Gentlemacs tumor dissociation kit

The human glioblastoma U87 cell line and mouse 3T3 fibroblast cells were obtained from ATCC and cultured per the vendor's instructions. The human breast cancer MCF‐7 parental cell line, the P‐gp‐overexpressing MCF‐7 TX400 subline, and the ABCG2‐overexpressing MCF‐7 MX100 subline were cultured in EMEM supplemented with 10% FBS, 100 U mL⁻¹ penicillin, 100 µg mL⁻¹ streptomycin, and 0.01 mg ml⁻¹ insulin (Sigma) as previously described.^[18 (link)
^] Cells were maintained in 5% CO₂ at 37 °C and tested to be free of mycoplasma (MycoAlert, Lonza).
GBM39 cells expressing luciferase were serially passaged in the flank of female J:NU mice (4‐5 weeks old, #0 0 7850, Jackson Laboratory). Tumors were dissociated using a gentleMACS tumor dissociation kit (Miltenyi Biotec) before in vitro culturing. Cells were cultured as recommended by the Mayo Clinic Brain Tumor Patient‐Derived Xenograft National Resource using the FBS cell culture protocol. Briefly, cells were cultured in DMEM containing 2.5% FBS, 100 U mL⁻¹ penicillin, and 100 µg mL⁻¹ streptomycin for 48 h, then media was exchanged to DMEM containing 10% FBS and antibiotics for at least 24 h before experimentation. Cells were dissociated and used in experiments, but not passaged serially. Assays were performed within 14 days of tumor dissociation.

Following the therapeutic schedule (as described in Figure 5a), the tumor tissue was removed and a single‐cell suspension was obtained using the gentleMACS Tumor Dissociation Kit (Miltenyi Biotec), according to the manufacturer's instructions. Then, CD45⁺ TILs were isolated using MACS beads (MicroBeads, Miltenyi Biotec). The isolated TILs were labeled with CD3 (PE labeled, Biolegend, San Diego, CA, USA), CD8 (FITC‐labeled, Biolegend), or CD4 (FITC‐labeled, Biolegend) antibodies. The cells were then analyzed using Guava easyCyte (EMD Millipore, Billerica, MA, USA).

Fresh tumor tissue resections were digested and processed into single cell suspension using gentleMACS tumor dissociation kit (Miltenyi Biotec). Tumor cells were then isolated by negative selection using a tumor cell isolation kit (Miltenyi Biotec). Adherent cells were passaged at least five times before being used for experiments. All cell lines were maintained in RPMI 1640 or DMEM GlutaMAX media (Thermo Fisher Scientific) supplemented with 10% HyClone fetal bovine serum (FBS) (GE Healthcare) and 1% Penicillin Streptomycin (PS) (Life Technologies). A tumor cell line and TIL culture from a breast cancer specimen was established as previously described.36 (link) Human osteosarcoma cell line U2OS (ATCC) was used in comparison to patient-derived sarcoma cell lines for relative adenosine production and ADO ectonucleotidase expression by flow cytometry.

Spheroids were prepared using patient-derived sarcoma cell lines. 1×10⁴ cells were seeded per well in 96-well ultra-low attachment plate with DMEM-F12 media containing 20% FBS and 1% PS for 5 days. 3×10⁴ CSFE-labeled autologous expanded TILs, at Effector:target ratio of 3:1, were added to the spheroid at day five with A_2BAR antagonism treatment at 12 µM. After 3 days, spheroid was removed and split into two groups—IN and OUT. IN indicates TILs infiltrated into the sphere, while OUT indicates TILs that did not infiltrate into the sphere. Spheroids were washed with PBS at least two times. GentleMACS tumor dissociation kit (Miltenyi Biotec) was used to digest the spheroids for FC analysis.

Three weeks post-implantation of Renca/luc cells, mice were sacrificed, lungs were harvested, and pulmonary metastases were precisely harvested with the assistance of fluorescence microscopy. Pulmonary metastatic lesions were dissociated to form a single-cell suspension using the gentleMACS™ Tumor Dissociation Kit (Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer's instructions. Tumor cells were purified from this suspension using a Tumor Cell Isolation Kit (Miltenyi Biotec), according to the manufacturer's instructions. This pulmonary metastatic cell subpopulation was termed Renca(HM)/luc and was maintained in RPMI 1640 medium supplemented with 10% FBS prior to again being intrarenally implanted, including subsequent isolation of pulmonary metastases (following an identical procedure to that described above). After each implantation, the interval to metastasis, tumor progression, and the interval to moribundity or demise were monitored.