples (see Table 1 for tissue donor information). Symbols under the dashed line were below the limit of detection. C, example of flow cytometry results showing Vδ2+ T cell expansion in the presence of 2.5 μmol/L BP compared with culture medium alone. Vα7.2+ MAIT cells were used as a control for nonresponsiveness to BP. D, quantification of Vδ2 T cell frequency from organoids compared to PBMCs as a positive control after 2 to 3 weeks of culture in the presence of BP. Five of 11 samples (45.5%) displayed Vδ2+ T cell expansion from the purified organoid fraction. Each symbol represents an independent expansion attempt using the indicated tissue samples. Dashed line indicates the threshold used to delineate expansion.

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    Figure 4.

    Breast organoid–derived Vδ2 γδ T cells produce IFNγ in response to a triple-negative breast carcinoma cell line pulsed with BP. A, Vδ2+ T cells were expanded in vitro from organoid preparations by exposure to BP. The T cells were coincubated with MDA-MB-468 breast carcinoma cells that were pulsed (right) or not pulsed (left) with BP, and intracellular IFNγ production was assessed by flow cytometry. Results shown are representative of two independent experiments. B, primary organoid-derived cells were coincubated with MDA-MB-468 cells that were pulsed (right) or not pulsed (left) with BP, and intracellular IFNγ production by Vδ2+ and Vδ2− T cells was assessed by flow cytometric analysis. Numbers shown in the gates are the percentage of IFNγ–expressing cells from the Vδ2+ or Vδ2− T cell populations. C, plot showing aggregated results for IFNγ production by primary Vδ2+ T cells from the indicated breast tissue samples. Normalized mean fluorescence intensity (MFI) for IFNγ was determined by dividing the IFNγ MFI of the Vδ2+ cells by the IFNγ MFI of the corresponding Vδ2− T cells in the same sample. Each symbol represents an independent experiment to assess IFNγ production by T cells from the indicated breast tissue samples. The dashed line represents a normalization ratio of 1. n.s., not significant; *, P = 0.0121 (Mann–Whitney test; plus BP to ex vivo); *, P = 0.0262 (Mann–Whitney test; plus BP to no BP).

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    Figure 5.

    Cytotoxic functions by breast-derived Vδ2 γδ T cells in response to a triple-negative carcinoma cell line. A, Vδ2+ T cells were expanded in vitro from breast tissue preparations by exposure to BP. The T cells were coincubated with MDA-MB-468 breast carcinoma cells that were pulsed (right) or not pulsed (left) with BP, and cell surface expression of CD107a (LAMP-1, a marker of recent cytotoxic activity) was assessed by flow cytometry after 4 hours. B, MDA-MB-468 cells were pulsed (black squares) or not pulsed (gray squares) with BP, and coincubated with in vitro–expanded Vδ2+ T cells at the indicated effector:target ratios. The plot shows cytotoxicity of the target cells as assessed by cell-surface upregulation of Annexin V. Similar results were obtained in five independent experiments. C, primary organoid cells were coincubated with MDA-MB-468 cells that were pulsed (right) or not pulsed (left) with BP, and CD107a expression by T cells was assessed by flow cytometry. Numbers in the gates indicate the percentage of CD107a-expressing cells from the Vδ2+ or Vδ2− T cell populations. The figure shows representative results from one out of seven independent experiments. D, plot showing aggregated results for cell-surface CD107a expression by primary T cells from the indicated breast tissue samples after exposure to MDA-MB-468 cells that were pulsed or not pulsed with BP, or by primary organoid-derived T cells that were not exposed to tumor cells ("ex vivo"). Each symbol represents an independent experiment to assess CD107a cell-surface expression by T cells from the indicated breast tissue samples. n.s., not significant; *, P = 0.0273; **, P = 0.0078 (Wilcoxon test).

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    Figure 6.

    Expression of tumor-associated ligands and receptors. A, flow cytometric analysis of MDA-MB-468 cells using antibodies specific for BTN3A1 (Vδ2+ T cell receptor ligand), or for the following NKG2D ligands: MICA and MICB, ULBP-1, ULBP-2 (antibody also cross-reacts with ULBP-5 and -6), ULBP-3, or ULBP-4. Filled histograms show staining with the specific mAb; dashed histograms show staining by an isotype control mAb. B, expression of NKG2D (an activating receptor that recognizes MICA/B) by Vδ2+ T cells, CD8α− T cells, and CD8α+ T cells from organoid preparations. Median fluorescence intensity (MFI) values for isotype control mAb staining are shown in italics, and specific mAb MFI values are shown in bold.

Tables

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  • Table 1.

    Human breast tissue donor information

    Sample IDAge, sex, raceElective procedurePathology report
    L625 A147 y, female, N/AProphylactic mastectomyN/A
    L625 B156 y, female, N/AProphylactic mastectomyN/A
    L625 C136 y, female, N/AReduction mammoplastyN/A
    L625 D128 y, female, N/AReduction mammoplastyN/A
    L625 G137 y, female, N/AProphylactic mastectomyN/A
    L625 I136 y, female, N/AReduction mammoplastyN/A
    L625 J118 y, female, BLReduction mammoplasty (macromastia)Normal
    L625 K141 y, female, WHReduction mammoplasty (macromastia)FPCI
    L625 L123 y, female, BLReduction mammoplasty (macromastia)Normal
    L625 M136 y, female, WHReduction mammoplasty (macromastia)Fibrosis; CLI; PASH
    L625 N138 y, female, BLReduction mammoplasty (macromastia)Normal
    L625 O146 y, female, WHReduction mammoplasty (macromastia)Normal
    L625 P142 y, female; WHReduction mammoplasty (macromastia)Fibrosis
    L625 Q137 y, female, BLReduction mammoplasty (macromastia)Normal
    L625 S150 y, female; BLReduction mammoplasty (macromastia)Normal
    L625 T124 y, female, WHReduction mammoplasty (macromastia)Normal
    L625 U151 y, female, BLReduction mammoplasty (macromastia)Normal
    L625 V127 y, female, WHReduction mammoplasty (macromastia)PASH
    L625 W119 y, female, BLReduction mammoplasty (macromastia)Fibrosis
    L625 X 133 y, female, WHReduction mammoplasty (macromastia)PASH
    L625 Y121 y, female, BLReduction mammoplasty (macromastia)Normal
    L625 Z144 y, female, WHReduction mammoplasty (macromastia)Normal
    L625 A236 y, female; WHReduction mammoplasty (macromastia)Normal
    • Abbreviations: CLI, chronic lobular inflammation; FPCI, focal periductal chronic inflammation; PASH, pseudoangiomatous stromal hyperplasia.

Additional Files

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  • Supplementary Data

    • Suppl Fig S1 - Flow cytometric analysis to provide further verification that EpCAM+ cells from organoid preps are epithelial cells.
    • Suppl Fig S2 - Further characterization of the phenotypes of T cells in organoid preps.
    • Suppl Fig S3 - Comparison of human Vd2+ T cell proliferation in response to two FDA-approved bisphosphonate drugs.
    • Suppl Fig S4 - IFN-gamma and IL-17 production by T cells in organoid preps.
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Cancer Prevention Research: 9 (4)
April 2016
Volume 9, Issue 4
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Analysis of Immune Cells from Human Mammary Ductal Epithelial Organoids Reveals Vδ2+ T Cells That Efficiently Target Breast Carcinoma Cells in the Presence of Bisphosphonate
Nicholas A. Zumwalde, Jill D. Haag, Deepak Sharma, Jennifer A. Mirrielees, Lee G. Wilke, Michael N. Gould and Jenny E. Gumperz
Cancer Prev Res April 1 2016 (9) (4) 305-316; DOI: 10.1158/1940-6207.CAPR-15-0370-T

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Analysis of Immune Cells from Human Mammary Ductal Epithelial Organoids Reveals Vδ2+ T Cells That Efficiently Target Breast Carcinoma Cells in the Presence of Bisphosphonate
Nicholas A. Zumwalde, Jill D. Haag, Deepak Sharma, Jennifer A. Mirrielees, Lee G. Wilke, Michael N. Gould and Jenny E. Gumperz
Cancer Prev Res April 1 2016 (9) (4) 305-316; DOI: 10.1158/1940-6207.CAPR-15-0370-T
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