Fig. 1

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