M0823

Tumor biopsy and blood DNA were isolated from fresh samples using TGuide M24 (Tiangen, Beijing, China). The purity and quantity of the total DNA were estimated by measuring absorbance at 260 nm (A260) and 280 nm (A280) with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA); the extracted DNA was considered pure and suitable for future experiments if the A260/A280 ratio was within 1.6–1.9. FUSCC NGS 511-gene panel sequencing [16 (link)] was conducted to detect somatic and germline mutations in both biopsy (n = 23) and ctDNA (n = 23) samples.
FFPE blocks of samples from 30 patients were retrieved, and 4–5-μm-thick samples on slides were used for IHC staining. IHC for TIM-3 (CST, Cat#45208T, 1/400 dilution), c-Myc (Abcam, Cat#ab32072, 1/200 dilution), STING (CST, Cat#13647S, 1/100 dilution), PD-L1 (Abcam, Cat#ab228462, 1/500 dilution) and CD31 (DAKO, Cat#M0823, 1/100 dilution) was performed on FFPE sections from 30 patients, with the staining status being independently assessed by two experienced pathologists. If multiple tissue sections from one patient were available, the highest score was used for classification.

Passage 1 EC and SMC were grown to confluence on plastic dishes or on chitosan/PCL membranes. After that, they were fixed with 4% PFA (paraformaldehyde) for 10 min, permeabilized with 0.05% Triton X-100 for 10 min, and blocked with 1% BSA (bovine serum albumin). The cells were stained with primary antibodies overnight at 4 °C, washed with PBS and incubated with secondary antibodies for 1 h at room temperature. The stained cells were analysed with an inverted fluorescence microscope (Nikon Ti-E) using Nikon AR software.
The following primary antibodies were used: anti-human CD31 (M0823, DAKO, 1:50), anti-α-SMA (DAKO, M0851, 1:50), anti-smooth muscle myosin heavy chain 11 (Abcam, ab82541, 1:500), anti-human CD90 (eBioscience, 14090982, 1:100), anti-Von Willebrand factor (Abcam, ab6994, 1:200), anti-fibronectin (Abcam, ab6328, 1:200), anti-elastin (Abcam, ab21610, 1:200), and anti-collagen IV (Life Span, 1:200).
The following secondary antibodies were used: Alexa Fluor 568 goat anti-mouse IgG1 (Life Technologies, A21124, 1:400), Alexa Fluor 488 goat anti-mouse IgG1 (Life Technologies, A21121, 1:400), Alexa Fluor 568 goat anti-mouse IgG2a (Life Technologies, A21134, 1:400), Alexa Fluor 568 goat anti-mouse IgG (H + L) (Life Technologies, A11031, 1:400), and Alexa Fluor 488 goat anti-mouse IgG (H + L) (Life Technologies, A11029, 1:400).

To demonstrate the presence of endothelial cells in the cocultures, CD31 immunofluorescence staining was conducted on half a sample fixed on day 10. After permeabilization, nonspecific protein binding was blocked before 1 hr of incubation with the primary anti‐CD31 antibody (0.13 mg/ml mouse anti‐human CD31, M0823, Dako), with or without anti‐NG2 chondroitin sulfate proteoglycan antibody (0.001 mg/ml, Merck Millipore, AB5320). After washing with 0.1% Tween in PBS, 1 hr of incubation with the secondary antibody (1:200, sheep anti‐mouse biotinylated, RPN1001v1, GE Healthcare) was performed. The samples were incubated with streptavidin Alexa Fluor 488, 2 mg/ml (Invitrogen), together with the anti‐α‐smooth muscle actin (α‐SMA) fluorescently labelled antibody (Clone 1A4, Cy3 0.5 μg/ml, C6198, Sigma Aldrich). The 5 mm Ø × 5 mm constructs were incubated overnight (4°C) with the antibodies. Finally, nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI) staining 100 ng/ml (Sigma) for 15 min at room temperature.

Skin samples were embedded in optimal cutting temperature (OCT; Miles Laboratories) compound and kept at −80°C. 7‐µm sections were prepared using a Bright OTF cryostat (Bright Instruments Ltd) and collected on Superfrost^® plus slides (ThermoFisher Scientific).
Immunofluorescence was carried out using the following primary antibodies rabbit anti‐SLC6A12 (dilution 1:100; catalogue #HPA034973; Sigma‐Aldrich), rabbit anti‐SLC6A6 (dilution 1:50; catalogue #HPA015028; Sigma‐Aldrich), rabbit anti‐SLC2A13 (dilution 1:1,000; catalogue #BMP026; MBL International), rabbit anti‐SLC5A3 (dilution 1:2000; catalogue #ABS518; Millipore), mouse anti‐e‐cadherin (dilution 1:500; catalogue #ab1416; Abcam), mouse anti‐K15 (dilution 1:1,000; catalogue #ab80522; Abcam), mouse anti‐CD31 (dilution 1:100; catalogue #M0823; Dako), and mouse anti‐vimentin (dilution 1:500; catalogue #M0725; Dako) as described in Supplementary Materials and Methods (Appendix S1). Finally, slides were left to dry overnight prior to imaging. Negative controls were included in every experiment by omission of the primary antibody.

Cells were two times washed with PBS before being fixed with 2% PFA at RT for 30 min. After another two washes with PBS, cells were permeabilized with 0.5% Triton in PBS for 15 min. Cells were washed again 3× in PBS and blocked with blocking buffer (5% donkey or goat serum in PBS) for 10 min. Cells were incubated with primary antibody (1:100 in blocking buffer) overnight at 4 °C. The next day, cells were washed with PBS, PBS + 0.05% Tween-20 and again with PBS before incubation with secondary antibody (1:500) in DAPI (1:5000) and 2% Human Serum for 60 min at RT in the dark. Cells were washed with PBS, PBS + 0.05% Tween-20 and PBS and stored in PBS at 4 °C until analysis. The following primary antibodies were used: mouse anti-human CD31 (M0823, Dako, Santa Clara, CA, USA), rabbit anti-human SM22α (ab14106, Abcam, Cambridge, MA, USA). Alexa Fluor 647 donkey anti-mouse (A31571, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 568 goat anti-rabbit (A21069, Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. The cells were analyzed using the EVOS FL cell imaging system (Life Technologies, Carlsbad, CA, USA).

After deparaffinisation, the sections (4 μm) were cut and incubated with primary antibodies against Ki‐67 (ab16667; Abcam, Cambridge, UK), heparan sulfate 6‐O‐sulfotransferase 1 (HS6ST1, bs‐10701R; Bioss Antibodies, Woburn, MA, USA), endothelial cell‐specific molecule 1 (ESM 1; ab103590; Abcam, Cambridge, UK), and the endothelial cell marker CD31 (M0823; Dako, Santa Clara, CA, USA). The target proteins were visualised using the VECTASTAIN Elite ABC system (Vector Laboratories) or the secondary antibodies (Alexa Fluor 488 and 568, Invitrogen) and Hoechst nuclear stain. The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology.