Optimization-based wavefront sensorless adaptive optics for multiphoton microscopy.

Optical aberrations have detrimental effects in multiphoton microscopy. These effects can be curtailed by implementing model-based wavefront sensorless adaptive optics, which only requires the addition of a wavefront shaping device, such as a deformable mirror (DM) to an existing microscope. The aberration correction is achieved by maximizing a suitable image quality metric. We implement a model-based aberration correction algorithm in a second-harmonic microscope. The tip, tilt, and defocus aberrations are removed from the basis functions used for the control of the DM, as these aberrations induce distortions in the acquired images. We compute the parameters of a quadratic polynomial that is used to model the image quality metric directly from experimental input-output measurements. Finally, we apply the aberration correction by maximizing the image quality metric using the least-squares estimate of the unknown aberration.

ß1 integrin inhibition elicits a prometastatic switch through the TGFß-miR-200-ZEB network in E-cadherin-positive triple-negative breast cancer.

Interactions with the extracellular matrix (ECM) through integrin adhesion receptors provide cancer cells with physical and chemical cues that act together with growth factors to support survival and proliferation. Antagonists that target integrins containing the ß1 subunit inhibit tumor growth and sensitize cells to irradiation or cytotoxic chemotherapy in preclinical breast cancer models and are under clinical investigation. We found that the loss of ß1 integrins attenuated breast tumor growth but markedly enhanced tumor cell dissemination to the lungs. When cultured in three-dimensional ECM scaffolds, antibodies that blocked ß1 integrin function or knockdown of ß1 switched the migratory behavior of human and mouse E-cadherin-positive triple-negative breast cancer (TNBC) cells from collective to single cell movement. This switch involved activation of the transforming growth factor-ß (TGFß) signaling network that led to a shift in the balance between miR-200 microRNAs and the transcription factor zinc finger E-box-binding homeobox 2 (ZEB2), resulting in suppressed transcription of the gene encoding E-cadherin. Reducing the abundance of a TGFß receptor, restoring the ZEB/miR-200 balance, or increasing the abundance of E-cadherin reestablished cohesion in ß1 integrin-deficient cells and reduced dissemination to the lungs without affecting growth of the primary tumor. These findings reveal that ß1 integrins control a signaling network that promotes an epithelial phenotype and suppresses dissemination and indicate that targeting ß1 integrins may have undesirable effects in TNBC.

Automated microinjection of cell-polymer suspensions in 3D ECM scaffolds for high-throughput quantitative cancer invasion screens.

Cell spheroids (CS) embedded in 3D extracellular matrix (ECM) serve as in vitro mimics for multicellular structures in vivo. Such cultures, started either from spontaneous cell aggregates or single cells dispersed in a gel are time consuming, applicable to restricted cell types only, prone to high variation, and do not allow CS formation with defined spatial distribution required for high-throughput imaging. Here, we describe a method where cell-polymer suspensions are microinjected as droplets into collagen gels and CS formation occurs within hours for a broad range of cell types. We have automated this method to produce CS arrays in fixed patterns with defined x-y-z spatial coordinates in 96 well plates and applied automated imaging and image analysis algorithms. Low intra- and inter-well variation of initial CS size and CS expansion indicates excellent reproducibility. Distinct cell migration patterns, including cohesive strand-like - and individual cell migration can be visualized and manipulated. A proof-of-principle chemical screen is performed identifying compounds that affect cancer cell invasion/migration. Finally, we demonstrate applicability to freshly isolated mouse breast and human sarcoma biopsy material - indicating potential for development of personalized cancer treatment strategies.

The regulation of MacMARCKS expression by integrin beta3.

Integrin-mediated adhesion regulates multiple signaling pathways. Our group previously showed that ectopic expression of different integrin beta-subunits in the neuroepithelial cell line GE11, has distinct effects on cell morphology, actin cytoskeletal organization, and on focal contact distribution. In this report we have investigated changes in gene transcription levels resulting from overexpression of the integrin beta3 subunit. We found that beta3 overexpression leads to the transcriptional downregulation of MARCKS related protein (MRP) resulting in a decreased expression of the MRP protein. Furthermore, we show that the Ras/MAPK pathway controls the basal level of MRP expression but beta3 overexpression bypasses this pathway downstream of ERK to downregulate MRP. Further studies indicate that a region of the cytoplasmic tail of beta3 containing part of the NITY motif is responsible for increased cell spreading and MRP downregulation. However, MRP overexpression failed to inhibit the beta3-induced increase in cell spreading while the knock down of MRP expression in GE11 cells did not increase cell spreading. We suggest that the downregulation of MRP by beta3 is not required for increased cell spreading but instead that MRP downregulation is a secondary effect of increased cell spreading.