Journal: Breast Cancer Research : BCR

Article Title: RHAMM regulates MMTV-PyMT-induced lung metastasis by connecting STING-dependent DNA damage sensing to interferon/STAT1 pro-apoptosis signaling

doi: 10.1186/s13058-023-01652-1

Figure Lengend Snippet: Rhamm -loss in MMTV-PyMT transgenic mice increases lung metastasis without affecting primary mammary tumor initiation, incidence or size. A Comparison of RHAMM mRNA expression in breast cancer subtypes (Breast Invasive Carcinoma, TCGA, PanCancer Atlas). RHAMM expression is significantly higher in basal and luminal B breast cancer subtypes than in luminal A comparators. B Rhamm- loss detected by the Mouse Diversity Genotyping Array (MDGA) genotyping was confirmed by ddPCR, which is the standard ultrasensitive method for verification of genotyping array-based CNV detection. Two additional ddPCR assays confirm another CNV deletion overlapping Ilk and Taf10 genes that is detected by MDGA CNV genotyping, confirming accuracy of the MDGA and consistency of the two detection methods. WT = Wildtype; Rh −/− = Rhamm −/− . C Western blot assays were performed using primary tumor lysates and RHAMM antibodies to the N-terminal sequence as described in Methods. Full-length RHAMM is expressed in Wildtype tumors (arrow) and is not detected in Rhamm −/− tumors. D Histology sections of primary tumors from 16-week-old mice. Tissue was paraffin-processed, stained for RHAMM protein and imaged with a Nikon confocal microscope as described in Methods. Results show heterogeneous RHAMM staining in primary tumors and also in the host microenvironment (arrows). The lack of staining in histology sections from Rhamm −/− tissue confirms the specificity of the anti-RHAMM antibody. E Whole mounts of mammary fat pads were prepared as described in Methods and show primary tumor initiation at 5 weeks in both Wildtype and Rhamm −/− mice. Detectable primary tumor masses of Wildtype and Rhamm −/− mice were measured weekly with calipers. Differences between genotypes are not statistically significant. Values are the Mean and S.E.M. n = 15 mice. F The lungs of Wildtype and Rhamm −/− mice were harvested and metastatic nodules identified in hematoxylin/eosin-stained histology sections. 100% of Rhamm −/− mice contain metastatic nodules in lungs in contrast to 58% of Wildtype mice, n = 11 mice/genotype. G Metastatic nodules were quantified from serial histology sections as described in Methods. Rhamm -loss significantly increases the number of metastatic colonies/lung compared to Wildtype. Values are the Mean and S.E.M. n = 15 mice. * p < 0.05

Article Snippet: Mouse diversity genotyping array (MDGA) hybridization was performed at the London Regional Genomics Centre (Robarts Research Institute, London, ON) according to instructions in the Affymetrix® Genome-Wide Human single nucleotide polymorphism (SNP) Nsp/Sty 6.0 manual (Affymetrix 2007; https://assests.thermofisher.com/TFS-Assets/LSG/manuals/snp6_atp_userguide.pdf ).

Techniques: Transgenic Assay, Comparison, Expressing, Western Blot, Sequencing, Staining, Microscopy

Journal: Breast Cancer Research : BCR

Article Title: RHAMM regulates MMTV-PyMT-induced lung metastasis by connecting STING-dependent DNA damage sensing to interferon/STAT1 pro-apoptosis signaling

doi: 10.1186/s13058-023-01652-1

Figure Lengend Snippet: Rhamm −/− tumor cells are resistant to DNA damage. A DNA fragmentation was quantified by comet assays as a measure of DNA damage using frozen samples of isolated, unstimulated Wildtype and Rhamm −/− primary tumor cells. Quantification of % DNA in tail and tail moment is not affected by Rhamm -loss consistent with similar mutation burdens in the two genotypes identified by the genotyping array. Values are the Mean and S.E.M of n = 3 cell samples/genotype and n = 21 wells/genotype. B The amount of ROS-induced DNA damage in Wildtype and Rhamm −/− tumors was quantified using ELISA as described in Methods. Results show that DNA from lung metastases of both genotypes carry more ROS-induced DNA damage that can cause point mutations and CNVs than primary tumors, which is consistent with the higher levels of ROS in lungs versus mammary fat pads. Values are the Mean and S.E.M. N = 4 biological replicates and 3 technical replicates. * p < 0.05, *** p < 0.001. C, D Cultures of Wildtype and Rhamm −/− primary tumor cells were exposed to H 2 O 2 ( C ) and cisplatin ( D ) to induce DNA damage. DNA damage was detected by gamma-H2AX staining, and cell death was quantified by Apoptag reactivity as described in Methods. Rhamm −/− tumor cells survive with more DNA damage than Wildtype comparators. Values are the Mean and S.E.M. n = 3 biological replicates * p < 0.05, ** p < 0.01

Article Snippet: Mouse diversity genotyping array (MDGA) hybridization was performed at the London Regional Genomics Centre (Robarts Research Institute, London, ON) according to instructions in the Affymetrix® Genome-Wide Human single nucleotide polymorphism (SNP) Nsp/Sty 6.0 manual (Affymetrix 2007; https://assests.thermofisher.com/TFS-Assets/LSG/manuals/snp6_atp_userguide.pdf ).

Techniques: Isolation, Mutagenesis, Enzyme-linked Immunosorbent Assay, Staining

Journal: Frontiers in Immunology

Article Title: A Humanized Mouse Strain That Develops Spontaneously Immune-Mediated Diabetes

doi: 10.3389/fimmu.2021.748679

Figure Lengend Snippet: Patients and healthy donors.

Article Snippet: Labeled targets were then hybridized overnight to Genechip ® Mouse Diversity Genotyping Array (Affymetrix, ref: 901615) at 49°C.

Techniques: