Journal: bioRxiv

Article Title: Invasive lobular carcinoma integrated multi-omics analysis reveals silencing of Arginosuccinate synthase and upregulation of nucleotide biosynthesis in tamoxifen resistance

doi: 10.1101/2025.01.16.633236

Figure Lengend Snippet: A. Phase contrast light microscopy images of parental and tamoxifen-resistant ILC cells with magnified images in the inserts. B. Growth kinetics of parental and tamoxifen-resistant MB134 (upper panel) and SUM44 (lower panel) cell lines. Fold change in growth normalized to day 0 at each time point over 72 hours. C. Dose response to tamoxifen in MB134 and MB134TAMR cells (upper panel) and SUM44 and SUM44TAMR cells (lower panel). Overnight cultures of exponentially growing cells were treated with vehicle or drugs for 5 days. IC 50 for TAM was calculated using GraphPad Prism 10. D. Transwell Boyden chamber assays comparing the migratory capabilities of parental vs. TAMR, MB134 (upper panel) and SUM44 (lower panel) cell lines. Area covered by migrated cells were quantified in the bar diagram. E. Representative image of western blot and densitometry analysis of ERα and HER2 levels in parental vs. TAMR ILC cells. GAPDH was used as loading control. Representative of three independent experiments is presented in the figures. Statistical differences between groups were evaluated using Student’s t-test. Significance levels are indicated as follows: **p <0.001, *p <0.05. P= Parental, TAMR = Tamoxifen Resistant

Article Snippet: ILC cell lines MDA-MB-134-VI (MB134) (ATCC, USA) and SUM44PE (SUM44) (Asterand, USA) were used to develop the tamoxifen-resistant (TAMR) cells by continuously exposing them to 100 to 500 nM of 4-hydroxy tamoxifen (4-OHT) for over 6 months.

Techniques: Light Microscopy, Western Blot, Control

Journal: bioRxiv

Article Title: Invasive lobular carcinoma integrated multi-omics analysis reveals silencing of Arginosuccinate synthase and upregulation of nucleotide biosynthesis in tamoxifen resistance

doi: 10.1101/2025.01.16.633236

Figure Lengend Snippet: A. Partial Least Squares Discriminant Analysis (PLS-DA) showing metabolic differences between parental and tamoxifen-resistant SUM44 cell pair. B. Variable Importance in Projection (VIP) plot highlighting the top 15 metabolites driving the separation between parental and resistant SUM44 cell pair. C. PLS-DA showing metabolic differences between parental and tamoxifen-resistant MB134 cell pair. D. VIP plot highlighting the top 15 metabolites driving the separation between parental and resistant MB134 cell pair. E. Overlap of altered pathways in parental vs. tamoxifen-resistant SUM44 and MB134 pairs, represented by impact scores. F. Venn diagram illustrating the number of shared and unique pathways altered in SUM44 and MB134 cell pairs, indicating common metabolic changes associated with tamoxifen resistance. G. Significantly deregulated KEGG pathways in parental vs. tamoxifen-resistant MB134 cells and, H. SUM44 cells as determined by RNA sequencing analysis of cell pairs in quadruplicate. I. Venn diagram showing the overlap of 52 deregulated pathways between parental and tamoxifen-resistant pairs of SUM44 and MB-134 cell lines. J. Venn diagram showing the overlap of metabolomic and transcriptomic data, identifying three mutually deregulated pathways in SUM44 and MB134 TAMR cells. K. Venn diagram showing the overlap of 15 genes within the three mutually deregulated pathways. L. Gene-metabolite interaction map illustrating interactions between three deregulated pathways. Circles represent genes and squares represent metabolites.

Article Snippet: ILC cell lines MDA-MB-134-VI (MB134) (ATCC, USA) and SUM44PE (SUM44) (Asterand, USA) were used to develop the tamoxifen-resistant (TAMR) cells by continuously exposing them to 100 to 500 nM of 4-hydroxy tamoxifen (4-OHT) for over 6 months.

Techniques: RNA Sequencing

Journal: bioRxiv

Article Title: Invasive lobular carcinoma integrated multi-omics analysis reveals silencing of Arginosuccinate synthase and upregulation of nucleotide biosynthesis in tamoxifen resistance

doi: 10.1101/2025.01.16.633236

Figure Lengend Snippet: A. Comparative expression of ASS1 in parental vs. parental and tamoxifen-resistant ILC cell lines as determined by change in Fragments Per Kilobase of transcript per Million mapped reads (FPKM) obtained from RNA seq data. B. qRT-PCR analysis showing ASS1 expression, and C. western blot and densitometry analysis showing ASS1 protein levels in MB134 and SUM44 cells, their respective tamoxifen resistant and LTED derivatives (LTEDA and LTEDB). D. Schematic diagram showing the CpG island in the ASS1 promoter. E. Analysis of the ASS1 promoter region using MS-PCR showing amplification of 150 bp methylated DNA in TAMR and LTED derivatives of ILC cell lines and 147 bp unmethylated DNA in parental ILC cell lines. F. qRT-PCR analysis showing ASS1 expression in MB134TAMR and SUM44TAMR cells that are either left untreated (UT) or treated with 5 μM 5-Aza-2’-deoxycytidine (dAzaC) for 120 hours. G. Western blot and densitometry analysis showing ASS1 protein levels in MB134TAMR and SUM44TAMR cells after 120 hours of dAzaC treatment (n=2 experiments). Error bars represent the standard deviation of triplicates. Significance levels are indicated as follows: ****p <0.0001, ***p <0.001, **p <0.01, *p <0.05.

Article Snippet: ILC cell lines MDA-MB-134-VI (MB134) (ATCC, USA) and SUM44PE (SUM44) (Asterand, USA) were used to develop the tamoxifen-resistant (TAMR) cells by continuously exposing them to 100 to 500 nM of 4-hydroxy tamoxifen (4-OHT) for over 6 months.

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot, Amplification, Methylation, Standard Deviation

Journal: bioRxiv

Article Title: Invasive lobular carcinoma integrated multi-omics analysis reveals silencing of Arginosuccinate synthase and upregulation of nucleotide biosynthesis in tamoxifen resistance

doi: 10.1101/2025.01.16.633236

Figure Lengend Snippet: MB134 and SUM44 cells were transduced with plasmids expressing shRNA targeting ASS1 or the corresponding empty vector to produce shASS1 and control (pLKO) cell lines. A. qRT-PCR analysis showing ASS1 expression, and B. western blot and densitometry analysis of ASS1 protein levels in ASS1 knockdown ( shASS1 ) vs. pLKO (control) MB134 and SUM44 cell lines. C. Dose response to tamoxifen and IC 50 of tamoxifen in ASS1 knockdown ( shASS1 ) vs. pLKO (control) MB134 (left panel) and SUM44 cell lines (right panel). D . Comparison of growth kinetics of shASS1 vs. pLKO derivatives of MB134 (left panel) and SUM44 cell lines (right panel). Fold change in growth is normalized to day 0 at each time point over 72 hours. E. Overall survival analysis of all breast cancer patients in relation to ASS1 expression using METABRIC data set. F . Overall survival analysis of ILC patients who received endocrine therapy in relation to ASS1 expression using METABRIC data set. G. Overall survival analysis of breast cancer patients who received endocrine therapy in relation to ASS1 expression using K-M plotter dataset. ****p <0.0001, ***p <0.001, **p <0.01, *p <0.05.

Article Snippet: ILC cell lines MDA-MB-134-VI (MB134) (ATCC, USA) and SUM44PE (SUM44) (Asterand, USA) were used to develop the tamoxifen-resistant (TAMR) cells by continuously exposing them to 100 to 500 nM of 4-hydroxy tamoxifen (4-OHT) for over 6 months.

Techniques: Transduction, Expressing, shRNA, Plasmid Preparation, Control, Quantitative RT-PCR, Western Blot, Knockdown, Comparison

Journal: bioRxiv

Article Title: Invasive lobular carcinoma integrated multi-omics analysis reveals silencing of Arginosuccinate synthase and upregulation of nucleotide biosynthesis in tamoxifen resistance

doi: 10.1101/2025.01.16.633236

Figure Lengend Snippet: A. Significantly enriched metabolic pathways in shASS1 vs. pLKO derivatives of MB134, and B. SUM44 cell lines. C. Changes in the levels of purine (dGMP, dGDP, GDP, GMP, IMP) and pyrimidine (UDP, UMP and CMP) nucleotides in parental (P) vs. tamoxifen-resistant (TAMR) MB134 cells. E. Changes in the levels of purine (ATP and ADP, dGTP, GDP) and F. pyrimidine (Cytosine, UMP) nucleotides in parental (P) vs. tamoxifen-resistant (TAMR) SUM44 cells. G. Expression levels of PRPS1 , H. PAICS, and I. DHODH in MB134 and SUM44 cell pairs as analyzed from RNA seq data. (FPKM: Fragments Per Kilobase Per Million reads). J. Representative picture of pCAD S1859 (pCAD) levels in parental and TAMR cells and K. in control (pLKO) vs. shASS1 derivatives of MB134 and SUM 44 cell pairs. Bar diagrams show average of more than one independent experiments (n=2). ***p <0.001, **p <0.01, *p <0.05.

Article Snippet: ILC cell lines MDA-MB-134-VI (MB134) (ATCC, USA) and SUM44PE (SUM44) (Asterand, USA) were used to develop the tamoxifen-resistant (TAMR) cells by continuously exposing them to 100 to 500 nM of 4-hydroxy tamoxifen (4-OHT) for over 6 months.

Techniques: Expressing, RNA Sequencing, Control