Hyaluronidase

The FLAG-tagged NO66 protein was examined by immunostaining as described previously [11 (link)]. In brief, paraffin-embedded mouse femoral sections from embryos (E15.5 and E18.5) or postnatal pups (P10) were deparaffinized and subjected to enzymatic digestion with hyaluronidase (2 mg/mL in phosphate buffer, pH 5.5 (MP Biomedicals). Primary antibody against the FLAG epitope (1 : 200; Chemicon) was incubated overnight at +4°C. The secondary antibodies, including 555 goat antimouse (1 : 1000; Molecular Probes/Invitrogen) or goat antimouse IgG linked with HRP (1 : 1000; Abcam), were incubated for 1 hr at room temperature. For immunofluorescence staining, slides were washed and mounted with antifade-Gold with 4′, 6-diamidino-2-phenylindole (DAPI; Molecular Probes) and then analyzed under a fluorescence microscope. For immunohistochemistry (IHC) staining, slides were washed and then stained using a DAB substrate kit (Vector Laboratories) following the manufacturer's protocol.

Organoid growth medium consists of base medium (ADMEM/F12 with 10% FBS, 1% penicillin/streptomycin, 1% Glutamax, and 1% HEPES) supplemented with 1X N2 (Sigma Aldrich, 17502048), 1X B-27 (Sigma Aldrich,17504044), 1mM N-Acetylcysteine (Sigma Aldrich, A7250) 50 ng/ml EGF (Life Technologies, PGH 0313), 100 ng/ml Noggin (Tonbo, 21-7075-U500), 10 mM nicotinamide (Sigma, N0636), 500 nM A 83-01 (Calbiochem, 616454-2MG), 10 μM SB202190 (Sigma 47067), and 0.01μM PGE2 (Sigma Aldrich, P5640).
Tissue digestion solution consists of 1.5 mg/ml collagenase (Millipore, 234155), 20 μg/ml hyaluronidase, (MP Biomedicals 100740) and 10 μM Ly27632 (Sigma Y0503).

To analyze the tumor immune microenvironment during the anticipated efficacious period of immune checkpoint activity, tumors were collected at day 14–17 after starting immune checkpoint inhibitor. Fresh tumors were dissociated into single cell suspensions with DNAse I (Invitrogen), collagenase Type IV (Sigma), and hyaluronidase (MP Biomedicals) for 1 h at room temperature using a dissociator (Miltenyi) with gentleMACS C-tubes. To remove calcium, cells were resuspended for 5 min in HBSS without calcium or magnesium (Gibco), then resuspended in 5 mM of EDTA for 30 min at room temperature. Next, cells were passed through a 70 μm filter before ACK lysing buffer (KD Medical Inc) was added to remove red blood cells before flow cytometry. Immediate staining was performed for surface marker expression to analyze with flow cytometry.

Tetraethyl orthosilicate (TEOS) was purchased from Acros Organics (TEOS A, Geel, Belgium) and Alfa Aesar (TEOS B, Lancashire, UK), both now Thermo Scientific (Waltham, MA, USA). Sodium Hyaluronan (HA, 5 kDa) was from Lifecore Biomedical (HA A, Chaska, MN, USA) and HTL Biomedical (HA B, Javené, France). Acetic acid (HOAc) was from EMD Millipore (Billerica, MA, USA), ammonium hydroxide (NH₄OH) was from Fisher Chemical (Fair Lawn, NJ, USA), and phosphate-buffered saline (PBS) was from Corning Life Sciences (Durham, NC, USA). Fluorescein was from Fluka Analytical, owned by Sigma Aldrich (St. Louis, MO, USA), fluorescein disodium was from Alfa Aesar, now Thermo Scientific (Waltham, MA, USA), green fluorescent protein (GFP) was from Novus Biologicals (Littleton, CO, USA), and blue dextran with molecular weights of 5597, 15,038, and 500,000 g/mol, referred to as 5000, 20,000, and 500,000 g/mol, respectively, was from Sigma Aldrich (St. Louis, MO, USA). Skin Irritation Test kit (SPI-200-SIT) was from MatTek (Ashland, MA, USA), thiazolyl blue tetrazolium bromide (MTT) was from Thermo Scientific (Waltham, MA, USA), and isopropanol was from VWR (Radnor, PA, USA). Hyaluronidase was from MP Biomedicals (Solon, OH, USA), and hydrochloric acid (HCl) was from Janssen Pharmaceuticals (Beerse, Belgium).