Normal cells repel WWOX-negative or -dysfunctional cancer cells via WWOX cell surface epitope 286-299

Metastatic cancer cells are frequently deficient in WWOX protein or express dysfunctional WWOX (designated WWOXd). Here, we determined that functional WWOX-expressing (WWOXf) cells migrate collectively and expel the individually migrating WWOXd cells. For return, WWOXd cells induces apoptosis of WWOXf cells from a remote distance. Survival of WWOXd from the cell-to-cell encounter is due to activation of the survival IκBα/ERK/WWOX signaling. Mechanistically, cell surface epitope WWOX286-299 (repl) in WWOXf repels the invading WWOXd to undergo retrograde migration. However, when epitope WWOX7-21 (gre) is exposed, WWOXf greets WWOXd to migrate forward for merge. WWOX binds membrane type II TGFβ receptor (TβRII), and TβRII IgG-pretreated WWOXf greet WWOXd to migrate forward and merge with each other. In contrast, TβRII IgG-pretreated WWOXd loses recognition by WWOXf, and WWOXf mediates apoptosis of WWOXd. The observatons suggest that normal cells can be activated to attack metastatic cancer cells. WWOXd cells are less efficient in generating Ca2+ influx and undergo non-apoptotic explosion in response to UV irradiation in room temperature. WWOXf cells exhibit bubbling cell death and Ca2+ influx effectively caused by UV or apoptotic stress. Together, membrane WWOX/TβRII complex is needed for cell-to-cell recognition, maintaining the efficacy of Ca2+ influx, and control of cell invasiveness.


File name: Supplementary Video 5
Description: UV/cold shock induces pop-out explosion death in L929R. L929R cells were exposed to UV 960 mJoule/cm 2 and subsequent cold shock at 4 o C for 5 min, and then subjected to imaging by time-lapse microscopy at room temperature. A picture was taken per 10 min.

File name: Supplementary Video 6
Description: Cell migration assay for glioblastoma U87-MG versus L929S cells. L929S (right chamber) and U87-MG (left chamber) were co-cultured in an insert (ibidi) using RPMI medium supplemented with 2% FBS, respectively. Time-lapse microscopy was then carried out at 37°C with 5% CO 2 . Each picture frame was taken per 10 minutes.

File name: Supplementary Video 7
Description: Cell migration assay for squamous cell carcinoma SCC9 versus SCC15 cells. SCC15 (right chamber) and SCC9 (left chamber) were co-cultured in an insert (ibidi) using DMEM-F12 medium supplemented with 2% FBS. Time-lapse microscopy was then carried out at 37°C with 5% CO 2 . Each picture frame was taken per 10 minutes.

File names: Supplementary Videos 8 and 9
Description: Sudden impact of MEF Wwox knockout by wild type cells leads to activation of the ectopic survival IkBa/ERK/WWOX signaling in the knockout cells. MEF Wwox knockout cells were transiently overexpressed with ECFP-IkBa, EGFP-ERK and DsRed-WWOX, and then cultured for 24 to 48 hr, followed by adding the wild type cells from suspension. Cell-to-cell impact induced the IkBa/ERK/WWOX signaling in the knockout cells (Video 8; shown as FRETc in artificial white color). Wild type cells were undergoing membrane blebbing and apoptosis (Video 9).

File names: Supplementary Videos 10 and 11
Description: The survival IkBa/ERK/WWOX signaling does not activate effectively in the MEF wild type cells upon sudden encountering with MEF Wwox knockout cells. MEF wild type cells were transiently overexpressed with ECFP-IkBa, EGFP-ERK and DsRed-WWOX and cultured for 24 to 48 hr, followed by adding the MEF Wwox knockout cells from suspension. Activation of the IkBa/ERK/WWOX signaling did not occur effectively in the wild type cells (Video 10; shown as FRETc). Apoptosis did not occurred in the wild type cells (Video 11).  Fig. 2a. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXf L929S cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 480 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 14
Description: Supplementary video for Suppl. Fig. 2a. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXd L929R cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 480 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 15
Description: Supplementary video for Suppl. Fig. 2b. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXf YMY normal human skin keratinocytes or epithelial cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 480 mJoule/cm 2 .The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.  Fig. 2b. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXd YMY neurofibromatosis NF1 skin cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 480 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 17
Description: Supplementary video for Suppl. Fig. 2c. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXf DU145 prostate cancer cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 480 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

Description: Supplementary video for Suppl. Fig. 2c. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells.
WWOXf MCF7 breast cancer cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml) and then treated with ceritinib (60 µM). The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 19
Description: Supplementary video for Suppl. Fig. 2c. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXf HCT116 colon cancer cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then treated with Na3VO4 (500 µM). The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 20
Description: Supplementary video for Suppl. Fig. 2c. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXf HCT116 colon cancer cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 720 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 22
Description: Supplementary video for Suppl. Fig. 2c. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXf SH-SY5Y cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 480 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 23
Description: Supplementary video for Suppl. Fig. 2c. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXf normal human skin cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 480 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 24
Description: Supplementary video for Suppl. Fig. 2c. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXf DU145 prostate cancer cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then treated with 0.035% H2O2. The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 25
Description: Supplementary video for Suppl. Fig. 2c. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXf mink lung Mv1Lu epithelial cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 480 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 26
Description: Supplementary video for Suppl. Fig. 2c. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXf NT2D1 cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 480 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

Description: Supplementary video for Suppl. Fig. 2c. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells.
WWOXf NT2D1 cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then treated with 0.035% H2O2. The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 29
Description: Supplementary video for Suppl. Fig. 2d. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXd B16F10 cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then treated with ceritinib (90 µM). The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 31
Description: Supplementary video for Suppl. Fig. 2d. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXd B16F10 cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 480 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 32
Description: Supplementary video for Suppl. Fig. 2d. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXd B16F10 cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then treated with an aliquot (10 µl) of a cocktail of proteinase inhibitor. The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 33
Description: Supplementary video for Suppl. Fig. 2d. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXd NB69 cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 480 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 34
Description: Supplementary video for Suppl. Fig. 2d. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXd U87-MG cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 480 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 35
Description: Supplementary video for Suppl. Fig. 2d. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXd L929R cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then treat with PMA (50 µM). The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 36
Description: Supplementary video for Suppl. Fig. 2d. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXd MDA-MB-231 cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 480 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 37
Description: Supplementary video for Suppl. Fig. 2d. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXd MDA-MB-435s cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 480 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 38
Description: Supplementary video for Suppl. Fig. 2d. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXd 4T1 cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 960 mJoule/cm 2 and then cols shock at 4 o C for 5 min. The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 39
Description: Supplementary video for Suppl. Fig. 2d. UV induces Ca2+ influx and BCD in WWOXf cells, but explosion in WWOXd cells. WWOXd L929R cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 720 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 40
Description: Supplementary video for Suppl. Fig. 5. Human tongue SCC4, 9, and 15 cells in migration, BCD and calcium influx. WWOXf SCC15 cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 960 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 41
Description: Supplementary video for Suppl. Fig. 5. Human tongue SCC4, 9, and 15 cells in migration, BCD and calcium influx. WWOXf SCC9 cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 960 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.

File name: Supplementary Video 42
Description: Supplementary video for Suppl. Fig. 5. Human tongue SCC4, 9, and 15 cells in migration, BCD and calcium influx. WWOXf SCC4 cells were stained with Fluo-8 (50 µM) and non-toxic levels of PI (2 µg/ml) and DAPI (10 µg/ml), and then exposed to UV 960 mJoule/cm 2 . The cells were subjected to time-lapse microscopy immediately at room temperature. 200x magnification. Each picture frame was taken per 2 minutes.