3 2D workstations housed in a 37°C, 5% CO2 incubators with image acquisition controlled by Fire-I software. 

We developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.2, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix.  The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity), and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 µm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix.  Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer.  J3D-DIAS 4.2 converts images to mathematical representations from which quantitative parameters are then derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. Our data on breast cancer cell lines suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.
Because histological sections of melanomas in patient biopsies suggest that melanoma cells migrate and coalesce to form tumors, we tested whether melanoma cells also underwent coalescence in a 3D Matrigel model. Using the J3D-DIAS 4.2 motion analysis system, we discovered that melanoma cells also underwent coalescence mediated by specialized cells in the 3D model. We then developed a screen of purified monoclonal antibodies (mAbs) from the Developmental Studies Hybridoma Bank (DSHB; http://dshb.biology.uiowa.edu/) collection. The DSHB is housed here in the Biology Department under the direction of David R. Soll. We selected mAbs against cell surface-associated molecules and identified two mAbs that blocked melanoma cell coalescence. These mAbs also blocked coalescence of tumorigenic cells derived from a breast tumor. These results add weight to the commonality of coalescence as a characteristic of tumorigenic cells, as well as the usefulness of the 3D Matrigel model and software for both elucidating the mechanisms regulating tumorigenesis and screening for potential anti-tumorigenesis mAbs.