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Human Platt neuroblastoma cells were transfected with the marker gene, bacterial lacZ, to track cells at the earliest stages after ectopic injection at two different sites in athymic nude mice. Three clones (LZPt-1,-2 and -3) of differing morphologies were analysed. All clones yielded large primary tumours subcutaneously or intradermally with similar latency. While LZPt-2 and -3 clones generated well-staining primary tumours, LZPt-1 cells yielded many non-staining tumours, indicating greater instability of lacZ expression for this clone in situ (stability of lacZ expression in culture was similar for all three clones). After s.c. or intradermal injections, tumour cells were tracked for 1 h to &gt; 3 weeks (palpable) to evaluate the topology and population expansion characteristics at the earliest times. From 1 h to 2 days, tumour cells were concentrated in central masses with 'crinkly hair' distributions emanating from the periphery. Between 3 and 7 days, these 'crinkly hair' patterns were cleared from the tissue, leaving dense ovoid patterns of tumour cells. These concentrations of cells expanded collectively, not by division of one or a few cells, but by division of many cells. For clone LZPt-1, cells stained well with X-gal for 2-3 days; by 7 days, most cells were non-staining. Evidence suggests that lacZ expression is turned off in these tumour cells, rather than a lacZ- cell type clonally dominating the population. For all three clones, tumour cells remained rounded and did not spread in any tissue environment at all time points, indicating very different matrix adhesion mechanisms operating in situ compared with their distinctive spreading patterns in culture. Angioneogenesis near primary tumours became evident by 2-3 days, leading to extensive vascularisation by 1-2 weeks. Overall, these studies indicate common tumour progression characteristics for three different clones of human neuroblastoma, insight into lacZ instability mechanisms operating in one of these clones and the earliest events in primary tumour formation for this tumour at two different ectopic sites.

Tumour progression of human neuroblastoma cells tagged with a lacZ marker gene: earliest events at ectopic injection sites