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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., Yu-An Chen et al characterize the role of cell surface epitopes of the tumor suppressor protein WWOX in cell–cell recognition. The find that cells expressing functional WWOX, repel cancer cells which express dysfunctional WWOX or lack this protein, in a manner mediated by WWOX epitope 286-299.

High-sensitivity hypothesis-free subcellular proteomics is challenging due to the limited sensitivity of mass spectrometry and the lack of amplification tools for proteins. Without such technology, it is not possible to discover proteins at specific locations of interest in cells or tissue samples. Here, we introduce a total-sync ultra-content microscopic system termed MicroscoopTM that integrates microscopy, optics, FPGA-based mechatronics, photochemistry, and deep learning or computer vision to enable high-content in situ photolabeling. MicroscoopTM photolabels proteins at user defined regions of interests (ROIs) under a microscope utilizing directed photochemistry in one field of view (FOV) at a time for tens of thousands of FOVs with similar morphological features. With this platform, we are able to photolabel proteins with biotin probes in cellular organelles, granules or cell-cell contact surfaces with a high precision at nanoscale resolution, and obtain sufficient amount of biotinylated proteins for mass spectrometry. We made a robust demonstration in the proteome mapping of human cellular nucleus from single-shot experiment to >1000 nuclear protein identification with > 90% specificity. Further data analysis revealed identification of a hundred of low protein copy number proteins and a high coverage of nuclear complexes. In proteome mapping of the nucleolus, we ranked proteins by order of abundance and revealed that 97 out of the top 100 proteins were annotated as nucleolar proteins. Unexpectedly, in mapping the stress granule (SG) proteome, a relatively low SG specificity (74%) were found in the top 50 abundant proteins, therefore we further characterize the proteins that have no prior SG annotation by immunostaining. Nine out of the thirteen unexplored proteins including PDLIM7, EIF3CL, YWHAE, RPSA, UGDH, DDX17, ANLN, PSMA6, and MCM2 were found to have SG patterns and co-localized with SG marker (G3BP1), raising our top 50 SG specificity to up to 92%. Together, our total-sync ultra-content microscopic platform enables hypothesis-free, de novo subcellular proteome mapping at user defined ROIs with high sensitivity and specificity, thereby broadly benefits the cell biology field in finding novel proteins or biomarkers. Citation Format: Jung-Chi Liao, Chih-Wei Chang, Yi-De Chen, Chantal Hoi Yin Cheung, Chia-Wen Chung, Hsiao-Jen Chang, Yong-Da Sie, You-Pi Liu, Yu-Chih Lin, Hsiang-Ju Kai, Weng Man Chong, Hsin-Yi Wu. Total-sync ultra-content microscopic opto-biotinylation enables high-sensitivity hypothesis-free subcellular protein discovery. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5638.

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-β assists WWOX- cells to migrate collectively forward and merge with WWOX+cells. Metastatic WWOX- cancer cells frequently secrete high levels of TGF-β, 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.

Rationale: Conventional 3-dimensional (3D) printing techniques cannot produce structures of the size at which individual cells interact. Objective: Here, we used multiphoton-excited 3D printing to generate a native-like extracellular matrix scaffold with submicron resolution and then seeded the scaffold with cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human-induced pluripotent stem cells to generate a human-induced pluripotent stem cell–derived cardiac muscle patch (hCMP), which was subsequently evaluated in a murine model of myocardial infarction. Methods and Results: The scaffold was seeded with ≈50 000 human-induced pluripotent stem cell–derived cardiomyocytes, smooth muscle cells, and endothelial cells (in a 2:1:1 ratio) to generate the hCMP, which began generating calcium transients and beating synchronously within 1 day of seeding; the speeds of contraction and relaxation and the peak amplitudes of the calcium transients increased significantly over the next 7 days. When tested in mice with surgically induced myocardial infarction, measurements of cardiac function, infarct size, apoptosis, both vascular and arteriole density, and cell proliferation at week 4 after treatment were significantly better in animals treated with the hCMPs than in animals treated with cell-free scaffolds, and the rate of cell engraftment in hCMP-treated animals was 24.5% at week 1 and 11.2% at week 4. Conclusions: Thus, the novel multiphoton-excited 3D printing technique produces extracellular matrix–based scaffolds with exceptional resolution and fidelity, and hCMPs fabricated with these scaffolds may significantly improve recovery from ischemic myocardial injury.

In this study, we implement temporal-focusing multiphoton selective excitation (TFMPSE) to light field microscopy (LFM), illuminating only the volume of interest, thus significantly reducing the background noise and providing higher contrast and accuracy for the light field image reconstruction; furthermore, offering higher penetration depth in scattering tissue via multiphoton. 3D human-skin in situ immunofluorescence images are used to demonstrate volumetric bioimaging capability. The volume rate of the TFMPSE-LFM can achieve around 100 volumes per second

Light field technique at a single shot can get the whole volume image of observed sample. Therefore, the original frame rate of the optical system can be taken as the volumetric image rate. For dynamically imaging whole micron-scale biosample, a light field microscope with temporal focusing illumination has been developed. In the light field microscope, the f-number of the microlens array (MLA) is adopted to match that of the objective; hence, the subimages via adjacent lenslets do not overlay each other. A three-dimensional (3D) deconvolution algorithm is utilized to deblur the out-of-focusing part. Conventional light field microscopy (LFM) illuminates whole volume sample even noninteresting parts; nevertheless, whole volume excitation causes even more damage on bio-sample and also increase the background noise from the out of range. Therefore, temporal focusing is integrated into the light field microscope for selecting the illumination volume. Herein, a slit on the back focal plane of the objective is utilized to control the axial excitation confinement for selecting the illumination volume. As a result, the developed light field microscope with the temporal focusing multiphoton illumination (TFMPI) can reconstruct 3D images within the selected volume, and the lateral resolution approaches to the theoretical value. Furthermore, the 3D Brownian motion of two-micron fluorescent beads is observed as the criterion of dynamic sample. With superior signal-to-noise ratio and less damage to tissue, the microscope is potential to provide volumetric imaging for vivo sample.

Observing dynamic micro-scale phenomena occurring at millisecond time scales, such as organism activity, micron particle flows, or any opaque object observation, requires volumetric microscopy techniques able to achieve high data acquisition rates while maintaining contrast so that measurement of fine micro-scale features is possible. In realizing this purpose, the light-field (LF) technique has already been used on three-dimensional (3D) scene capturing and even for microscopic visualizations. In studying the ability and feasibility of 3D surface morphology reconstruction via LF microscopy, we adopted a lab-made LF microscope and integrated a four-dimensional Fourier slice algorithm and a Markov random field propagation algorithm. Furthermore, for numerical comparison and quantized analysis, the Tenengrad function was utilized to calculate the average contrast of the region of interest. Reflective US Air Force targets and 3D photolithography-made micro-scaffolds coated with 50 nm nickel thin films were adopted for system alignment and calibration. The experimental results demonstrate that the developed LF microscope with the signal processing algorithms can observe the 3D surface morphology of opaque microstructures with one snapshot, and has been preliminary applied to Brownian motion observation with 30 Hz volumetric image rate.

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.

Malignant breast cancer MDA-MB-231 cells are highly metastatic and their cell-cell contact is fairly loose. Accordingly, they migrate individually. Upon stably expressing with ERK, the MDA-MB-231 cells lost individual migration and underwent migration in a collective manner. No apparent loss of speed was observed due to ectopic ERK. Suppression of ERK phosphorylation by U0126 abolished ERK-induced collective migration. Similarly, dominant negative ERK with K72R mutation failed to induce collective migration, indicating that ERK phosphorylation is needed for its induction of collective migration. MDA-MB-231 is a WWOX-negative cell line due to epigenetic modification in the promoter. Ectopic ERK induced the expression of endogenous WWOX. Stimulation of cells with antibody against Ser14 phosphorylation in WWOX abolished the ERK-induced collective migration. In parallel, transiently overexpressed WWOX increased cell-to-cell adhesion and their collective migration. These observations suggest that ERK-induced c...

In this study, three-dimensional (3D) multi-component microstructures were precisely fabricated via multiphoton excited photochemistry using a femtosecond laser direct-writing system with proposed repetition positioning and vector scanning techniques. Extracellular matrix (ECM) proteins, such as fibronectin (FN), are difficult to stack and form 3D structures larger than several-hundred microns in height due to the nature of their protein structure. Herein, to fabricate complex 3D microstructures with FN, a 3D scaffold was designed and formed from bovine serum albumin (BSA), after which human FN was inserted at specific locations on the BSA scaffold; in this manner, the fabricated ECM microstructure can guide cells in a 3D environment. A human breast cancer cell line, MDA-MB-231, was used to investigate the behavior of cell migration and adhesion on the fabricated human FN and BSA protein structures. Experimental results indicate that many cells are not able to attach or climb on a 3D structure’s inclined plane without FN support; hence, the influence of cell growth in a 3D context with FN should being taken into consideration. This 3D multi-protein fabrication technique holds potential for cell studies in designed complex 3D ECM scaffolds.

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.

In this study, the light diffraction of temporal focusing multiphoton excitation microscopy (TFMPEM) and the excitation patterning of nonlinear structured-illumination microscopy (NSIM) can be simultaneously and accurately implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TFMPEM are remarkably improved through the second-order NSIM and projected structured light, respectively. The experimental results demonstrate that the lateral and axial resolutions are enhanced from 397 nm to 168 nm (2.4-fold) and from 2.33 μm to 1.22 μm (1.9-fold), respectively, in full width at the half maximum. Furthermore, a three-dimensionally rendered image of a cytoskeleton cell featuring ~25 nm microtubules is improved, with other microtubules at a distance near the lateral resolution of 168 nm also able to be distinguished.

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.