Fig 2: In vivo tumorigenesis and immune status under the interleukin (IL)-6-deficient condition. CT26 murine colon cancer cells (1 × 106) were intradermally injected into wild-type and IL-6-/- BALB/c mice (day 0). (a) Tumor size was monitored for 13 days after injection. Means and SD (n = 5) of the tumor volume are indicated. *P < 0.05 by Student's t-test (b) Representative images of tumors at day 15. (c) H&E and immunohistochemistry (IHC) staining of tumor tissues collected at day 14 were performed using anti-CD3 and anti-CD11c antibodies. Representative images are shown. Bar represents 100 µm. (d) Tumor-infiltrating immune cells of wild-type and IL-6-/- mice at day 12 were evaluated by flow cytometry using anti-CD45, anti-CD4, anti-CD8, anti-CD11c and anti-I-Ad mAbs. Percentages of CD45+, CD4+ T, CD8+ T and CD11c+I-Adhigh dendritic cells were calculated. Means and SD (n = 5) are indicated. *P < 0.05 by Student's t-test.

Fig 3: Expression levels of MHC class I and PD-L1 on CT26 cells in vitro and in vivo. (a) CT26 cells were stimulated with IFN-? (50 ng/mL) for 24 h in vitro. Surface expression levels of MHC class I and PD-L1 on CT26 cells were evaluated by flow cytometry using anti-H-2Kd and anti-PD-L1 mAbs. Representative profiles are shown. Mean fluorescence intensities (MFI) for the expression levels of H-2Kd and PD-L1 were calculated. Means and SD (n = 3) are indicated. *P < 0.05 by Student's t-test. (b) GFP-transduced CT26 murine colon cancer cells (1 × 106) were intradermally injected into wild-type and IL-6-/- BALB/c mice (day 0). Surface expression levels of MHC class I and PD-L1 on GFP + CD45- CT26 cells at day 27 were evaluated by flow cytometry using anti-H-2Kd and anti-PD-L1 mAbs. Representative profiles are shown. MFI for the expression levels of H-2Kd and PD-L1 were calculated. Means and SD (n = 3) are indicated. *P < 0.05 by Student's t-test. (c) Surface expression levels of MHC class I and PD-L1 on GFP - CD45+ cells and GFP - CD45+ CD11c+ cells at day 27 were evaluated by flow cytometry using anti-H-2Kd and anti-PD-L1 mAbs. Representative profiles are shown. MFI for the expression levels of H-2Kd and PD-L1 were calculated. Means and SD (n = 3) are indicated. *P < 0.05 by Student's t-test.

Fig 4: Milk-derived extracellular vesicles (mEVs) survive gastrointestinal environment and reach colon immune cells.(A) Morphology and size distribution of bovine mEVs (B-mEVs) and liposomes before and after going through the ex vivo digestive system. Three independent experiments were performed in triplicate. (B) Representative IVIS images of healthy mice (top) and dissected colons (bottom) at 12 hours after oral administration of mEVs. (C) Fluorescence microscopy imaging of colon cryosections showing B-mEVs in red (Cy5-labeled), DC2.4 dendritic cells (DCs) strained with CD11c in green (top), and macrophages stained with CD68 in green (bottom). Scale bars, 100 µm. Colons were dissected from healthy mice as in (B). (D to G) Fluorescence-activated cell sorting (FACS) analysis showing cellular uptake of B-mEVs by colon immune cells in healthy and DSS-induced colon impaired mice (n = 3 mice per group). Control, healthy mice received free dye orally; Cy5-B-mEVs, healthy mice received Cy5-labeled B-mEVs orally; DSS-Cy5-B-mEVs, DSS-induced colon impaired mice received Cy5-labeled B-mEVs orally. *P < 0.05 and ****P < 0.0001, one-way ANOVA.