Epcam

Anti-CD49f, c-Kit and EpCam antibodies were obtained from BioLegend (San Diego, CA); Anti-CD10, Muc1 and Thy1 antibodies were obtained from BD Biosciences (San Jose, CA); and anti-keratin 14 and keratin 19 antibodies were purchased from Neomarkers/ThermoScientific (Fremont, CA). Detailed information on the clones and conjugates are provided in Supplementary Table 3. Muc1 antibody was custom labelled using the PacificBlue Antibody Labeling Kit (Invitrogen, Carlsbad, CA). Polybrene/hexadimethrine bromide (H9268), α-L-Fucosidase from bovine kidney (F5884), β-(1→3,4,6)-Galactosidase (G1288), Hyaluronidase (H3506) and neuraminidase (type III) from Vibrio cholera (N7885) were purchased from Sigma-Aldrich (St Louis, MO).

EVs used in Western blotting analysis were purified from patient plasma using ExoQuick Ultra (System Biosciences, EQULTRA-20A-1), following the manufacturer’s instruction. Final elutes were dissolved in RIPA buffer (1/10 of elute volume). Signals were detected and quantitated with the LI-COR Odyssey system. To calculate marker ratios, intensities of protein bands of each marker were divided by signals of CD9 bands of the same patient. To rescale the marker ratios, the median value of each marker ratio in the whole patient cohort was set to be 100; then all other values were rescaled accordingly. Antibodies used were CD9, Cell Signaling Technology 13174; VGLUT2, Cell Signaling Technology 71555; GPC4, R&D Systems, Bio-Techne, MAB9195; MUC1, BD Biosciences 555925; EGFR, Cell Signaling Technology 4267; EPCAM, BioLegend 118201; CD44v6, Thermo Fisher Scientific BMS125; CD14, BioLegend 367101; annexin A11, Thermo Fisher Scientific MA5-25052; and sLeX, BD Biosciences 551344.

The protocol for isolation of IEC cells was based on the previously described method with minor modifications. In brief, the small and large intestines were harvested individually from treated mice and rinsed extensively with RPMI-1640 media (from Lonza) after Peyer’s patches were removed (for small intestine). The rinsed intestines were opened longitudinally and macerated; the tissue pieces were shaken gently in RPMI-1640 containing 2 mM EDTA and 10% fetal calf serum. The tissue preparations were passed through 70-µm mesh filters, and the resulting single-cell suspensions were applied to Percoll (from Sigma) density gradients of 25%, 40%, and 75%. After centrifugation at 2000×g for 20 min, the interface between the 25% and 40% layers was collected to obtain IECs. The cells were stained using antibodies for either epithelial cell adhesion molecule (EpCAM, from Biolegend) or CD45 (from Biolegend) and nucleic acid dye (Via-Probe, from BD Biosiences). The Via-Probe⁻/CD45⁻/EpCAM⁺ IEC were sorted using BD FACSMelody^TM Cell Sorter (BD Biosciences) for further biomedical analysis [55 (link), 56 (link)].

Tumor cell suspensions were stained with specific antibodies against EPCAM and CD45 (BioLegend, San Diego, CA). Proportions of EPCAM+CD45- tumor cells in the final cell suspension were assessed by flow cytometry using BD FACS Aria and FlowJo software.