A Computer-Assisted 3D Model for Analyzing the Aggregation of Tumorigenic Cells Reveals Specialized Behaviors and Unique Cell Types that Facilitate Aggregate Coalescence

Abstract

We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, 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. Images are converted to mathematical representations from which quantitative parameters can be 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. We report here that cells from tumorigenic lines and tumors undergo rapid coalescence in 3D, mediated by specific cell types that we have named “facilitators” and “probes.” A third cell type, the “dervish”, is capable of rapid movement through the gel and does not adhere to it. These cell types have never before been described. Our data 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.

Melanoma cells undergo aggressive coalescence in a 3D Matrigel model that is repressed by anti-CD44

“…Attachment of facilitators to probes or between probes of different aggregates initiates additional cells to extend along the attachment to form a multicellular cable that then contracts, always pulling the smaller aggregate into the larger one [19]. A similar mechanism was observed in the mediation of aggregate-aggregate coalescence in 3D melanoma cell preparations.…”

“…In this model, non-tumorigenic cell lines and fresh breast tissue cell culture did not undergo coalescence [18, 19]. In the process of coalescence in these preparations, single cells in Matrigel grew into clonal islands during a precoalescence period.…”

“…Eventually nests can spread horizontally along the skin, as well as vertically towards the dermis [40, 41]. These observations suggest that aggregate coalescence and the mediation of coalescence between aggregates by cables of specialized cells, which we demonstrated in 3D Matrigel models of breast tumor-derived cell lines in vitro [18, 19], may indeed occur in the progression of melanomas in vivo . Moreover, there are reasons to believe that melanoma cells may be more aggressive in behavior than breast cancer cells in the process of metastasis.…”

“…It is demonstrated that melanoma cells from fresh tumors or an established melanoma cell line indeed undergo aggregate coalescence and that coalescence of aggregates is mediated by the formation of cell bridges formed by behaviorally distinct cells, as is the case for tumorigenic breast cancer-derived cell bridges [18, 19]. However, the results also demonstrate behavioral characteristics specific to melanoma cells, both fresh cells from tumors secondary melanoma cell cultures and an established melanoma cell line.…”

Evaluation by quantitative image analysis of anticancer drug activity on multicellular spheroids grown in 3D matrices

“…Powerful methods for reconstructing and live 3D in vivo analysis of the process of cancer cell aggregation and growth have already been documented, however, they rely on the use of sophisticated microscopy and/or image processing tools (14,15). The approach reported in the present study aims to provide a simple, robust pipeline to do so based on technologies that are readily available to many investigators.…”