Fig 2: THP‐1 dramatic polarization toward an M1‐like phenotype with Sepiapterin treatment in the 3D Flipwell coculture system. (A) Confocal images of THP‐1 cells cocultured with Caco‐2:HT‐29 in the 3D Flipwells and stained for pan macrophage marker CD68 (red) and CD80 (green). Pan macrophage CD68 marker is present in both the SEP treated sample and the untreated control. CD80 marker, typical of the M1 polarization phenotype, is more prominently present in the SEP treated sample with long pseudopodia extending outward typical of the M1 phenotype. (B) Confocal images of THP‐1 cells cocultured with Caco‐2:HT‐29 in the 3D Flipwell and stained for pan macrophage marker CD68 and CD163 to validate phenotypic polarization toward M1 phenotype and away from M2 phenotype. Scale bar = 25 µm.

Fig 3: Uptake of Bif.BEVs in lung cancer cells and their regulation of PD-L1 expression.a Immunofluorescent detection of uptake of PKH67 (green) stained Bif.BEVs (5 µg/ml) by LL/2 cells after 24 h post treatment with endocytosis inhibitors. DAPI (blue) was used to visualize nuclei. The scale bar is 20 µm. b Quantification of fluorescence intensity of images from 2a by ImageJ analysis using 17 different areas. Statistical comparisons of medians were conducted using the Kruskal-Wallis test. For all box plots, the central line inside each box represents the median (50th percentile). The box bounds correspond to the first quartile (Q1, 25th percentile) and third quartile (Q3, 75th percentile). The whiskers extend to the minimum and maximum values. Exact p values are indicated in the figure. c Western blot analysis of anti-LTA in LL/2 cells after pre-treatment with endocytosis inhibitor and Bif.BEVs (5 ug/ml). GAPDH was used as internal control. d Western blot analysis of PDL-1, CD80, PVR/CD155 in lung cancer cells (A549, H460, LL/2) along with immortal noncancerous lung cells BEAS-2B after treatment with either Bif.BEVs (5 µg/ml) or cell lysates (5 µg/ml). GAPDH and actin were used as internal controls. e Western blot analysis of TLR-4, TLR-7 and NF-κB after treatment with Bif.BEVs (5 µg/ml). Actin was used as internal loading control. f Western blot analysis of NF-κB and PD-L1 after pre-treatment with NF-κB inhibitor for 2 h and Bif.BEVs (5 µg/ml) for 48 h. All Western blots except using BEAS-2B (due to slow growth) have been repeated at least twice in independent experiments. Source data are provided as a Source Data file.