Microenvironmentally-driven Plasticity of CD44 isoform expression determines Engraftment and Stem-like Phenotype in CRC cell lines

Environmental conditions modulate CD133/CD44 phenotypes and tumorigenic potential of CRC subpopulations. The identification of fibroblasts as drivers of cancer-specific CD44 expression profile and plasticity sheds light on the limitation of per se dynamic surface antigens as biomarkers. It can also explain the location of putative CD133/CD44-positive CRC CSC in the perivascular niche, which is likely to comprise cancer-associated fibroblasts. The LS1034 in vitro/in vivo model is a valuable tool to unravel the mechanism of stromal-induced CD44v8-10 expression and identify further therapeutically relevant, mutual interrelations between microenvironment and tumorigenic phenotype.

CD133/CD44 profiles of 20 CRC cell lines were monitored; three models with distinct surface patterns in vitro were systematically examined. CD133/CD44 subpopulations were isolated by FACS and analyzed upon in vitro growth and/or in limiting dilution engraftment studies. The experimental setup included biomarker analyses on the protein (flow cytometry, Western blotting, immunofluorescence) and mRNA levels (RT-/qPCR) as well as CD44 gene sequencing.

In general, we found that (i) the in vitro CD133/CD44 pattern never determined engraftment and (ii) the CD133/CD44 population distributions harmonized under in vivo conditions. The LS1034 cell line appeared as a unique model due to its de novo in vivo presentation of CD44. CD44v8-10 was identified as main transcript, which was stronger expressed in primary human CRC than in normal colon tissues. Biomarker pattern of LS1034 cells in vivo reflected secondary engraftment: the tumorigenic potential was highest in CD133+/CD44+, intermediate in CD133+/CD44- and entirely lost in CD133-/CD44- subfractions. Both CD44+ and CD44- LS1034 cells gave rise to tumorigenic and non-tumorigenic progeny and were convertible - but only as long as they expressed CD133 in vivo. The highly tumorigenic CD133+/CD44(v8-10)+ LS1034 cells were localized in well-oxygenated perivascular but not hypoxic regions. From a multitude of putative modulators, only the direct interaction with stromal fibroblasts triggered an essential, in vivo-like enhancement of CD44v8-10 presentation in vitro.