Theranostic biomarkers for putative cancer stem-like cells (CSC) in colorectal cancer (CRC) are of particular interest in translational research to develop patient-individualized treatment strategies. Surface proteins still under debate are CD44 and CD133. The structural and functional diversity of these antigens, as well as their plasticity, has only just begun to be understood. Our study aimed to gain novel insight into the plasticity of CD133/CD44, thereby proving the hypothesis of marker-associated tumorigenic and non-tumorigenic phenotypes to be environmentally driven./nMethods: 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./nResults: 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./nConclusion: 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.

中文翻译：

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CD44亚型表达的微环境驱动可塑性决定了CRC细胞系中的移入和茎样表型。

大肠癌（CRC）中假定的癌干样细胞（CSC）的治疗诊断生物标志物在转化研究中特别感兴趣，以开发患者个性化的治疗策略。仍在争论的表面蛋白是CD44和CD133。这些抗原的结构和功能多样性以及可塑性才刚刚开始被理解。我们的研究旨在获得对CD133 / CD44可塑性的新颖见解，从而证明环境相关的标志物相关致瘤和非致瘤表型的假说。/n方法：监测20个CRC细胞系的CD133 / CD44谱；三种模式具有不同的表面图案在体外被系统地检查了。通过FACS分离CD133 / CD44亚群，并在体外生长和/或有限稀释度植入研究中进行分析。实验设置包括对蛋白质的生物标记分析（流式细胞仪，蛋白质印迹，免疫荧光）和mRNA水平（RT- / qPCR）以及CD44基因测序。/n结果：总的来说，我们发现（i）在体外CD133 / CD44模式从未确定植入，并且（ii）在体内条件下， CD133 / CD44群体分布是一致的。LS1034细胞系由于其从头体内CD44表达而表现出独特的模型。CD44v8-10被鉴定为主要转录物，其在原代人CRC中的表达强于正常结肠组织。LS1034细胞的生物标志物模式的体内反射的次级移植：致瘤潜力在CD133最高⁺ / CD44 ⁺，中间在CD133 ⁺ / CD44 ^-和完全失去了在CD133 ^- / CD44 ^-亚。CD44 ⁺和CD44 ^- LS1034细胞均可产生致瘤和非致瘤后代，并且可转化-但只要它们在体内表达CD133 。高致癌性CD133 ⁺ / CD44（v8-10）⁺LS1034细胞位于充氧的血管周围，而不是缺氧区域。从假定的调制器的群众，只有用基质成纤维细胞的直接相互作用引发的一个重要，体内般增强CD44v8-10呈现的体外./n结论：环境条件调节CD133 / CD44表型与CRC亚群的致瘤性。将成纤维细胞鉴定为癌症特异性CD44表达谱和可塑性的驱动器，这揭示了本身作为生物标志物的动态表面抗原的局限性。它也可以解释假定的CD133 / CD44阳性CRC CSC在血管周位中的位置，该位点可能包含与癌症相关的成纤维细胞。LS1034体外/体内 该模型是揭示基质诱导的CD44v8-10表达机制并鉴定微环境与致瘤表型之间进一步治疗相关的相互关系的有价值的工具。