Isolation and analysis of tumor-infiltrating leukocytes (TILs)

CT26 tumors were collected and cut into pieces of similar weight. Tumor pieces were distributed into different tubes containing MACS Tissue Storage Solution and stored for 24 h or 48 h (two replicates per condition). After each storage period, tumors were dissociated using the Tumor Dissociation Kit, mouse and the gentleMACS Octo Dissociator with Heaters. TIL populations from the obtained cell suspensions were analyzed by flow cytometry. Percentages refer to pre-gating strategies.

CT26 tumors were collected and cut into pieces of similar weight. Tumor pieces were distributed into different tubes containing MACS Tissue Storage Solution and stored for 24 h or 48 h (two replicates per condition). After each storage period, tumors were dissociated using the Tumor Dissociation Kit, mouse and the gentleMACS Octo Dissociator with Heaters. TIL populations from the obtained cell suspensions were analyzed by flow cytometry. Percentages refer to pre-gating strategies.

B16-F10 tumors were collected and dissociated using the gentleMACS Octo Dissociator in the presence (A) or absence (B) of Tumor Dissociation Kit enzymes. Cells were subsequently labeled with REAfinity™ Antibodies. Gated populations show viable cells.

TILs were prepared by combining mechanical and enzymatic dissociation of tumor tissue with the gentleMACS Dissociator. Tumor samples from two donors were processed with both tumor dissociation methods in parallel to allow for direct comparison. Cell culture supernants were analyzed for IFN-γ by ELISA. Data are means±SEM of three experimental replicates (*p < 0.05; ***p < 0.001; two-way ANOVA with Bonferroni post-test correction).

Xenograft tumors were induced by injection of human colon tumor cells into NOD/SCID/IL-2rγ^null (NSG™) mice. Additionally, human T cells were adoptively transferred into the mice and re-directed to the tumor by a bispecific antibody. Human CD45⁺ TILs were isolated from dissociated xenograft tumors with the CD45 (TIL) MicroBeads in combination with the autoMACS Pro Separator. Cells were stained with a PE-conjugated CD45 antibody and analyzed by flow cytometry. The positive fraction contained the magnetically labeled CD45⁺ TILs. Gates denote the tumor cell populations. Blue curve: before separation; orange curve: after separation, negative fraction (dot plots not shown); red curve: after separation, positive fraction. 

During separation, target cells are magnetically labeled and retained within the column, while unlabeled cells flow through. After a washing step, the column is removed from the magnetic field of the separator and target cells areeluted from the column. MicroBeads are then removed through a short incubation with the REAlease Bead Release Reagent. Optionally, the REAlease Biotin Complex can then be removed through a short incubation with the REAlease Release Reagent.

Human CD45⁺ TILs were isolated from a dissociated ovarian carcinoma sample using the REAlease CD45 (TIL) MicroBead Kit. Isolated cells were fluorescently stained with CD45-VioBlue® and analyzed by flow cytometry using the MACSQuant® X. The efficiency of biotin complex release was determined by re-applying the isolated cells to a second MACS Column. The ratio between the numbers of cells in the flow-through and the total number of cells applied to the second column allowed the calculation of the efficiency of magnetic labeling removal. The efficient removal of all labels was shown by using Anti-Biotin-APC to analyze the cells by flow cytometry for the presence of REAlease Biotin Complex.

Tumors derived from B16-F10 (left and center columns) or B16-OVA (right column) melanoma cells were inoculated subcutaneously into the right flank of C57BL/6 mice. After 11-15 days, tumors were collected and dissociated using the gentleMACS Octo Dissociator and the mouse Tumor Dissociation Kit (TDK) optimized for epitope preservation. CD4⁺, CD8⁺, and pan T cells were isolated using the mouse CD4 (TIL) MicroBeads, CD8 (TIL) MicroBeads, and the CD4/CD8 (TIL) MicroBeads, respectively. Frequencies of tumor-infiltrating T cells among living cells before or after isolation are shown for different tumor models, as assessed by flow cytometry. 

Syngeneic mouse tumors derived from B16-F10 melanoma cells showed TIL frequencies of 2–4% among total viable cells after dissociation. CT26.WT cell–derived colon carcinoma contained 15–21%, and 4T1 cell–derived breast carcinoma 32–37% TILs. TILs were enriched to purities above 90% at yields above 70% for CT26.WT and 4T1 tumors, and purities above 80% at high yields above 95% for B16-F10 tumors.

CT26 mouse tumor (1 g) was dissociated with the gentleMACS Octo Dissociator with Heaters and the Tumor Dissociation Kit, mouse and CD45⁺ TILs were pre-enriched using CD45 (TIL) MicroBeads, mouse. NK cells (CD45⁺, CD11b^–, and anti-NKP46⁺) were sorted with the MACSQuant Tyto Cell Sorter. The negative fraction was further sorted in a new cartridge for B cells (CD45⁺, CD11b^–, anti-NKP46^–, and CD19⁺). TILs were analyzed using the MACSQuant Analyzer 10.