Mice Model: 8-week-old male mice were used in all experiments.Trem1 knockout mice on a C57BL/6 genetic background were generated as described in our previous report (Nguyen-Lefebvre AT, Ajith A, Portik-Dobos V, Horuzsko DD, Arbab AS, Dzutsev A, et al. The innate immune receptor TREM-1 promotes liver injury and fibrosis. J Clin Invest. 2018;128(11):4870-83). Breeding of Trem1 heterozygous parents (Trem1+/-) yielded Trem1-/- and Trem1+/+ offspring. PCR genotyping of offspring was performed based on the product sizes, 300 and 506 bp for the Trem1+/+ and mutant alleles, respectively. The ratio of Trem1-/-, Trem1+/- , and Trem1+/+ offspring did not deviate significantly from the expected 1:2:1 ratio. All animals were grossly normal, with normal weight and appearance. The expression of Trem1 was analyzed by PCR amplification of a 152-bp product using forward (5'-CGCCTGGTGGTGACCAAGGG-3') and reverse (5'-ACAACCGCAGTGGGCTTGGG-3') primers. Trem1+/+ littermate controls were used as the control for the in vivo tumorigenesis studies. Cell culture:The murine melanoma cell line B16-F10 (ATCC, cat. no. CRL-6475) was maintained in RPMI media (STEMCELL Technologies, cat. no. 36750) supplemented with 10% heat-inactivated FBS and penicillin-streptomycin. Tumor Model:8-week-old Trem1+/+ or Trem1-/- mice were subcutaneously injected with syngeneic 105 B16F10 murine melanoma cells. Mice were euthanized on the 22nd day post tumor cell injection. Freshly harvested tumors from Trem1+/+ or Trem1-/- mice were processed into a single-cell suspension using the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotech, cat. no. 130-096-427) in combination with the tumor dissociation kit (Miltenyi Biotech, cat. no. 130-095-929).Single-cell RNA sequencing sample preparation: Freshly harvested tumors from Trem1+/+ and Trem1-/- mice were pooled separately into 2 groups to maximize the isolation of tumor infiltrating myeloid cells. Tumors were gently dissociated using the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotech). Single-cell suspensions of tumor cells were purified by incubation with CD45(Miltenyi Biotec, cat. no. 130-110-618) Microbeads as per the manufacturer's instructions to enrich the tumor-infiltrating cells. 7,000-8,000 live myeloid cells were loaded onto the Chromium Controller (10x Genomics), and scRNA-seq libraries were generated using Chromium Next GEM Single Cell 3' Reagent kit v3.1 (10x Genomics) according to the manufacturer's protocol. The libraries were sequenced on NextSeq 500 (Illumina) using Mid Output v2.5 (150 cycles) kit (Illumina) with 28 bp (Read 1), 8 bp (Indexing Run), and 91 bp (RNA Read 2) to collect approximately 23K mean reads per cell at the range of 1,294,379 median genes per cell. Reads from the raw fastq files were mapped to the mm10 mouse genome reference by STAR aligner linked with the Cell Ranger v5.0.1 pipeline to output clusters representing the cell populations and the upregulated genes annotated as the cell surface markers in each cell population. The output was imported into Loupe Browser v6.0 (10x Genomics) for visualization along with further analysis.Freshly harvested tumors from Trem1+/+ and Trem1-/- mice were pooled separately into 2 groups to maximize the isolation of tumor infiltrating myeloid cells. Tumors were gently dissociated using the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotech). Single-cell suspensions of tumor cells were purified by incubation with CD45, CD11b Microbeads (Miltenyi Biotec, cat. no. 130-049-601) as per the manufacturer's instructions to enrich the tumor-infiltrating myeloid cells. Quality and quantity of cells were assessed under the microscope by trypan blue staining, and dead cells were removed using the Dead Cell Removal Kit (STEMCELL, cat. no. 17899) as per the manufacturer's instructions. Nuclei acid extraction was performed following the manufacturer's instructions (10x Genomics, as part of the Chromium Single Cell 3' v3.1). Tocapture approximately 5,000 targeted cells, 7,000-8,000 live myeloid cells were loaded onto theChromium Controller (10x Genomics), and scRNA-seq libraries were generated using ChromiumNext GEM Single Cell 3' Reagent kit v3.1 (10x Genomics) according to the manufacturer'sprotocol.