Hydrogels

For swelling studies, OPF 35K, OPF 10K, OPF 3K and OPF 1K hydrogel composites encapsulating gelatin microparticles were prepared in the same manner as would be used for cell encapsulation. Briefly, 0.1 g of OPF and 0.05 g of the crosslinking agent poly(ethylene glycol) diacrylate (PEG-DA; nominal MW 3400, Nektar Therapeutics, Huntsville, AL) and 0.0219 g of microparticles were combined in 578 μl of phosphate buffer saline (PBS). Equal volumes (46.8 μl) of the thermal radical initiators, 25 mM ammonium persulfate (APS) and 25 mM N,N,N′,N′-tetramethylethylenediamine (TEMED) in PBS, were then added. After gentle mixing, the suspension was quickly injected into Teflon molds (6 mm diameter, 1 mm thickness) followed by incubation at 37°C for 8 min. Hydrogel composites were transferred to PBS and cultured statically at 37°C for 4 weeks. At day 1, 7, 14, 21 and 28, the swelling ratio and sol fraction of OPF hydrogel composites were then determined by the following equations.
Here, W_i, W_s, and W_d represent the weight of dried hydrogel composites after crosslinking, the weight of hydrogel composites after swelling in PBS, and the weight of dried hydrogel composites after swelling, respectively. The swelling ratio is defined as the fractional increase in the weight of the hydrogel due to water absorption. The sol fraction represents the fraction of the polymer following a crosslinking reaction that is not part of a crosslinked network. A decrease in sol fraction over time reflects polymer loss and characterizes the extent of hydrogel degradation.

Fresh tumor specimens (murine and human patients) were received in media (DMEM) on ice and minced in a 10cm dish (on ice) using sterile forceps and scalpel. Minced tumor was resuspended in DMEM (4.5 mM glucose, 100 mM Na pyruvate, 1:100 penicillin-streptomycin) (Corning CellGro, Manassas, VA) +10% FBS (Gemini Bio-Products, West Sacramento, CA), 100 U/mL collagenase type IV (Life Technologies, Carlsbad, CA), and 15 mM HEPES (Life Technologies, Carlsbad, CA), except for CT26 tumors that were prepared in RPMI. Samples were pelleted and resuspended in 10–20 mL media. Red blood cells were removed from visibly bloody samples using RBC Lysis Buffer (Boston Bio-Products, Ashland, MA). Samples were pelleted and then resuspended in fresh DMEM +10% FBS and strained over 100 μm filter and 40 μm filters to generate S1 (>100 μm), S2 (40–100 μm), and S3 (<40 μm) spheroid fractions, which were subsequently maintained in in ultra low-attachment tissue culture plates. S2 fractions were used for ex vivo culture. An aliquot of the S2 fraction was pelleted and resuspended in type I rat tail collagen (Corning, Corning, NY) at a concentration of 2.5 mg/mL following addition of 10× PBS with phenol red with pH adjusted using NaOH. pH 7.0–7.5 confirmed using PANPEHA Whatman paper (Sigma-Aldrich, St. Louis, MO). The spheroid-collagen mixture was then injected into the center gel region of the 3D microfluidic culture device. Collagen hydrogels containing PDOTS/MDOTS were hydrated with media with or without indicated therapeutic monoclonal antibodies after 30 minutes at 37°C. MDOTS were treated with isotype control IgG (10 μg/mL, clone 2A3) or anti-PD-1 (0.1, 1.0, 10 μg/mL, clone RMP1-14). Monoclonal rat-anti-mouse-CCL2 (5 μg/mL, clone 123616, R&D Systems) was used for CCL2 neutralization in MDOTS. PDOTS were treated with anti-PD-1 (pembrolizumab, 250 μg/mL), anti-CTLA-4 (ipilimumab, 50 μg/mL), or combination (250 μg/mL pembrolizumab + 50 μg/mL ipilimumab). For indicated PDOTS studies, anti-human PD-L1 (atezolizumab) was used at 600 μg/mL (1:100) alongside recombinant human interferon-gamma (200 ng/mL) obtained from R&D Systems (285-IF). Doses were selected (1:100 dilutions of stock concentrations used clinically) to correspond to reported peak plasma concentrations of each drug following administration of 10mg/kg (FDA CDER application). In select experiments, PDOTS were treated with InVivoMAb human IgG isotype control (BioXCell). For spheroid cultures lacking immune cells, MC38 or CT26 cells (1 × 10⁶) were seeded in low attachment conditions for 24 hours and were filtered (as above). The S2 fraction was pelleted and resuspended in collagen (as above) prior to microfluidic culture (see Supplementary Video 3).