Cancer initiating cells (CICs) have been shown to be responsible for primary tumor formation, drug resistance and metastatic dissemination. We previously demonstrated that the subset of CD133+ lung CICs co-expressing the chemokine receptor CXCR4, is resistant to chemotherapy and endowed with high metastasis initiating ability using in vivo experimental metastasis assay.
To investigate the role of the subset of CD133+CXCR4+ cells in the in vivo spontaneous tumor dissemination process, we analyzed lungs of mice bearing subcutaneous implants of lung cancer patients derived xenografts (PDXs). Using FACS and Real Time PCR analysis a small fraction of lung disseminated tumor cells (DTCs: 0.001% to 0.01%) was detected in 9/11 different PDX models, likely remaining at single cells state as suggested by the lack of histologically apparent metastasis. In particular, compared to parental subcutaneous tumors, lung DTCs were enriched for CD133+CXCR4+ CICs not expressing the epithelial antigen EpCAM. Modifying an innovative cancer cells culture method that allows the recovery of highly purified and viable tumor cells (Kondo J. et al. 2011, PNAS), we also generated murine lung tissue spheroids that were 100 fold enriched in DTCs. Real Time PCR analysis of lung spheroids consistently revealed an up-regulation of stemness and EMT-associated genes compared to the parental tumor, in accordance with the enrichment for disseminating CD133+CXCR4+EpCAM- CICs subset observed by FACS. Subcutaneous injection in SCID mice of lung spheroids containing approximately 200 DTCs, generated tumors resembling the original PDX, but also showing an increased fraction of CD133+ stem like cells and particularly of the CD133+CXCR4+EpCAM- subset. Moreover, cells recovered from DTCs-originated tumors were able to grow in vitro as spheres in anchorage independent condition, up-regulated EMT-genes and showed an enhanced tumorigenic potential and increased ability to disseminate to murine lung compared with original PDX in in vivo serial transplantation assay, indicating that the fraction of CD133+CXCR4+EpCAM- CICs enriched in DTCs was able to efficiently originate tumors with distinctive traits. We also found that the fraction of CD133+ cells and particularly the CD133+CXCR4+EpCAM- subset were increased in patient's metastatic lymph nodes compared to primary tumors, confirming in a clinical setting the role of CD133+CXCR4+EpCAM- cells as metastasis initiating cells.
Our data functionally proves that lung DTCs contain a specific subset of tumor initiating cells that is able to generate tumors showing distinct properties and enrichment in the mesenchymal-like CICs compartment. The relevance of this finding is confirmed in clinical setting, identifying a new possible target for novel therapies aimed at interfering with metastatic dissemination, which represents the main reason for lung cancer poor outcome..
Citation Format: Giulia Bertolini, Massimo Moro, Monica Tortoreto, Roberto Caserini, Ugo Pastorino, Luca Roz, Gabriella Sozzi. Disseminated cells from primary lung cancers contain a distinct cancer initiating subpopulation with mesenchymal-like features. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3898. doi:10.1158/1538-7445.AM2014-3898