Extended Data Fig. 9: Generation of antibodies against hFWE^Win isoforms (hFWE2 and hFWE4) or the common hFWE N terminus and expression in human cancers.

a, Immunocytochemistry of hFWE isoforms in paraffin-embedded and sectioned human MCF-7 cells using novel anti-hFWE antibodies. The specificity was validated by immunocytochemistry of MCF-7 hFWE^KO cells with or without overexpression of individual hFWE isoforms. Cells were stained with anti-hFWE-N-term antibody or anti-hFWE^Win antibody. Immunocytochemistry results confirm antibody specificity in paraffin sections with MCF-7 hFWE^KO cells with or without overexpression of individual hFWE isoforms. The anti-hFWE^Win antibody does not stain sections with hFWE^KO cells, and recognizes specifically hFWE2 and hFWE4, but not the hFWE^Lose isoforms hFWE1 and hFWE3 (row 1). The anti-hFWE-N-term antibody is also negative for staining in control hFWE^KO cells but detected positive membrane staining for each of the four hFWE isoforms (row 2; n = 3). b, Immunohistochemistry of hFWE proteins in sections from clinical breast cancer samples (n = 3). Arrows indicate membrane staining. c, Immunofluorescence staining of hFWE proteins in human SCC tissue shows an abundance of hFWE^Win isoforms within defined cancer lesions, whereas the anti-hFWE-N-term antibody stained both stroma and cancer tissue (top; n = 3) when compared with normal skin (bottom; n = 3). d, Tumour volumes were measured and analysed for each of the 16 combinations of hFWEX–IRES–GFP expression in MCF-7 hFWE^KO xenografted cells and hFWE isoform expression within Fwe^KO mouse mammary tissue (n = 3). In vivo bioluminescence imaging (using the IVIS system) was used to detect and measure the fluorescence of GFP⁺ cancer cells in tumours resulting from all genetic combinations at 28 days post-implantation. Substantially reduced tumour growth was observed when mammary tissue expressed hFWE^Win isoforms and cancer cells expressed hFWE^Lose isoforms. By contrast, tumour growth is strongly promoted when mammary tissue expresses hFWE^Lose isoforms and cancer cells express hFWE^Win isoforms (n = 3). e, Tumorigenic potential of HCT-116 (colon origin) or MCF-7 (breast origin) hFWE^KO cells overexpressing hFWE2 in recipient Fwe^WT and Fwe^KO mice. Tumour growth of both Fwe^KO breast and colon cancer cell lines overexpressing the Win isoform hFWE2 was greater in Fwe^WT mice than in Fwe^KO mice. Photos of resected tumours are shown. f, Tumour volumes were measured every week over 42 days for groups shown in a. hFWE^KO colon- or breast-derived tumours overexpressing hFWE2 showed significantly higher growth in Fwe^WT mice than in Fwe^KO mice (n = 5, P values shown, one-tailed t-test, mean ± s.d.). g, At the conclusion of tumour growth experiments, mice were examined for the presence or absence of metastases in inguinal lymph nodes (ILN), axillary lymph nodes (ALN), colon, pancreas, prostate, lung, and liver. Heat map scale indicates the probability of metastasis. Results show a marked reduction in the metastatic potential of both breast and colon cancer hFWE^KO cells overexpressing hFWE2 when xenografted into Fwe^KO mice as compared to Fwe^WT mice (n = 5 each group).

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