Using unique computer-assisted 3D reconstruction software, it was previously demonstrated that tumorigenic cell lines derived from breast tumors, when seeded in a 3D Matrigel model, grew as clonal aggregates which, after approximately 100 hours, underwent coalescence mediated by specialized cells, eventually forming a highly structured large spheroid. Non-tumorigenic cells did not undergo coalescence. Because histological sections of melanomas forming in patients suggest that melanoma cells migrate and coalesce to form tumors, we tested whether they also underwent coalescence in a 3D Matrigel model. Melanoma cells exiting fragments of three independent melanomas or from secondary cultures derived from them, and cells from the melanoma line HTB-66, all underwent coalescence mediated by specialized cells in the 3D model. Normal melanocytes did not. However, coalescence of melanoma cells differed from that of breast-derived tumorigenic cell lines in that they 1) coalesced immediately, 2) underwent coalescence as individual cells as well as aggregates, 3) underwent coalescence far faster and 4) ultimately formed long, flat, fenestrated aggregates that were extremely dynamic. A screen of 51 purified monoclonal antibodies (mAbs) targeting cell surface-associated molecules revealed that two mAbs, anti-beta 1 integrin/(CD29) and anti-CD44, blocked melanoma cell coalescence. They 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 investigating the mechanisms regulating tumorigenesis and screening for potential anti-tumorigenesis mAbs.

One possible approach to normalize mutant cells that are metastatic and tumorigenic, is to upregulate a functionally similar homolog of the mutated gene. Here we have explored this hypothesis by generating an overexpressor of TPTE2 (TPIP), a homolog of PTEN, in PTEN−/− mutants, the latter generated by targeted mutagenesis of a human epithelial cell line. Overexpression of TPTE2 normalized phenotypic changes associated with the PTEN mutation. The PTEN−/−-associated changes rescued by overexpressing TPTE2 included 1) accelerated wound healing in the presence or absence of added growth factors (GFs), 2) increased division rates on a 2D substrate in the presence of GFs, 3) adhesion and viability on a 2D substrate in the absence of GFs, 4) viability in a 3D Matrigel model in the absence of GFs and substrate adhesion 5) loss of apoptosis-associated annexin V cell surface binding sites. The results justify further exploration into the possibility that upregulating TPTE2 by a drug may reverse metastatic and tumorigenic phenotypes mediated in part by a mutation in PTEN. This strategy may also be applicable to other tumorigenic mutations in which a homolog to the mutated gene is present and can substitute functionally.