mouse model  (Simmler Inc)

Journal: bioRxiv

Article Title: TUMOR–PRE-ADIPOCYTE CROSSTALK SUSTAINS BREAST CANCER GROWTH VIA RET SIGNALLING

doi: 10.64898/2026.05.05.722993

Figure Lengend Snippet: (A) H&E-staining shows smaller adipocytes in the proximity of breast tumor in patients. Representative pictures indicating regions of the breast adipose tissue in distant (Distant AT) or adjacent (Adjacent AT) locations to the tumor mass are shown. BC patient ID is indicated on the right corner of the picture. Images were acquired at 10x or 400x magnification. (B) Analysis of average adipocyte area in Distant (>2000 mm) and Adjacent (<2000 mm) regions of the adipose tissue (AT) next to tumor lesion in BC patient biopsies. Each dot represents an individual sample (n=12). Data are presented as means. Unpaired Student’s t test was used; **p=0.001. (C) H&E-staining shows smaller adipocytes in the proximity of mammary tumor in the mouse EO771-tumor model. Representative pictures of either regions of the mammary adjacent adipose tissue (Adjacent AT) or tumor-free adipose tissue (Ctrl AT) are shown. Images were acquired at 10x or 400x magnification. (D) Quantification of adipocyte area in mouse mammary samples from the EO771 model is shown in the graph bars. Each dot represents an individual animal (n=4-5). Data are presented as median ± SD. Mann-Whitney test was used; *p=0.0159. (E-F) Representative images of IF for PLIN1-staining in tumor-adjacent and control areas of adipose tissue in the human breast and mouse model. PLIN1 (red) labels lipid droplet membranes. Nuclei DNA (DNA) were counterstained with DAPI (blue). Images were acquired at 10x magnification. Scale bars=100 μm. (G-H) Hallmark gene set analysis (MSigDB) of RNA-seq data from tumor-adjacent adipose tissue (Adjacent AT) compared with control adipose tissue (Ctrl AT) in the EO771 mouse mammary tumor model from independent animals (n=4). Gene set enrichment significance was assessed using Fisher’s exact test, with p-values corrected for multiple testing using the Benjamini–Hochberg false discovery rate (FDR). Heatmaps show the distribution of gene expression for each experimental group (left), and the expression levels of selected genes representing mature adipocyte markers (right). (I, L) Schematic representation of the experimental set-up of treatments with conditioned medium (CM) after which cultures were subsequently used for analysis. In a Set up a. cultures of terminal differentiated white adipocytes (WA) were treated for 3 days with CM from EO771 cells. In a Set up b. cultures of pre-adipocytes (PAs) under differentiation process were treated for 9 days with CM. (J, M) After treatments lipid content was determined by oil-red staining in the adipocyte cultures and quantified as shown in graphic bars. Representative pictures of oil-red-stained plates are shown for each condition. Images were acquired at 20× magnification. Each dot represents an individual well (Set up a. n=30 from 3 independent experiments, N=3; Set up b. n=14, N=2). Data is presented as mean ± SD. For statistic unpaired Student’s t test was used; corresponding p-values are indicated on bar graphs. (K, N) Either tumoral CM-treated cultures of terminal differentiated WA (Set up a.) or PAs under differentiation process (Set up b.) were used for the analysis of expression of specific adipocyte differentiation markers by RT-qPCR ( Adipoq, Pparg for WA; Dlk1, Pdgfra for PA). Each dot represents an individual well (n=9-18) from 3 independent experiments (N=3). Data is presented as mean ± SD. Unpaired Student’s t or Mann-Whitney tests were used when compared conditions; p-values are indicated on bar graphs. No CM: no CM was added. T CM: Tumoral CM from EO771 cultures. Schemes from experimental procedures along figures were prepared using ServierMedical Art ( https://smart.servier.com/ ), licensed under a creative common Attribution 3.0 unported license.

Article Snippet: In the EO771-tumor mouse model, the transcriptomic analysis of mammary peri-tumoral PA-like AT, identified differentially secreted factors (absolute log2FC >1, p-value <0.05; secreted proteins from Human Protein Atlas), among which GDNF, the ligand for the RET oncogene, was prominently induced ( Figure S2A, B, SI ).

Techniques: Staining, MANN-WHITNEY, Control, RNA Sequencing, Gene Expression, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: TUMOR–PRE-ADIPOCYTE CROSSTALK SUSTAINS BREAST CANCER GROWTH VIA RET SIGNALLING

doi: 10.64898/2026.05.05.722993

Figure Lengend Snippet: (A) Scheme representing the in vivo EO771-tumor model where the co-injection of EO771 mammary tumor cells together with PA cells was performed. (B) Representative curves from 2 independent experiments (N=2) showing the tumor growth of the mouse allografts generated by co-injection of both cell types, tumor cell line E0771 and PA cells. Data are presented as mean ± SEM. Each dot in the curve represents the mean (EO771, n=5; EO771+PA, n=6; PA, n=3). Statistics were calculated using One-way ANOVA followed by Tukey’s multiple comparison test at end point (Day 23); p-values are indicated on bar graphics. (C) IHC for a proliferation marker (BrdU) on tumor slides are shown with the corresponding quantification. Data is presented as mean ± SD. Each dot in the curve represents an individual animal (EO771, n=10; EO771+PA, n=9) from 2 independent experiments (N=2). Statistics were calculated using Unpaired Student’s t test; p-values are indicated on bar graphics. Images were acquired at 10× magnification. (D) Schematic representation of the experimental set-up of the transwell co-culture system. Cultures of either differentiated WA or PA cells were co-cultured (Cc) with EO771 tumor cells and subsequently used for the analysis of tumor cell counting. (E) Proliferation was assessed by crystal violet staining followed by absorbance measurement at 590nm after Cc with differentiated WA or PA. Each dot in the plot represents an individual well (n=6-9) from 3 independent experiments (N=3). Statistics were calculated using One-way ANOVA followed by Tukey’s multiple comparison test; p-values are indicated on bar graphics.

Article Snippet: In the EO771-tumor mouse model, the transcriptomic analysis of mammary peri-tumoral PA-like AT, identified differentially secreted factors (absolute log2FC >1, p-value <0.05; secreted proteins from Human Protein Atlas), among which GDNF, the ligand for the RET oncogene, was prominently induced ( Figure S2A, B, SI ).

Techniques: In Vivo, Injection, Generated, Comparison, Marker, Co-Culture Assay, Cell Culture, Cell Counting, Staining

Journal: bioRxiv

Article Title: TUMOR–PRE-ADIPOCYTE CROSSTALK SUSTAINS BREAST CANCER GROWTH VIA RET SIGNALLING

doi: 10.64898/2026.05.05.722993

Figure Lengend Snippet: (A) Representative pictures of RET-staining by IHC in breast tumor tissue cores from patients indicating low, moderate and high levels of RET-positive cells. BC patient ID is indicated on the right corner. Images were acquired at 40x magnification. (B) Higher levels of RET expression within tumors are positively correlated with grater difference in the size of adjacent adipocytes in the BC patient cohort. Correlation analysis of the difference in median adipocyte area (D adipocyte area) and tumoral RET expression (by intensity of RET-positive cells in pixels) is shown. Each dot represents an individual sample (n=12). Pearson correlation test was used; *p=0.0225. (C) Representative images showing GFP-driven fluorescence (GFP) in EO771 cultures Cas9-expressing (Cas9-GFP cassette) cells respect to control (Parental) are shown. Images were acquired at 400x magnification. (D) EO771 RET-WT and RET-KO mammary tumor cells (sg1 RET) were analyzed for RET protein expression by WB with specific antibodies, confirming the absence of RET expression in the RET-KO cells. A lysate from MCF7 human breast cancer cells was added as positive control of RET expression. (E) Scheme of RET-KO EO771-tumor allograft model. In this case, RET-WT or RET-KO EO771 tumor cell lines (sg1 RET) were injected into female mice to generate EO771-derived tumors. (F) Scheme showing a dissection and anatomical location of either tumor (T) or tumor adjacent adipose tissue (Adjacent AT) used for posterior analysis. Fluorescence images (GFP) confirmed GFP-positive tumor cells forming the tumor mass. Images were acquired at 1x magnification. (G) In vivo tumor growth of RET-WT or RET-KO EO771-tumor allografts (sg1RET). Tumor volumes were measured after mammary fat pad injection of EO771 into female mice (RET-WT, n=8; RET-KO, n=8). Data are presented as mean ± SEM. Each dot in the curve represents the mean of a group of animals from a representative experiment from a total of 8 independent experiments (N=8). In the bar chart, each dot represents an animal. Statistics were calculated using two-tailed unpaired Student’s t test at end point (Day 24); *p=0.0106. (H) BrdU incorporation followed by specific staining were performed on tumor tissue from the EO771-tumor model from 2 independent in vivo experiments (N=2). Representative pictures are shown with the indicated quantifications in the graph bars. Each dot represents an individual animal (n=8). Data is presented as mean ± SD. Unpaired Student’s t test was used; **p=0.0074. Images were acquired at 10× magnification. (I) H&E- and PLIN1-staining were performed on tumor and adjacent tissue from the EO771-tumor model. Representative pictures are shown with the indicated quantifications of the adipocyte area in the graph bars. Each dot represents an individual animal (n=16, N=8). Data are presented as median ± SD. Unpaired Student’s t test was used; *p=0.0468. Images were acquired at 10× magnification. Scale bars=100 μm. (J) Analysis of expression of specific adipocyte differentiation markers ( Adipoq, Pparg , for WA) were performed by RT-qPCR on samples of Adjacent AT from EO771 RET-WT or RET-KO EO771-tumor bearing animals. Each dot represents an individual animal (n=5-21, N=4). Data is presented as mean ± SEM. Unpaired Student’s t or Mann-Whitney tests were used when compared conditions; p-values are indicated on bar graphics. (K) WB analysis showing the expression for DLK1 at protein level on samples of Adjacent AT from EO771 RET-WT or RET-KO EO771-tumor bearing animals. Corresponding quantification is shown. Each dot represents an individual animal (n=9, N=2). Unpaired Student’s t test was used; *p=0.0106.

Article Snippet: In the EO771-tumor mouse model, the transcriptomic analysis of mammary peri-tumoral PA-like AT, identified differentially secreted factors (absolute log2FC >1, p-value <0.05; secreted proteins from Human Protein Atlas), among which GDNF, the ligand for the RET oncogene, was prominently induced ( Figure S2A, B, SI ).

Techniques: Staining, Expressing, Fluorescence, Control, Positive Control, Injection, Derivative Assay, Dissection, In Vivo, Two Tailed Test, BrdU Incorporation Assay, Quantitative RT-PCR, MANN-WHITNEY

Journal: bioRxiv

Article Title: TUMOR–PRE-ADIPOCYTE CROSSTALK SUSTAINS BREAST CANCER GROWTH VIA RET SIGNALLING

doi: 10.64898/2026.05.05.722993

Figure Lengend Snippet: (A) Volcano plot analysis showing the genes encoding soluble factors which are significantly up-regulated in the RET-expressing RET/MTB transgenic glands. p-value <0.01 were considered and calculated by Two-sample t test and log2FC cutoff ± 1. (B) PDGFs levels were confirmed in tumor tissue from the EO771-tumor mouse model (sg1RET and sg2RET) by WB. WB results are representatives of at least three independent animals. Corresponding quantification is shown. Each dot represents an individual animal (n=9-10, 2 independent experiments). Data is presented as mean ± SD. Unpaired Student’s t test was used; *p=0.0172. As control, RET levels in lysates of EO771-tumor tissue (n=4) were analyzed by WB. (C) Correlation analysis of PDGFB expression and tumoral RET in tumor tissue patient samples from the new cohort. Each dot represents an individual sample (n=14). Pearson correlation test was used; **p=0.001. (D) MCF7 cultures were stimulated with GDNF (25 ng/ml) in absence (DMSO) or presence of RET inhibitor BLU-667 (BLU, 2mM), lysates were obtained and analyzed for PDGF ligands by WB. Results are representatives of at least 3 independent experiments. Corresponding quantification is shown. Each dot represents an individual well (n=3). Data is presented as mean ± SD. One-way ANOVA test was used followed by Tukey’s multiple comparation test p-values are indicated on bar graphics. (E) Scheme of breast cancer cells conditioned medium (CM) treatments on pre-adipocyte (PA). CM from MCF7 (MCF7 CM) previously stimulated with GDNF (25 ng/ml) after treatments with RET inhibitor (BLU, 2mM) was used to treat PA cultures. As control of PDGFR activation, a condition with PDGF-B ligand (PDGF-BB, 0,1 mg/ml) stimulation was performed. (F) Levels of activation of PDGFRA and ERK measured by WB in the PA-treated cultures as indicated. Duplicates are shown in the blots. Arrowhead indicates the correct band. *: unspecific band. (G) A scheme showing experiments where PA cultures were treated with CM from EO771 RET-WT or RET-KO tumor cells (EO771 CM). (H) Levels of activation of ERK were measured (including a time course) by WB in the PA-treated cultures as indicated. The results are shown for two independent EO771 RET-KO cell lines. (I) MTS assays were performed to assess changes in viability in PA-treated cultures with EO771 RET-WT or RET-KO cells CM (48 hours). Each dot represents an individual well (n=4). Data is presented as mean ± SD. One-way ANOVA test was used followed by Tukey’s multiple comparation test; p-values are indicated on bar graphics.

Article Snippet: In the EO771-tumor mouse model, the transcriptomic analysis of mammary peri-tumoral PA-like AT, identified differentially secreted factors (absolute log2FC >1, p-value <0.05; secreted proteins from Human Protein Atlas), among which GDNF, the ligand for the RET oncogene, was prominently induced ( Figure S2A, B, SI ).

Techniques: Expressing, Transgenic Assay, Control, Activation Assay