In vivo enrichment of cancer stem cells in histone deacetylase inhibitor resistant xenograft of lung cancer in mouse model

Objectives Cancer stem cells (CSCs) are usually resistant to chemotherapy and radiotherapy and responsible for tumor relapse. Although CSCs can be generated in vitro by non-adherent suspension culture in serum-free medium, the biologic characteristic of tumorspheres was far from CSCs in real tumors due to the distinct environmental factors, such as stroma cells, inflammation and hypoxia within tumor, which may profoundly reprogram CSCs. Histone deacetylase inhibitors (HDACIs) may induce epithelial-to-mesenchymal transition (EMT) and CSC-enriched characteristics during treatment. This study aimed to develop an in vivo CSCs-screening system using suberoylanilide hydroxamic acid (SAHA) to enrich the CSC population to facilitate the isolation of CSCs from solid tumors.

Methods We established a H1299 cell clone (NSCLC) which stably expressed a triple-fused reporter gene (DsRedm-Fluc-tTKsr39) under the control of CMV promoter. Tumor cells were tracked by in vivo luminescence imaging after continuous SAHA treatment of tumor-bearing mice. Using the Aldefluor assay, viable CSCs were isolated from xenograft tumors which co-express the reporter gene and ALDH activity. The stem cell phenotype of CSCs was assessed by a cell invasion assay, analyzing CSC-related gene expression, and immunohistochemical staining for ALDH1A1 in vitro.

Results Tumor tissues from SAHA-treated mice had increased CSC population which is enriched by continuous administration of SAHA (4.14% vs 1.50% of control). The microarray analysis showed that genes involved in the maintenance of CSCs, self-renewal and metastasis were upregulated in these isolated CSCs. In addition, the invasive ability of these SAHA-induced CSCs was also significantly elevated.

Conclusions We isolated and characterized CSCs by using in vivo reporter gene imaging and Aldefluor assay. These results may improve understanding of the molecular mechanism of CSCs and facilitate the discovery of specific targets of CSCs.