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Heterogeneity of circulating epithelial tumour cells from individual patients with respect to expression profiles and clonal growth (sphere formation) in breast cancer.

Pizon M, Zimon D, Carl S, Pachmann U, Pachmann K, Camara O - Ecancermedicalscience (2013)

Bottom Line: Here we present data that a variable fraction among the circulating tumour cells detected by the Maintrac(®) approach expresses mRNA of the stem cell gene NANOG and of the adhesion molecule vimentin and is capable of forming tumour spheres, a property ascribed to tumour-initiating cells (TICs).Although no epithelial cell adhesion molecule (EpCAM)-positive cells expressing stem cell genes or the adhesion molecule vimentin was detected before surgery, 10%-20% of the cells were found to be positive for mRNA of these genes after surgery.Here we show that among the peripherally circulating tumour cells, a variable fraction is able to express stem cell and adhesion properties and can be grown into tumour spheres, a property ascribed to cells capable of initiating tumours and metastases.

View Article: PubMed Central - PubMed

Affiliation: Transfusion Center Bayreuth D-95448, Germany.

ABSTRACT

Background: The detection of tumour cells circulating in the peripheral blood of patients with breast cancer is a sign that cells have been able to leave the primary tumour and survive in the circulation. However, in order to form metastases, they require additional properties such as the ability to adhere, self-renew, and grow. Here we present data that a variable fraction among the circulating tumour cells detected by the Maintrac(®) approach expresses mRNA of the stem cell gene NANOG and of the adhesion molecule vimentin and is capable of forming tumour spheres, a property ascribed to tumour-initiating cells (TICs).

Patients and methods: Between ten and 50 circulating epithelial antigen-positive cells detected by the Maintrac approach were selected randomly from each of 20 patients with breast cancer before and after surgery and were isolated using automated capillary aspiration and deposited individually onto slides for expression profiling. In addition, the circulating tumour cells were cultured without isolation among the white blood cells from 39 patients with breast cancer in different stages of disease using culture methods favouring growth of epithelial cells.

Results: Although no epithelial cell adhesion molecule (EpCAM)-positive cells expressing stem cell genes or the adhesion molecule vimentin was detected before surgery, 10%-20% of the cells were found to be positive for mRNA of these genes after surgery. Tumour spheres from circulating cells of 39 patients with different stages of breast cancer were grown without previous isolation in a fraction increasing with the aggressivity of the tumour.

Summary: Here we show that among the peripherally circulating tumour cells, a variable fraction is able to express stem cell and adhesion properties and can be grown into tumour spheres, a property ascribed to cells capable of initiating tumours and metastases.

No MeSH data available.


Related in: MedlinePlus

Gel analysis of expression profiles of 10–12 cells from three patients isolated before and after surgery. Each lane is the profile derived from a single cell.
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figure2: Gel analysis of expression profiles of 10–12 cells from three patients isolated before and after surgery. Each lane is the profile derived from a single cell.

Mentions: For expression analysis, all white blood cells comprising the tumour suspect cells from 1 mL of blood were stained with green fluorescence labelled anti-EpCAM. Vital epithelial cells (10–12) from 20 breast cancer patients before and after surgery stained with membrane EpCAM and excluding PI as an avitality marker were selected individually, deposited one by one, and analysed for the mRNA expression of EpCAM, human epidermal growth factor receptor 2 (HER2)/neu, vimentin, and NANOG (homeobox protein), and the two housekeeping genes Gremlin and RPL 13 A. In 20/20 cases, cells showed increased expression after surgery as compared to before surgery. Three typical comparisons are shown in Figures 2a–c. In a patient at the end of preoperative chemotherapy, expression of at least one housekeeping gene was seen in all of the ten surface EpCAM-positive cells randomly picked before surgery. However, clear mRNA expression of EpCAM was detected only in one cell in which expression of the stem cell gene NANOG was also seen. After surgery, in contrast, EpCAM mRNA was detected in four out of ten cells, HER2/neu expression in six out of ten cells, vimentin (an adhesion molecule, which is characteristic for epithelial-mesenchymal transition) in three out of ten cells, and NANOG in one out of ten cells, indicating that the cells present after surgery showed a higher activation status of genes relevant for growth activity and adhesion than those before surgery. This was also true for the expression of HER2/ neu, although the primary tumour had been classified as HER2/neu-negative. The increase in gene expression was even more obvious in the second patient, in whom prior to surgery for invasive ductal carcinoma (pT1c N1a (1/49) L1 V0 G3 R0, M0 ER: 90%, PR: 20%, HER-2/ neu-negative) all of the 12 randomly picked surface EpCAM-positive cells tested showed no relevant expression of any of the genes. After surgery, circulating numbers of tumour suspect cells had increased by fourfold, and a clear expression of both tested housekeeping genes was seen in ten out of ten cells selected. EpCAM mRNA was now clearly detected in six out of ten cells, HER2/neu in eight out of ten cells, vimentin in six out of ten cells, and NANOG in three out of ten cells. EpCAM and vimentin were coexpressed in three out of ten cells, EpCAM and HER2/neu in five out of ten cells, NANOG and HER2/neu in two out of ten cells, and vimentin and NANOG in two out of ten cells.


Heterogeneity of circulating epithelial tumour cells from individual patients with respect to expression profiles and clonal growth (sphere formation) in breast cancer.

Pizon M, Zimon D, Carl S, Pachmann U, Pachmann K, Camara O - Ecancermedicalscience (2013)

Gel analysis of expression profiles of 10–12 cells from three patients isolated before and after surgery. Each lane is the profile derived from a single cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750196&req=5

figure2: Gel analysis of expression profiles of 10–12 cells from three patients isolated before and after surgery. Each lane is the profile derived from a single cell.
Mentions: For expression analysis, all white blood cells comprising the tumour suspect cells from 1 mL of blood were stained with green fluorescence labelled anti-EpCAM. Vital epithelial cells (10–12) from 20 breast cancer patients before and after surgery stained with membrane EpCAM and excluding PI as an avitality marker were selected individually, deposited one by one, and analysed for the mRNA expression of EpCAM, human epidermal growth factor receptor 2 (HER2)/neu, vimentin, and NANOG (homeobox protein), and the two housekeeping genes Gremlin and RPL 13 A. In 20/20 cases, cells showed increased expression after surgery as compared to before surgery. Three typical comparisons are shown in Figures 2a–c. In a patient at the end of preoperative chemotherapy, expression of at least one housekeeping gene was seen in all of the ten surface EpCAM-positive cells randomly picked before surgery. However, clear mRNA expression of EpCAM was detected only in one cell in which expression of the stem cell gene NANOG was also seen. After surgery, in contrast, EpCAM mRNA was detected in four out of ten cells, HER2/neu expression in six out of ten cells, vimentin (an adhesion molecule, which is characteristic for epithelial-mesenchymal transition) in three out of ten cells, and NANOG in one out of ten cells, indicating that the cells present after surgery showed a higher activation status of genes relevant for growth activity and adhesion than those before surgery. This was also true for the expression of HER2/ neu, although the primary tumour had been classified as HER2/neu-negative. The increase in gene expression was even more obvious in the second patient, in whom prior to surgery for invasive ductal carcinoma (pT1c N1a (1/49) L1 V0 G3 R0, M0 ER: 90%, PR: 20%, HER-2/ neu-negative) all of the 12 randomly picked surface EpCAM-positive cells tested showed no relevant expression of any of the genes. After surgery, circulating numbers of tumour suspect cells had increased by fourfold, and a clear expression of both tested housekeeping genes was seen in ten out of ten cells selected. EpCAM mRNA was now clearly detected in six out of ten cells, HER2/neu in eight out of ten cells, vimentin in six out of ten cells, and NANOG in three out of ten cells. EpCAM and vimentin were coexpressed in three out of ten cells, EpCAM and HER2/neu in five out of ten cells, NANOG and HER2/neu in two out of ten cells, and vimentin and NANOG in two out of ten cells.

Bottom Line: Here we present data that a variable fraction among the circulating tumour cells detected by the Maintrac(®) approach expresses mRNA of the stem cell gene NANOG and of the adhesion molecule vimentin and is capable of forming tumour spheres, a property ascribed to tumour-initiating cells (TICs).Although no epithelial cell adhesion molecule (EpCAM)-positive cells expressing stem cell genes or the adhesion molecule vimentin was detected before surgery, 10%-20% of the cells were found to be positive for mRNA of these genes after surgery.Here we show that among the peripherally circulating tumour cells, a variable fraction is able to express stem cell and adhesion properties and can be grown into tumour spheres, a property ascribed to cells capable of initiating tumours and metastases.

View Article: PubMed Central - PubMed

Affiliation: Transfusion Center Bayreuth D-95448, Germany.

ABSTRACT

Background: The detection of tumour cells circulating in the peripheral blood of patients with breast cancer is a sign that cells have been able to leave the primary tumour and survive in the circulation. However, in order to form metastases, they require additional properties such as the ability to adhere, self-renew, and grow. Here we present data that a variable fraction among the circulating tumour cells detected by the Maintrac(®) approach expresses mRNA of the stem cell gene NANOG and of the adhesion molecule vimentin and is capable of forming tumour spheres, a property ascribed to tumour-initiating cells (TICs).

Patients and methods: Between ten and 50 circulating epithelial antigen-positive cells detected by the Maintrac approach were selected randomly from each of 20 patients with breast cancer before and after surgery and were isolated using automated capillary aspiration and deposited individually onto slides for expression profiling. In addition, the circulating tumour cells were cultured without isolation among the white blood cells from 39 patients with breast cancer in different stages of disease using culture methods favouring growth of epithelial cells.

Results: Although no epithelial cell adhesion molecule (EpCAM)-positive cells expressing stem cell genes or the adhesion molecule vimentin was detected before surgery, 10%-20% of the cells were found to be positive for mRNA of these genes after surgery. Tumour spheres from circulating cells of 39 patients with different stages of breast cancer were grown without previous isolation in a fraction increasing with the aggressivity of the tumour.

Summary: Here we show that among the peripherally circulating tumour cells, a variable fraction is able to express stem cell and adhesion properties and can be grown into tumour spheres, a property ascribed to cells capable of initiating tumours and metastases.

No MeSH data available.


Related in: MedlinePlus