Fig. 3: Characterisation of the new in vivo traceable non-melanogenic murine melanoma cell line 4599.NC.
From: A preclinical pipeline to evaluate migrastatics as therapeutic agents in metastatic melanoma
a/top Sketch of the lentiviral radionuclide-fluorescence fusion reporter gene construct. a/bottom Immunoblot analysis of lentivirally transduced and sorted 4599.NC cells compared to parental 4599 cells. b Confocal micrographs showing fusion reporter expression and overlap with the plasma membrane stain wheat germ agglutinin (WGA) conjugated to Alexa488. Representative cells are shown; scale bar = 10 µm. c Radionuclide reporter function as quantified by uptake of the radioactive NIS substrate [99mTc]TcO4−. ‘Ref’ indicates a fusion reporter reference cell line as previously described.26 Specificity of uptake was demonstrated by abolished radiotracer uptake in the presence of the competitive substrate perchlorate; error bars are SD, N = 3. d 4599.NC cells were intradermally administered to 5-week-old male NSG mice to establish orthotopic tumours (N = 4 animals). Three weeks post administration animals were imaged by [99mTc]TcO4−-afforded NIS-SPECT/CT clearly indicating cancerous tissues (primary tumour: solid arrow; metastases: dashed arrows) alongside signals stemming from organs expressing NIS endogenously (thyroid and salivary glands (T/G), lachrymal glands (L), stomach (S), and lower in intestine (I) and testes (Te)); none of the latter interfered with the primary tumour or metastases in this model. To assess NIS specificity in vivo, animals received the NIS co-substrate perchlorate intraperitoneally 40 min before animals were re-imaged (48 h after the first imaging session); remaining signals in kidney and bladder (B) reflect radiotracer excretion routes. For corresponding tumour growth curves and ex vivo γ-counting results see Fig. S2. For growth comparison with tumours established from parental 4599 cells see Fig. S2b. e Harvested tumour tissues presented with red fluorescence stemming from reporter expression, which not only guided dissection, but enabled histological assessment of tumour tissues. A typical confocal micrograph of one animal from a cohort of N = 4 is shown; scale bar = 25 µm. f Hematoxilin and eosin staining and (g) phospho-MLC2 immunohistochemistry of adjacent tumour sections from the same tumour as in (e/f); in (f/g) the red dashed line indicated the tumour front while large blood vessels are encircled with purple dots; scale bars = 200 µm.
