Circulating tumour cells demonstrate an altered response to hypoxia and an aggressive phenotype

Abstract

BACKGROUND: Tumours contain hypoxic regions that select for an aggressive cell phenotype; tumour hypoxia induces metastasisassociated genes. Treatment refractory patients with metastatic cancer show increased numbers of circulating tumour cells (CTCs), which are also associated with disease progression. The aim of this study was to examine the as yet unknown relationship between hypoxia and CTCs. METHODS: We generated human MDA-MB-231 orthotopic xenografts and, using a new technology, isolated viable human CTCs from murine blood. The CTCs and parental MDA-MB-231 cells were incubated at 21 and 0.2% (hypoxia) oxygen, respectively. Colony formation was assayed and levels of hypoxia-and anoxia-inducible factors were measured. Xenografts generated from CTCs and parental cells were compared. RESULTS: MDA-MB-231 xenografts used to generate CTCs were hypoxic, expressing hypoxia factors: hypoxia-inducible factor1 alpha (HIF1a) and glucose transporter protein type 1 (GLUT1), and anoxia-induced factors: activating transcription factor 3 and 4 (ATF3 and ATF4). Parental MDA-MB-231 cells induced ATF3 in hypoxia, whereas CTCs expressed it constitutively. Asparagine synthetase (ASNS) expression was also higher in CTCs. Hypoxia induced ATF4 and the HIF1a target gene apelin in CTCs, but not in parental cells. Hypoxia induced lower levels of carbonic anhydrase IX (CAIX), GLUT1 and BCL2/adenovirus E1B 19-KD protein-interacting protein 3 (BNIP3) proteins in CTCs than in parental cells, supporting an altered hypoxia response. In chronic hypoxia, CTCs demonstrated greater colony formation than parental cells. Xenografts generated from CTCs were larger and heavier, and metastasised faster than MDA-MB-231 xenografts. CONCLUSION: CTCs show an altered hypoxia response and an enhanced aggressive phenotype in vitro and in vivo.

Targeting Tumor Hypoxia: Suppression of Breast Tumor Growth and Metastasis by Novel Carbonic Anhydrase IX Inhibitors

“…In addition to this syngeneic mouse model, we selected the MDA-MB-231 human breast tumor cell line, as previous studies have shown this model to be hypoxic and to have hypoxia-inducible levels of CAIX (40,41). We confirmed the effect of hypoxia on CAIX expression in these cell lines to validate them as appropriate models for subsequent in vivo studies.…”

Asparagine synthetase expression and its potential prognostic value in patients with NK/T cell lymphoma

“…When cancer spreads from the primary tumor to distant sites, cells enter into the bloodstream and exist in suspension until they reach a metastatic site. Patrikainen et al (33) and Ameri et al (34) observed that PC-3 prostate cancer cells and human MDA-MB-231 breast cancer cells which have a strong potential for distant metastasis and an increased capacity for colony formation exhibit an elevated expression of ASNS. Its increased abundance in metastasizing cells suggests that ASNS activity is beneficial for cancer cell survival once they detach from the primary tumor and enter the bloodstream.…”

The Interrelating Dynamics of Hypoxic Tumor Microenvironments and Cancer Cell Phenotypes in Cancer Metastasis

“…Diminished homologous recombination may lead to genetic instability by shifting the balance between the high-fidelity homologous recombination and the error-prone NHEJ pathways of double-strand break repair in primary tumor cells [83]. It was recently demonstrated that CTC that were released from the primary tumor show an altered phenotype compared to the parental tumor cells [85]. These CTC show a hypoxic phenotype with overexpression of HIF-1 alpha, asparagine synthetase and GLUT1.…”

“…Cancer cell survival in the bone marrow environment was supported by Src facilitating CXCL12-CXCR4-AKT signalling and conferring resistance to TRAIL [45]. Second, cell lines-especially MDA-MB-231 cells in breast cancer-with their own specific differentiation status and metastatic potential [97] were frequently used as cellular models, e.g., for cellular adaptation strategies and site specific dissemination [60,85,98,99]. These experiments additionally show that differentiated cells are one source to produce tumors.…”