Neutralization solution

Organotypic cultures were prepared as follows: Nine volumes of rat tail collagen I (3.8 mg/mL; Advanced BioMatrix; San Diego, CA, USA), 1 volume of neutralization solution (Advanced BioMatrix), 1.2 volumes of Matrigel^® (9.8 mg/mL, Corning^®; Corning, NY, USA), and 7.3 volumes of 2× serum-free DMEM medium were mixed. CAF cells (5 × 10⁵ cells/mL) were resuspended in the collagen/Matrigel^® solution and 900 µL of this mixture were pipetted into each well of a 24-well plate. DMEM media, supplemented with 10% FBS, was added on top of the gels, once collagen had polymerized, and was changed every 2–3 days. To allow the CAF cells to remodel the matrix, they were grown within collagen/Matrigel gels for 5 days. On day 5, NCI-H661 tumor cells (5 × 10⁵ cells/900 µL) were seeded on top of each gel and allowed to adhere to collagen gel surface overnight. The following day, the gels were carefully detached from the wells and were placed onto collagen-coated nylon NET filters (Millipore, Billerica, MA, USA), supported by metal grids. The grids were placed into 60-mm plates and RPMI 1640 media, supplemented with 10% FBS, was added to reach the underside of each metal grid, establishing an air–liquid interface. The media was changed every 2–3 days for the next 21 days.

Immune enhanced tumor organoids (iTOs) were created by encapsulating ~ 10 × 10⁶ tumor cells and ~ 30 × 10⁶ immune cells counted with a Nucleocount NC-200 (Chemometec, Denmark) per 1 mL of a 3:1 mixture of 3 mg/mL methacrylated collagen (Advanced Biomatrix, USA) suspended in 20 mM acetic acid (Advanced Biomatrix, USA) and 1 mg/mL thiolated hyaluronic acid (Advanced Biomatrix, USA) suspended in 0.1% w/v irgacure photo-initiator (Advanced Biomatrix, USA) in autoclaved DO water. The hydrogel mixture was neutralized with 95ul of Neutralization Solution (Advanced Biomatrix, USA) per 1 mL of 3 mg/mL collagen utilized. This leads to a final concentration of 2.25 mg/mL collagen and 0.25 mg/mL collagen in the organoid. 10µL of the mixture (equal to 1 × 10⁵ 4T1, 3 × 10⁵ immune or 4 × 10⁵ combined cells) was pipetted into individual wells in a 48 well plate (Falcon, USA) coated with 120 µL PDMS (DOW Chemical, USA) to prevent cell outgrowth outside the organoids. The hydrogel mixture was exposed to 13.2 J/m³ UVA radiation for two and a half seconds to initiate the crosslinking (gelation) of the hydrogel to form the individual organoids. Pervious work with this hydrogel system has demonstrated that no phenotypic and viability changes in cells are observed^{19 (link),26 (link),38 (link)}. Control organoids were also created with either the immune cells or the tumor cells only.

Collagen gels were prepared as follow: Nine volumes of 4 mg/mL rat tail type I collagen (Advanced Biomatrix, San Diego, CA, USA) were mixed with 1 volume of neutralization solution (Advanced Biomatrix) and 12 volumes of 2× serum-free DMEM medium. CAFs were trypsinized and incorporated in the collagen solution at a final density of 2.5 × 10⁵ cells/mL. Five hundred microliters of the cell/collagen mixture was added into each well of a 24-well plate and allowed to polymerize for 1 h at 37 °C. DMEM supplemented with 10% of serum was added on top of each gel. Twelve days later, collagen gel was released and diameter was measured. Acellular collagen gels were used as negative control.