Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Lipid associated macrophage marker genes are increased in obese tumor-adjacent mammary adipose tissue (mAT Tum-adj ) and positively correlate with tumor mass. B) Schematic for magnetically sorting CD11b + cells from E0771 tumors using Miltenyi Octomacs. C) Trem2 , Cd36 , and Cd9 relative gene expression by RT-PCR in tumor CD11b + fraction. C) Anatomical schematic of subcutaneous mAT Tum-adj and contralateral mammary adipose tissue (mAT Contra ). D - F) Trem2 , Plin2 , Cd36 relative gene expression by RT-PCR from whole tissue lysates of mAT Contra and mAT Tum-adj . G-H) Correlation analysis of tumor mass vs. Trem2 and Plin2 gene expression in mAT Contra and mAT Tum-adj with linear regression fit. Figures D-F, solid bar = mAT Contra , hashed bar = mAT Tum-adj . One-way ANOVA tests were used to compare tumor LAM marker gene expression among groups. Two-way ANOVA with Tukey’s multiple comparison tests were used to compare groups for all mAT gene expression. Simple linear regression analysis was applied to correlation plots and the p value and coefficient of determination (r 2 ) is displayed on each graph. All data plotted as mean ± SEM for 8-12 mice per group. *denotes diet effect, †denotes tissue effect (mAT Contra vs. mAT Tum-adj ), **p<0.01, ***p<0.001, ****p<0.0001; †p<0.05, †††p<0.001, ††††p<0.0001. & C were created with Biorender.com.

Article Snippet: Recombinant mouse TREM2 protein (R&D Systems; cat. #1729- T2) was reconstituted according to the manufacturer specifications and diluted to a range of 0-10 ng/mL for the standard curve.

Techniques: Marker, Expressing, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Trem2 ablation attenuates tumor growth, decreases lipid associated macrophages, and increases adipocyte hypertrophy in a chow-fed postmenopausal breast cancer model. A) Schematic of study design for chow-fed postmenopausal breast cancer model in Trem2 +/+ and Trem2 -/- mice. B) sTREM2 (ng/mL) in plasma from female Trem2 +/+ and Trem2 -/- mice measured by ELISA. C) Blood glucose (mg/dL) measured during 14-week time point intra-peritoneal glucose tolerance test (ipGTT) and corresponding integrated area under the curve (iAUC). D) Tumor volume (mm 3 ) over time measured every 1-2 days. E) Tumor mass (g) at study endpoint. F) Relative gene expression by RT-PCR of LAM and macrophage markers from oAT whole tissue lysates. G) Adipocyte sizing frequency in tumor-adjacent mammary adipose tissue (mAT Tum-adj ). Diameter: small = 25-49μm, medium = 50-69μm, large = 70-99μm, very large = 100-150μm, extremely large = >150μm. H-I) Representative image of Trem2 +/+ and Trem2 -/- mAT Tum-adj stained with hematoxylin and eosin. Scale bar represents 100μm. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare blood glucose over time in the ipGTT. Two-way ANOVA with Sidak’s multiple comparison test was used to compare tumor volumes over time between genotypes. Unpaired two-tailed t-tests were used to compare groups for sTREM2 ELISA, iAUC, tumor mass, LAM gene expression, and each binning diameter size in adipocyte sizing analysis. All data plotted as mean ± SEM for 10-13 mice per group. *denotes genotype effect (Trem2 +/+ vs. Trem2 -/- ) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. was created with Biorender.com.

Article Snippet: Recombinant mouse TREM2 protein (R&D Systems; cat. #1729- T2) was reconstituted according to the manufacturer specifications and diluted to a range of 0-10 ng/mL for the standard curve.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Lean, but not obese or weight loss, Trem2 -/- mice exhibit decreased tumor growth. A) Body weight (g) measured weekly with dashed lines to indicate time of diet switch and E0771 PD-L1 cell injection. For diet groups and genotypes, light green = lean Trem2 +/+ , dark green = lean Trem2 -/- , light blue = obese Trem2 +/+ , dark blue = obese Trem2 -/- , light pink = weight loss Trem2 +/+ , and maroon = weight loss Trem2 -/- . B) sTREM2 (ng/mL) in plasma from Trem2 +/+ and Trem2 -/- mice measured by ELISA.C) Tumor mass (g) at study endpoint. D-F) Tumor volume (mm 3 ) over time for lean, obese, and weight loss Trem2 +/+ and Trem2 -/- mice, respectively. G) Diagram of tumor and tumor-adjacent mammary adipose tissue CD45 + cell sorting and processing for single cell RNA Sequencing. Mixed effects analysis with uncorrected Fisher’s LSD test was used compare tumor volumes over time between genotypes in the lean group (one missing value in lean Trem2 +/+ group at day 28). Two-way ANOVA with Sidak’s multiple comparison test was used to compare tumor volumes over time between genotypes in obese and weight loss groups. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare tumor mass between genotypes in lean, obese, and weight loss groups. *denotes genotype effect (Trem2 +/+ vs. Trem2 -/- ), †denotes diet effect, *p<0.05, **p<0.01, ****p<0.0001 for genotype differences (Trem2 +/+ vs. Trem2 -/- ) and ††††p<0.0001 for diet differences. All data plotted as mean ± SEM for n=4-23 per group. Figure G was created with Biorender.com.

Article Snippet: Recombinant mouse TREM2 protein (R&D Systems; cat. #1729- T2) was reconstituted according to the manufacturer specifications and diluted to a range of 0-10 ng/mL for the standard curve.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, FACS, RNA Sequencing Assay, Comparison

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Myeloid populations in tumors and tumor-adjacent mammary adipose tissues (mAT Tum-adj ) analyzed by single-cell RNA sequencing. A-B) Uniform Manifold Approximation and Projection (UMAP) of tumor (A) and mAT Tum-adj (B) myeloid cell subclusters from merged conditions labeled broadly by cell type category and colored by high-resolution cell type identities. C-D) Myeloid cell subcluster proportions expressed as frequency (%) split by diet-genotype groups for tumor (C) and mAT Tum-adj (D). E-F) Trem2 expression in lean, obese, and weight loss Trem2 +/+ mice in tumor (E) and mAT Tum-adj (F). G) Dot plot of the top 5 differentially expressed genes (DEGs) between lean Trem2 +/+ and lean Trem2 -/- tumor Macro_C1qc subclusters. Genes include H2-Eb1 , H2-Ab1 , H2-Aa , Cd74 , and Trem2 . Log fold change is shown for all 6 diet-genotype groups. H) Dot plot of H2-Eb1 , H2-Ab1 , H2-Aa , Cd74 , and Trem2 log fold change in the mAT Tum-adj LAM subcluster shown in all 6 diet-genotype groups.

Article Snippet: Recombinant mouse TREM2 protein (R&D Systems; cat. #1729- T2) was reconstituted according to the manufacturer specifications and diluted to a range of 0-10 ng/mL for the standard curve.

Techniques: RNA Sequencing Assay, Labeling, Expressing

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Visualization and binning of Tumor T cell clones reveals expansion of highly clonal CD4 + Th1 cells in lean Trem2 -/- tumors. A) Stacked bar graph of tumor T cell subclusters indicating cells per gram of tumor across all 6 diet-genotype groups. B) Tumor T cell subcluster proportions expressed as frequency (%) across all 6 diet-genotype groups. C) Uniform Manifold Approximation and Projection (UMAP) of tumor T cell subclusters from merged conditions colored by high-resolution cell type identities. D-I) Tumor T cell clones with a clonal population of two or more visualized by mapping clonal cell counts, indicated by color, onto the tumor T cell UMAP. The data is split as labeled by diet and genotype. J) Stacked bar graph showing the distribution of tumor T cell subclusters, binned as single cells or clonal low, clonal moderate, or clonal high (low = x ≤ 12 (median); moderate = 13 ≤ x ≤ 79 (3 rd quartile cutoff); high = x >79) shown for all 6 diet-genotype groups.

Article Snippet: Recombinant mouse TREM2 protein (R&D Systems; cat. #1729- T2) was reconstituted according to the manufacturer specifications and diluted to a range of 0-10 ng/mL for the standard curve.

Techniques: Clone Assay, Labeling

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Obese mice do not respond to αPD-1 therapy and do not exhibit an increase of T cell activation markers in tumor-adjacent mammary adipose tissue (mAT Tum-adj ) or tumor. A) Schematic of study design for lean, obese, and weight loss breast cancer model treated with IgG and αPD-1. During the last two weeks of tumor monitoring, mice were injected with either αPD-1 or IgG isotype control. B) Tumor mass (g) at study endpoint. C-E) Tumor volume (mm 3 ) over time for lean, obese, and weight loss IgG and αPD-1 treated mice with dashed line to indicate start of antibody treatment. F) Schematic of isolating the stromal vascular fraction from the mAT Tum-adj and subsequently isolating a pooled sample of CD4 + and CD8 + T cells using the magnetic Miltenyi Octomacs microbead sorting system. G) T cell activation markers, Pdcd1 , Tigit , Tox , and Lag3 relative gene expression by RT-PCR from magnetically sorted CD4 + and CD8 + T cells from the mAT Tum-adj . H) Cd8a , Pdcd1 , Lag3 , and Trem2 gene expression by RT-PCR from whole tumor lysates. Due to the response to αPD-1 treatment in the lean mice, only two tumors from the lean αPD-1 group could be analyzed by RT-PCR. For diet groups, solid colors: green = lean IgG, blue = obese IgG, pink = weight loss IgG; open circles: dark green = lean αPD-1, dark blue = obese αPD-1, maroon = weight loss αPD-1. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare groups for tumor mass. Mixed effects analysis was used for statistical analysis of tumor volumes over time. Two-way ANOVA with Sidak’s multiple comparison test was used to compare groups in the T cell activation marker gene expression analysis. All data plotted as mean ± SEM for 2-6 mice per group. *denotes antibody effect (IgG vs. αPD-1), †denotes diet effect, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; †p<0.05, ††p<0.01. , F, & H were created with Biorender.com.

Article Snippet: Recombinant mouse TREM2 protein (R&D Systems; cat. #1729- T2) was reconstituted according to the manufacturer specifications and diluted to a range of 0-10 ng/mL for the standard curve.

Techniques: Activation Assay, Injection, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Comparison, Marker