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 TGFß receptor (TßRII), and TßRII IgG-pretreated WWOXf greet WWOXd to migrate forward and merge with each other. In contrast, TßRII 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/TßRII complex is needed for cell-to-cell recognition, maintaining the efficacy of Ca2+ influx, and control of cell invasiveness.

Strategies by which WWOX-deficient metastatic cancer cells utilize to survive via dodging, compromising, and causing damage to WWOX-positive normal microenvironment.

Proapoptotic tumor suppressor WWOX is upregulated in the early stage of cancer initiation, which probably provides limitation to cancer growth and progression. Later, WWOX protein is reduced to enhance cancer cell growth, migration, invasiveness and metastasis. To understand how WWOX works in controlling cancer progression, here we demonstrate that apoptotic stress mediated by ectopic WWOX stimulated cancer cells to secrete basic fibroblast growth factor (bFGF) in order to support capillary microtubule formation. This event may occur in the cancer initiation stage. Later, when WWOX loss occurs in cancer cells, hyaluronidase production is then increased in the cancer cells to facilitate metastasis. We determined that inhibition of membrane hyaluronidase Tyr216-phosphorylated Hyal-2 by antibody suppresses cancer growth in vivo. WWOX-negative (WWOX-) cells dodged WWOX+cells in the microenvironment by migrating individually backward to avoid physical contacts and yet significantly upregulating the redox activity of WWOX+parental cells or other WWOX+cell types for causing apoptosis. Upon detecting the presence of WWOX+cells from a distance, WWOX- cells exhibit activation of MIF, Hyal-2, Eph, and Wnt pathways, which converges to MEK/ERK signaling and enables WWOX- cells to evade WWOX+cells. Inhibition of each pathway by antibody or specific chemicals enables WWOX- cells to merge with WWOX+cells. In addition, exogenous TGF-ß assists WWOX- cells to migrate collectively forward and merge with WWOX+cells. Metastatic WWOX- cancer cells frequently secrete high levels of TGF-ß, which conceivably assists them to merge with WWOX+cells in target organs and secure a new home base in the WWOX+microenvironment. Together, loss of WWOX allows cancer cells to develop strategies to dodge, compromise and even kill WWOX-positive cells in microenvironment.

Fast and improved bioimaging via temporal focusing multiphoton excitation microscopy with binary digital-micromirror-device holography.

Conventional temporal focusing-based multiphoton excitation microscopy (TFMPEM) can offer widefield optical sectioning with an axial excitation confinement of a few microns. To improve the axial confinement of TFMPEM, a binary computer-generated Fourier hologram (CGFH) via a digital-micromirror-device (DMD) was implemented to intrinsically improve the axial confinement by filling the back-focal aperture of the objective lens. Experimental results show that the excitation focal volume can be condensed and the axial confinement improved about 24% according to the DMD holography. In addition, pseudouniform MPE can be achieved using two complementary CGFHs with rapid pulse-width modulation switching via the DMD. Furthermore, bioimaging of CV-1 in origin with SV40 genes-7 cells demonstrates that the TFMPEM with binary DMD holography can improve image quality by enhancing axial excitation confinement and rejecting out-of-focus excitation.

Temporal focusing-based widefield multiphoton microscopy with spatially modulated illumination for biotissue imaging.

A developed temporal focusing-based multiphoton excitation microscope (TFMPEM) has a digital micromirror device (DMD) which is adopted not only as a blazed grating for light spatial dispersion but also for patterned illumination simultaneously. Herein, the TFMPEM has been extended to implement spatially modulated illumination at structured frequency and orientation to increase the beam coverage at the back-focal aperture of the objective lens. The axial excitation confinement (AEC) of TFMPEM can be condensed from 3.0 µm to 1.5 µm for a 50 % improvement. By using the TFMPEM with HiLo technique as two structured illuminations at the same spatial frequency but different orientation, reconstructed biotissue images according to the condensed AEC structured illumination are shown obviously superior in contrast and better scattering suppression. Picture: TPEF images of the eosin-stained mouse cerebellar cortex by conventional TFMPEM (left), and the TFMPEM with HiLo technique as 1.09 µm-1 spatially modulated illumination at 90° (center) and 0° (right) orientations.

Temporal focusing-based multiphoton excitation microscopy via digital micromirror device.

This Letter presents an enhanced temporal focusing-based multiphoton excitation (MPE) microscope in which the conventional diffraction grating is replaced by a digital micromirror device (DMD). Experimental results from imaging a thin fluorescence film show that the 4.0 µm axial resolution of the microscope is comparable with that of a setup incorporating a 600 lines/mm grating; hence, the optical sectioning ability of the proposed setup is demonstrated. Similar to a grating, the DMD diffracts illuminating light frequencies for temporal focusing; additionally, it generates arbitrary patterns. Since the DMD is placed on the image-conjugate plane of the objective lens' focal plane, the MPE pattern can be projected on the focal plane precisely.