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

Ameri K, Luong R, Zhang H, Powell AA, Montgomery KD, Espinosa I, Bouley DM, Harris AL, Jeffrey SS - Br. J. Cancer (2010)

Bottom Line: Treatment refractory patients with metastatic cancer show increased numbers of circulating tumour cells (CTCs), which are also associated with disease progression.Xenografts generated from CTCs and parental cells were compared.Xenografts generated from CTCs were larger and heavier, and metastasised faster than MDA-MB-231 xenografts.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Stanford University School of Medicine, Stanford, CA 94305-5494, USA. kurosh.ameri@gmail.com

ABSTRACT

Background: Tumours contain hypoxic regions that select for an aggressive cell phenotype; tumour hypoxia induces metastasis-associated 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 (HIF1alpha) 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 HIF1alpha 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.

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Circulating tumour cells (CTCs), isolated and cultured from murine blood, demonstrate a distinct response to hypoxia compared with parental MDA-MB-231cells. (Ai) Freshly captured CTCs are cells with attached magnetic beads as indicated by white arrows. The magnetic beads are individual small dark circles, as can be seen in the control; CTCs covered with magnetic beads appear as dark clusters of beads on the cell membrane with translucent centres, as indicated by white arrows; lack of CTCs in blood samples obtained from six control mice without xenografts. (Aii) The magnetically captured CTCs in culture on day 10 after capture and after day 60. (B–F) Induction of HIF1α in hypoxic MDA-MB-231 cells and CTCs. (B) Expression of the anoxia factor ATF3 in CTCs and MDA-MB-231 cells. Graphs demonstrate densitometry measurement of ATF3 expression normalised to actin expression from three experiments. (C) Induction of the anoxia-induced factor ATF4 in CTCs compared with MDA-MB-231 cells. Graphs demonstrate densitometry measurements of ATF4 expression normalised to actin expression from three experiments. (D) Expression of ATF4 and its target gene ASNS in CTCs, compared with parental MDA-MB-231 cells. (E and F) Differences between CTCs and parental MDA-MB-231 cells with respect to induction of HIF1α target genes. (E) Induction of apelin in hypoxic CTCs compared with parental MDA-MB-231. Hypoxic induction of (E) GLUT1 and (F) CAIX, BNIP3 in CTCs, compared with parental MDA-MB-231 cells. Experiments were performed at least three or four times with three or four different extracts. N, normoxia; H, hypoxia.
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fig3: Circulating tumour cells (CTCs), isolated and cultured from murine blood, demonstrate a distinct response to hypoxia compared with parental MDA-MB-231cells. (Ai) Freshly captured CTCs are cells with attached magnetic beads as indicated by white arrows. The magnetic beads are individual small dark circles, as can be seen in the control; CTCs covered with magnetic beads appear as dark clusters of beads on the cell membrane with translucent centres, as indicated by white arrows; lack of CTCs in blood samples obtained from six control mice without xenografts. (Aii) The magnetically captured CTCs in culture on day 10 after capture and after day 60. (B–F) Induction of HIF1α in hypoxic MDA-MB-231 cells and CTCs. (B) Expression of the anoxia factor ATF3 in CTCs and MDA-MB-231 cells. Graphs demonstrate densitometry measurement of ATF3 expression normalised to actin expression from three experiments. (C) Induction of the anoxia-induced factor ATF4 in CTCs compared with MDA-MB-231 cells. Graphs demonstrate densitometry measurements of ATF4 expression normalised to actin expression from three experiments. (D) Expression of ATF4 and its target gene ASNS in CTCs, compared with parental MDA-MB-231 cells. (E and F) Differences between CTCs and parental MDA-MB-231 cells with respect to induction of HIF1α target genes. (E) Induction of apelin in hypoxic CTCs compared with parental MDA-MB-231. Hypoxic induction of (E) GLUT1 and (F) CAIX, BNIP3 in CTCs, compared with parental MDA-MB-231 cells. Experiments were performed at least three or four times with three or four different extracts. N, normoxia; H, hypoxia.

Mentions: To determine whether CTCs have an altered hypoxia response, they had to be isolated and grown in culture. We demonstrated that our MDA-MB-231 xenograft model shed CTCs into mouse circulation using a new technology that enabled viable human CTC capture from mouse blood (Talasaz et al, 2009). Magnetic beads attached to the epithelial cell adhesion molecule antigen on CTCs appeared as dark clusters of beads on the cell membrane surrounding translucent cells. In contrast, there were no dark cell/magnetic bead clusters visible in the blood of control mice. Instead, only small dark circles representing individual magnetic beads were observed in control mouse blood (Figure 3Ai). Captured CTCs were viable and could be subsequently grown in culture (Figure 3Aii).


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

Ameri K, Luong R, Zhang H, Powell AA, Montgomery KD, Espinosa I, Bouley DM, Harris AL, Jeffrey SS - Br. J. Cancer (2010)

Circulating tumour cells (CTCs), isolated and cultured from murine blood, demonstrate a distinct response to hypoxia compared with parental MDA-MB-231cells. (Ai) Freshly captured CTCs are cells with attached magnetic beads as indicated by white arrows. The magnetic beads are individual small dark circles, as can be seen in the control; CTCs covered with magnetic beads appear as dark clusters of beads on the cell membrane with translucent centres, as indicated by white arrows; lack of CTCs in blood samples obtained from six control mice without xenografts. (Aii) The magnetically captured CTCs in culture on day 10 after capture and after day 60. (B–F) Induction of HIF1α in hypoxic MDA-MB-231 cells and CTCs. (B) Expression of the anoxia factor ATF3 in CTCs and MDA-MB-231 cells. Graphs demonstrate densitometry measurement of ATF3 expression normalised to actin expression from three experiments. (C) Induction of the anoxia-induced factor ATF4 in CTCs compared with MDA-MB-231 cells. Graphs demonstrate densitometry measurements of ATF4 expression normalised to actin expression from three experiments. (D) Expression of ATF4 and its target gene ASNS in CTCs, compared with parental MDA-MB-231 cells. (E and F) Differences between CTCs and parental MDA-MB-231 cells with respect to induction of HIF1α target genes. (E) Induction of apelin in hypoxic CTCs compared with parental MDA-MB-231. Hypoxic induction of (E) GLUT1 and (F) CAIX, BNIP3 in CTCs, compared with parental MDA-MB-231 cells. Experiments were performed at least three or four times with three or four different extracts. N, normoxia; H, hypoxia.
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fig3: Circulating tumour cells (CTCs), isolated and cultured from murine blood, demonstrate a distinct response to hypoxia compared with parental MDA-MB-231cells. (Ai) Freshly captured CTCs are cells with attached magnetic beads as indicated by white arrows. The magnetic beads are individual small dark circles, as can be seen in the control; CTCs covered with magnetic beads appear as dark clusters of beads on the cell membrane with translucent centres, as indicated by white arrows; lack of CTCs in blood samples obtained from six control mice without xenografts. (Aii) The magnetically captured CTCs in culture on day 10 after capture and after day 60. (B–F) Induction of HIF1α in hypoxic MDA-MB-231 cells and CTCs. (B) Expression of the anoxia factor ATF3 in CTCs and MDA-MB-231 cells. Graphs demonstrate densitometry measurement of ATF3 expression normalised to actin expression from three experiments. (C) Induction of the anoxia-induced factor ATF4 in CTCs compared with MDA-MB-231 cells. Graphs demonstrate densitometry measurements of ATF4 expression normalised to actin expression from three experiments. (D) Expression of ATF4 and its target gene ASNS in CTCs, compared with parental MDA-MB-231 cells. (E and F) Differences between CTCs and parental MDA-MB-231 cells with respect to induction of HIF1α target genes. (E) Induction of apelin in hypoxic CTCs compared with parental MDA-MB-231. Hypoxic induction of (E) GLUT1 and (F) CAIX, BNIP3 in CTCs, compared with parental MDA-MB-231 cells. Experiments were performed at least three or four times with three or four different extracts. N, normoxia; H, hypoxia.
Mentions: To determine whether CTCs have an altered hypoxia response, they had to be isolated and grown in culture. We demonstrated that our MDA-MB-231 xenograft model shed CTCs into mouse circulation using a new technology that enabled viable human CTC capture from mouse blood (Talasaz et al, 2009). Magnetic beads attached to the epithelial cell adhesion molecule antigen on CTCs appeared as dark clusters of beads on the cell membrane surrounding translucent cells. In contrast, there were no dark cell/magnetic bead clusters visible in the blood of control mice. Instead, only small dark circles representing individual magnetic beads were observed in control mouse blood (Figure 3Ai). Captured CTCs were viable and could be subsequently grown in culture (Figure 3Aii).

Bottom Line: Treatment refractory patients with metastatic cancer show increased numbers of circulating tumour cells (CTCs), which are also associated with disease progression.Xenografts generated from CTCs and parental cells were compared.Xenografts generated from CTCs were larger and heavier, and metastasised faster than MDA-MB-231 xenografts.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Stanford University School of Medicine, Stanford, CA 94305-5494, USA. kurosh.ameri@gmail.com

ABSTRACT

Background: Tumours contain hypoxic regions that select for an aggressive cell phenotype; tumour hypoxia induces metastasis-associated 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 (HIF1alpha) 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 HIF1alpha 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.

Show MeSH
Related in: MedlinePlus