title: Sample 6
organism: Mus musculus
description: Protocols: Tumor samples were harvested into cold PBS and stored on ice until processing. Tumors were transferred to GentleMACS C Tubes (Miltenyi Biotec, #103-093-237) containing 2.5 mL of enzymatic digestion mixture consisting of RPMI 1640, 2% FBS and 0.05 mg/mL DNase I (Sigma-Aldrich, #10104159001) and 0.25 mg/mL Liberase™ TL (Roche, #5401020001). Tumors were minced into small pieces using scissors. The tubes were then placed on a GentleMACS Octo Dissociator (Miltenyi Biotec) using the manufacturer's recommended tumor dissociation protocol, followed by incubation for 30 min at 37°C. Following incubation, the cell suspension was filtered through 70 μm MACS® SmartStrainers (Miltenyi Biotec, #130-110-96) and washed with RPMI 1640 containing 2% FBS. The samples were centrifuged and resuspended in pZerve™ freezing medium (Sigma-Aldrich, #Z1653) and stored for single-cell RNA sequencing analysis.  Frozen tumor single-cell suspensions were thawed and washed with RPMI 1640 supplemented with 10% FBS. Following centrifugation, cells were washed once with PBS. To block non-specific binding, normal Mouse IgG (1:50 dilution in FACS buffer, vendor, # ) was added to the cells and incubated for 10 minutes at 4°C. After one PBS wash, cells were resuspended with the antibody staining mix and incubated for 30 minutes at 4°C. Following two additional washes, cells were resuspended in FACS buffer.  Cell sorting was performed using a 100 µm nozzle with cells separated into two distinct populations: CD45 + CD3 + NK1.1 + cells (T and NK cells) and CD45 + CD3 - NK1.1 - (myeloid cells). This sorting strategy was employed to enriched for the rare cDC1 subset within the myeloid population. Sorted cells were collected in PBS containing 1% Bovine Serum Albumin (Sigma-Aldrich, #126615-25ML). For each sample, myeloid cells and NK/T cells were mixed at a ratio of 5:1 (myeloid cells:T/NK cells) immediately prior to processing for single-cell RNA sequencing. For tumor cell inoculation, KPC cells were dissociated using TrypLE (Thermo Fisher, #12605-010). The cells were then resuspended at a concentration of 3x10 6 /ml in a mixture composed of 50% RPMI 1640 (Gibco, #42401-018) and 50% Matrigel (Corning, #354230). The cell suspension (100 μl/mouse) was subsequently injected into the left flank of anaesthetized C57BL/6J mice. Therapy injection was initiated when tumors reached an average volume of 200-250 mm 3 . Mice were randomized into treatment groups either one day before or on the day of the first treatment administration. Both molecules were formulated in Histidine buffer (Bichsel, customized formulation for Roche Glycart AG) on the day of administration. FAP-CD40 was administered at a dose of 13.3 mg/kg via intraperitoneal injection (200 μl), while PD1-IL2v was delivered intravenously (200 μl) at a dose of 0.5 mg/kg. The molecules were produced and purified at Roche Innovation Center Zurich (RICZ) and Roche Innovation Center Munich (RICM). Tumors were collected for downstream analysis on day 10  with mice anaesthetized prior to tumor collection. Tumor samples were processed according to the protocols described in the “Dissociation of tumor samples” and “Sample preparation for single-cell RNA sequencing” sections. A total of 16 samples were analyzed (from 4 treatment groups: vehicle, FAP-CD40, PD1-IL2v and FAP-CD40 + PD1-IL2v), with 4 mice per group. The cell number and viability of sorted and pooled cells were determined using a Nexcelom Cellometer Auto 2000. A total of approx. 20,000 viable cells per sample were loaded into the 10x Genomics Chromium Connect automated platform and library preparation was performed according to the manufacturer's instructions (Chromium NextGEM Automated Single Cell 5&#39; Reagent Kit v2). 14 and 16 PCR cycles were used for cDNA and library amplification respectively. The concentration of the cDNAs and the resulting final libraries were quantified using an Invitrogen Qubit fluorometer (High Sensitivity dsDNA Assay) and the respective fragment size distribution was determined using an Agilent 2100 Bioanalyzer automated electrophoresis instrument.