Extended Data Fig. 5: Expression of NMDA receptor subunits and staining of proteins indicative of aspartate signaling in patients with breast cancer.
From: Aspartate signalling drives lung metastasis via alternative translation
a. RMA-normalized mRNA expression levels of NMDA-receptor-related genes in breast cancer-derived lung metastases vs breast cancer-derived bone/brain/liver metastases, based on patient biopsies (GSE14018). N = 16 lung metastases and n = 20 non-lung metastases samples (n = 8 bone metastases, n = 7 brain metastases, n = 5 liver metastases). Box hinges indicate the 1st/3rd quartiles of the corresponding data, while box mid-lines represent medians, and whiskers span the range of the data excluding outliers (data points more than 1.5 x IQR away from the 1st/3rd quartiles, where IQR = inter-quartile range), with the latter indicated in red. Individual data points are indicated by the white dots. Statistics show P-values based on differential expression analysis with limma. b. Top: DAPI and PanCK (left), GRIN2D and membrane marker (right) detected in metastatic and adjacent lungs from the UPTIDER breast cancer patient 2026 shown in Fig. 5e, assessed by multiplex immunohistochemistry. Blue = DAPI nuclear staining, red = GRIN2D, green = membrane marker ATP1A1 (scale bars = 100 μm). Membrane marker has been added to identify surface localization of GRIN2D, the same exact representative image was used in Fig. 5e. Bottom: representative image of H&E staining of the whole lung from patient 2026, scale bar = 2 mm. H&E and multiplex immunohistochemistry stainings originate from non-consecutive cuts. c. Quantification of GRIN2D, Hypusine, DOHH, nuclear pCREB, nuclear pSMAD3, COL1A1 and COL6A1 intensities in tissues derived from the UPTIDER breast cancer patients. For GRIN2D, Hypusine and DOHH, fluorescence intensity values per single cell are depicted, across 5 independent regions. For pCREB and pSMAD3, fluorescence intensity values per nucleus are depicted, across 5 independent regions. For COL1A1 and COL6A1, mean fluorescence intensities per unit area are depicted, across 5 independent regions. Two-way ANOVA, with P values for the tissue type (metastatic vs adjacent) factor shown above the graphs. All single comparisons per patients were significant based on unpaired two-tailed t-test with Welch correction, except for COL1A1 and COL6A1, for which single comparisons per patients were significant in 7 out of 10 measurements based on unpaired two-tailed t-test with Welch correction. Representative images are shown in Fig. 5e and Extended Data Fig. 6. d. Representative images of linearized collagen based on Picrosirius Red staining and polarized light microscopy detected in metastatic and adjacent lungs from the UPTIDER breast cancer patient 2026, quantified in Fig. 5f. Red color mostly indicates thick collagen I fibers and green color mostly indicates thin collagen III fibers. A representative image of n = 5 patients is shown. Scale bars = 1 mm.
