a, The potential effect of individual hFWE isoforms on intracellular calcium levels was investigated. Live-cell imaging was performed to detect levels of Fluo-4AM in MCF-7 hFWEKO cells and MCF-7 hFWEKO cells overexpressing single hFWE isoforms (n = 3 biologically independent experiments, ANOVA showed no significant differences, mean ± s.d.). b, Live-cell imaging at 5-s intervals over 1 min showed constant levels of Fluo-4AM in MCF-7 hFWEKO cells and MCF-7 hFWEKO cells overexpressing single hFWE isoforms (n = 3 biologically independent experiments, ANOVA showed no significant differences, mean ± s.d.). c, hFWE-mediated cell competition assay carried out in the presence or absence of the calcium chelator BAPTA-AM. Treatment with BAPTA-AM did not affect the ability of MCF-7 hFWEKO cells expressing hFWE2 to kill co-cultured MCF-7 hFWEKO cells expressing hFWE1 (n = 3 biologically independent experiments, P values shown, two-tailed t-test found no significant differences, mean ± s.d.). d, Laser-capture microdissection was performed in increments of 200 µm in distance perpendicular to the defined boundary of breast tissue tumour mass. Representative images are shown for breast cancer tissue section before and after laser capture. hFWELose isoforms were upregulated in tumour-adjacent host tissue nearest to breast cancer (0–400 μm) or colon cancer (0–200 μm). hFWEWin isoforms were highly enriched within breast or colon tumour tissue but not adjacent tissue (n = 3 biologically independent experiments, mean ± s.d., fold change calculated relative to the external reference of the expression of hFWEWin isoforms in normal breast tissue, P values shown, two-tailed t-test). e, qPCR array was used to examine the expression of 354 genes involved in apoptotic pathways in samples of laser-captured tumour-adjacent host tissue. Gene expression heat map shows that host tissue in the immediate vicinity of cancer (0–400 μm) displayed upregulation of genes involved in apoptotic pathways. Host tissue farther away (400–1,000 μm) from the tumour edge displayed basal levels of apoptosis-related genes. The heat map shows apoptotic genes that were induced (red), suppressed (blue) or unchanged (yellow) in expression. Black represents unsuccessful runs (n = 3). f, FISH shows the expression of hFWEWin isoforms in FFPE samples of breast cancer tissue (top) and normal breast tissue (bottom). First column, H&E staining; second column, control DAPI staining; third column, expression of hFWEWin isoforms within tumour tissue; fourth column, overlay of DAPI and hFWEWin isoforms. Magnified images below each panel show expression of hFWEWin isoforms specifically in tumour tissue, as these isoforms are poorly expressed in the stromal tissue surrounding the tumour and normal breast tissue (n = 3 for all staining). g, Schematic depicting the process of epithelial cell isolation from normal breast, colon, and lung tissue. Expression of the four hFWE isoforms was compared in epithelia versus total tissue by qPCR analysis (n = 3 biologically independent experiments, all statistically significant P values shown, two-tailed t-test, mean ± s.d.). h, IHC staining of FFPE samples of breast cancer with newly developed antibodies against hFWE. Breast cancer and stromal regions are shown in the H&E images (left). The anti-hFWEWin antibody is specific to Win isoforms hFWE2 and hFWE4 whereas the anti-hFWE-N-term antibody targets the common N terminus and recognizes all four isoforms. Immunohistochemistry staining shows the abundance of hFWEWin isoforms within the breast cancer samples in human tumours. From this, we conclude that hFWEWin proteins are expressed in the tumour but not in the stroma. To demonstrate the expression of Lose isoforms in the stroma, we compared the staining of all hFWE isoforms using our N-terminal-specific antibody with the poor expression of hFWEWin in the stroma. The anti-hFWE-N-term antibody shows strong immunoreactivity and equal distribution of staining in the tumour and stromal tissue. We find no positive signal for hFWEWin isoforms in the stroma, but we find strong expression of total hFWE (hFWEWin and hFWELose combined) in the stroma near the tumour. From this, we can conclude that Lose isoforms are expressed in the stroma. This experiment was repeated independently three times with similar results. Source Data