Tumor isolation kit

Single‐cell suspensions from mouse and human pancreatic cancer tissues were obtained using the tumor isolation kits (Miltenyi Biotec, mouse,130‐096‐730; human, 130‐095‐929;) and the gentle MACS Dissociator (Miltenyi Biotec, 130‐093‐235) after the tumors were cut into small pieces with diameter around 2–4 mm. T cells from the spleen of OT‐I mice were enriched using the mouse Pan T cell isolation kit II (Miltenyi Biotec, 130‐095‐130) after spleens were minsed by a 3‐mL syringe over a cell strainer with 40‐µm pore size (Fisher Scientific, 22‐363‐547) and lysed by RBC Lysis Buffer (BioLegend, 420301). Autologous T cells from human pancreatic tumor samples were enriched using the human Pan T cell isolation kit (Miltenyi Biotec, 130‐096‐535) after tumor tissues were digested into single cells by the tumor isolation kits (Miltenyi Biotec, 130‐095‐929). Mouse tumor‐associated macrophages were isolated from mouse cell suspensions using the mouse anti‐F4/80 MicroBeads (Miltenyi Biotec, 130‐110‐443).

3D HYGTIC model was constructed as has been previously described (20 (link)). Briefly, PDTF from human tumor fragments, or tumor cells sorted from murine TME (from cell clumps below Percoll layer) by tumor isolation kit (Miltenyi Biotec, Cat: 130-110-187) (MDTF), were collected for 3D tumor-microspheres construction. 5μl GelMA-PEO mixture containing 40,000 cells/μl was pipetted to make microspheres in BIOFLOAT™ 96-well plate (faCellitate, Cat: F202003), and was cultured and grown till day 7 in organoid culture medium. TIL was separated and T cells were isolated as aforementioned. Isolated T cells were cultured in T cells expanding medium (TEM, which was prepared as follows: ImmunoCult-XF T Cell Expansion Medium (10981, Stemcell) supplemented with 1000 IU/mL Recombinant IL-2 (human: 200-02; mice: 212-12; PeproTech). 0.5×10⁶ expanded T cells after 2 days culturing in TEM were added to each 3D tumor-microsphere containing PDTF or MDTF and set up as 3D-HYGTIC, with 50ng/ml mIL-2 and no CD3/28 addition. For sorted TIL above-mentioned (Supplementary Table S2 sheet2), 3D-HYGTIC was constructed without TIL expansion to exclude its interference on proliferation monitoring by Ki67 and CFSE.

Tumors were harvested and mechanically dissociated into fragments within 2 h. Subsequently, the tumor tissue was disrupted to prepare single cells using a tumor isolation kit (Miltenyi Biotec) following the manufacturer’s instructions. The cell suspension was lysed with a 70 μm cell filter to remove red blood cells. Tumor-infiltrating leukocytes were isolated through gradient centrifugation using a 40%/80% Percoll (GE Healthcare) solution. Subsequently, the collected cells were blocked with Fc block (anti-mouse CD16/32, BioLegend) on ice for 30 min. The samples were first stained for surface markers for lymphoid immune populations before intracellular staining. For FoxP3 and intracellular staining, True-Nuclear Transcription Factor Buffer Set 424,401 (BioLegend) was used following the manufacturer’s instructions. The following antibodies and stain kit were purchased from BioLegend: APC-Cy7 anti-mouse CD45, Alexa Fluor 700 Hamster anti-mouse CD3e, FITC anti-mouse CD4, PerCP-Cy5.5 anti-mouse CD8a, BV786 anti-mouse CD45R/B220, PE anti-mouse NK-1.1, BV650 anti-mouse IFN-γ, Alexa Fluor 647 anti-mouse Foxp3 and fixable viability stain 510. PE-CYN7 anti-mouse granzyme B was obtained from Thermo Fisher Scientific.