Suppression and Activation of the Malignant Phenotype by Extracellular Matrix in Xenograft Models of Bladder Cancer: A Model for Tumor Cell Dormancy. Homo sapiens

A major problem in cancer research is the lack of a tractable model for delayed metastasis. Herein we show that cancer cells suppressed by SISgel, a gel-forming normal ECM material derived from Small Intestine Submucosa (SIS), in flank xenografts show properties of suppression and re-activation that are very similar to normal delayed metastasis and suggest these suppressed cells can serve as a novel model for developing therapeutics to target micrometastases or suppressed cancer cells. Co-injection with SISgel suppressed the malignant phenotype of highly invasive J82 bladder cancer cells and highly metastatic JB-V bladder cancer cells in nude mouse flank xenografts. Cells could remain viable up to 120 days without forming tumors and appeared much more highly differentiated and less atypical than tumors from cells co-injected with Matrigel. In 40% of SISgel xenografts, growth resumed in the malignant phenotype after a period of suppression or dormancy for at least 30 days and was more likely with implantation of 3 million or more cells. Ordinary Type I collagen did not suppress malignant growth, and tumors developed about as well with collagen as with Matrigel. A clear signal in gene expression over different cell lines was not seen by transcriptome microarray analysis, but in contrast, Reverse Phase Protein Analysis of 250 proteins across 4 cell lines identified Integrin Linked Kinase (ILK) signaling that was functionally confirmed by an ILK inhibitor. We suggest that cancer cells suppressed on SISgel could serve as a model for dormancy and re-awakening to allow for the identification of therapeutic targets for treating micrometastases. Overall design: Earlier we demonstrated that the phenotype of bladder cancer cells was radically different in 3-dimensional organotypic culture when grown on a normal extracellular matrix preparation (SISgel) as compared to that observed on a cancer-modulated permissive extracellular matrix preparation (Matrigel). SISgel is a gel-forming material derived from acellular small intestine submucosa, whereas Matrigel is a basement membrane preparation obtained from a mouse sarcoma. On Matrigel the bladder cancer cells recapitulated the phenotype reported for the original tumor; in sharp contrast, most of the malignant properties were lost when the cells were grown on SISgel. Cell lines derived from papillomas formed a layered structure reminiscent of normal urothelium, whereas cell lines derived from higher grade tumors formed a noninvasive layer of cells. These findings suggested that growth of cancer cells on normal ECM could provide a model to investigate the phenomenon of suppression of malignancy by normal ECM in metastasis and recurrence. In this study we explored whether the phenotypic suppression seen in organotypic culture of bladder cancer cells on SISgel also is observed in vivo. Positive findings support the use of SISgel as a model for investigations of the dormant or suppressed tumor cell phenotype and of mechanisms by which the normal ECM exerts an inhibitory influence on tumorigenesis and metastasis. The findings strongly suggest that interactions of cancer cells with normal ECM play an important role in recurrence and metastasis and further suggest that targeting suppressed cells could represent a heretofore unexploited point of vulnerability in cancer therapy.

癌症研究领域的一大核心难题，在于缺乏可用于研究延迟性转移的易处理模型。本研究证实，在裸鼠肋腹部皮下异种移植瘤模型中，经小肠黏膜下层（Small Intestine Submucosa, SIS）来源的成胶状正常细胞外基质（extracellular matrix, ECM）材料SISgel处理的癌细胞，其增殖抑制与重新激活的特性与典型的延迟性转移高度相似；我们提出，此类受抑制的癌细胞可作为一种新型模型，用于开发靶向微转移灶或休眠癌细胞的治疗手段。将SISgel与癌细胞共注射，可显著抑制高侵袭性J82膀胱癌细胞与高转移性JB-V膀胱癌细胞在裸鼠肋腹部皮下异种移植瘤中的恶性表型。此类癌细胞可存活长达120天而不形成肿瘤，且与共注射基质胶（Matrigel）的肿瘤相比，其细胞分化程度更高、异型性更低。在40%的SISgel异种移植瘤中，经过至少30天的抑制或休眠期后，肿瘤会重新恢复恶性表型；且当植入细胞数达300万及以上时，该现象的发生概率更高。普通I型胶原无法抑制恶性增殖，其促瘤效果与基质胶相当。转录组微阵列分析未在不同细胞系中检测到统一的基因表达特征，但与之相反，针对4种细胞系的250种蛋白进行的反相蛋白质阵列分析，成功鉴定出整合素连接激酶（Integrin Linked Kinase, ILK）信号通路的异常，且该结果通过ILK抑制剂得到了功能验证。我们认为，在SISgel上受抑制的癌细胞可作为肿瘤休眠与复醒的研究模型，助力鉴定靶向治疗微转移的药物靶点。实验整体设计：此前我们已证实，膀胱癌细胞在三维器官型培养体系中，于正常细胞外基质制剂SISgel上的生长表型，与在肿瘤源性的促瘤细胞外基质制剂基质胶（Matrigel）上的表型存在显著差异。SISgel是一种源自脱细胞小肠黏膜下层的成胶材料，而基质胶则是从小鼠肉瘤中提取的基底膜制剂。在基质胶上，膀胱癌细胞可重现原发肿瘤的表型；与之形成鲜明对比的是，当细胞培养于SISgel上时，其绝大多数恶性特性均会消失。源自乳头状瘤的细胞系可形成类似正常尿路上皮的分层结构，而源自高级别肿瘤的细胞系则形成非侵袭性的细胞层。上述发现提示，癌细胞在正常细胞外基质上的生长模式，可构建用于研究正常细胞外基质抑制肿瘤转移与复发中恶性表型的模型。本研究旨在探究：膀胱癌细胞在SISgel上的器官型培养体系中观察到的表型抑制现象，是否同样可在活体动物体内重现。若实验结果为阳性，则支持将SISgel作为研究休眠/受抑制肿瘤细胞表型，以及正常细胞外基质抑制肿瘤发生与转移机制的模型。本研究结果强烈表明，癌细胞与正常细胞外基质的相互作用在肿瘤复发与转移中发挥关键作用，同时提示靶向受抑制的癌细胞，可成为癌症治疗中一种此前未被发掘的脆弱靶点。