Limits...
Downregulation of CXCR4 in Metastasized Breast Cancer Cells and Implication in Their Dormancy.

Nobutani K, Shimono Y, Mizutani K, Ueda Y, Suzuki T, Kitayama M, Minami A, Momose K, Miyawaki K, Akashi K, Azuma T, Takai Y - PLoS ONE (2015)

Bottom Line: In the gene expression analysis of the orthotopic cancer cells by a single-cell multiplex real-time quantitative reverse transcription PCR followed by flow cytometric analysis, restrained cellular proliferation was associated with downregulation of the chemokine receptor CXCR4.In the immunohistological and flow cytometric analyses, the expression level of CXCR4 in the metastasized cancer cells was decreased compared with that in the cancer cells in orthotopic tumors, although the expression level of the CXCR4 ligand CXCL12 was not reduced in the lung.These findings indicate that CXCR4 is downregulated in metastasized breast cancer cells and implicated in their dormancy.

View Article: PubMed Central - PubMed

Affiliation: Division of Pathogenetic Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan; Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan; Division of Molecular and Cellular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan.

ABSTRACT
Our understanding of the mechanism of cancer dormancy is emerging, but the underlying mechanisms are not fully understood. Here we analyzed mouse xenograft tumors derived from human breast cancer tissue and the human breast cancer cell line MDA-MB-231 to identify the molecules associated with cancer dormancy. In immunohistological examination using the proliferation marker Ki-67, the tumors included both proliferating and dormant cancer cells, but the number of dormant cells was remarkably increased when they metastasized to the lung. In the gene expression analysis of the orthotopic cancer cells by a single-cell multiplex real-time quantitative reverse transcription PCR followed by flow cytometric analysis, restrained cellular proliferation was associated with downregulation of the chemokine receptor CXCR4. In the immunohistological and flow cytometric analyses, the expression level of CXCR4 in the metastasized cancer cells was decreased compared with that in the cancer cells in orthotopic tumors, although the expression level of the CXCR4 ligand CXCL12 was not reduced in the lung. In addition, the proliferation of the metastasized cancer cells was further decreased by the CXCR4 antagonist administration. In the ex vivo culture of the metastasized cancer cells, the expression level of CXCR4 was increased, and in the xenotransplantation of ex vivo cultured cancer cells, the expression level of CXCR4 was again decreased in the metastasized cancer cells in the lung. These findings indicate that CXCR4 is downregulated in metastasized breast cancer cells and implicated in their dormancy.

No MeSH data available.


Related in: MedlinePlus

Dormancy of breast cancer cells in the xenograft tumor-bearing mice.(A) Immunofluorescent images for the endothelial cell marker, CD31, and MDA-MB-231 breast cancer cells in the orthotopic tumor and metastatic lesions in the lung. The arrowhead indicates the metastatic tumor. Green: ZsGreen1; red: CD31; blue: nucleus. Scale bars: 100 μm for the low power field; 10 μm for the high power field. Representative images are shown. (B) Immunofluorescent images for Ki-67 in the MDA-MB-231 breast cancer cells in the orthotopic tumor and metastatic lesions in the lung. The arrowheads indicate the metastatic tumor lesions in the lung. Green: human leukocyte antigen (HLA)-A, B, C; red: Ki-67; blue: nucleus. Scale bars: 100 μm for the low power field; 10 μm for the high power field. Representative images are shown.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4470829&req=5

pone.0130032.g001: Dormancy of breast cancer cells in the xenograft tumor-bearing mice.(A) Immunofluorescent images for the endothelial cell marker, CD31, and MDA-MB-231 breast cancer cells in the orthotopic tumor and metastatic lesions in the lung. The arrowhead indicates the metastatic tumor. Green: ZsGreen1; red: CD31; blue: nucleus. Scale bars: 100 μm for the low power field; 10 μm for the high power field. Representative images are shown. (B) Immunofluorescent images for Ki-67 in the MDA-MB-231 breast cancer cells in the orthotopic tumor and metastatic lesions in the lung. The arrowheads indicate the metastatic tumor lesions in the lung. Green: human leukocyte antigen (HLA)-A, B, C; red: Ki-67; blue: nucleus. Scale bars: 100 μm for the low power field; 10 μm for the high power field. Representative images are shown.

Mentions: To examine the characteristics of dormant breast cancer cells, we analyzed xenograft tumors in mice that we generated by orthotopic xenotransplantation of human breast cancer tissues obtained from breast cancer patients and the human breast cancer cell line MDA-MB-231 [9]. In the following experiments, we used one of the PDX lines and one from the cell line that allowed us to detect and isolate the cancer cells by the green fluorescence protein ZsGreen1 that was transduced to the cell line or the expression of the HLA-A, B, C (Figs 1, 3, 4 and 5; S1, S2 and S3 Figs). The human breast cancer PDX was generated from the ER (+)/PR (+)/HER2 (-) breast cancer and the MDA-MB-231 cells showed triple-negative breast cancer phenotype [9,22]. The growth rate of the PDX was much lower than that of the cell line-derived xenografts: the PDX and cell line-derived xenografts reached approximately 1–2 cm in about 3 months and 5–6 weeks, respectively. In the cell line-derived tumor model, when the tumors grew to these sizes, a small number of the cancer cells spontaneously metastasized to the lung and located at the perivascular area (Fig 1A and 1B). The metastatic cancer cells in the lung were also detected by flow cytometry (S1 Fig). Essentially the same results were obtained in the human PDX model (S2A and S2B Fig). In the cell line-derived tumor model, the metastatic cancer cells in the lung were present solely or in small clusters in contrast to the cancer cells in the orthotopic tumors, in which a large number of the cancer cells were densely packed in the tumors (Fig 1A and 1B). More than 80% of the cancer cells in the orthotopic tumors excluding those in the necrotizing tissue were positively stained for anti-Ki-67 pAb, although there was a small fraction of Ki-67-negative cancer cells (Fig 1B and Table 1). In contrast, consistent with a previous report [5], cancer cells not stained with the anti-Ki-67 pAb were dominant in the lung, and less than 20% of the metastasized cancer cells in the lung were Ki-67-positive (Fig 1B and Table 1). The Ki-67-positive normal lung cells were rarely observed (data not shown). Essentially the same results were obtained in the PDX model (S2B Fig and Table 1). Therefore, cancer cells in the orthotopic tumor are prone to be in the proliferating state, whereas the metastasized cancer cells in the lung tend to be in the dormant state. These findings suggest that the proliferating activity of the cancer cells is heterogeneous, and that the number of the proliferating cancer cells changes depending on the change of the tumor environment.


Downregulation of CXCR4 in Metastasized Breast Cancer Cells and Implication in Their Dormancy.

Nobutani K, Shimono Y, Mizutani K, Ueda Y, Suzuki T, Kitayama M, Minami A, Momose K, Miyawaki K, Akashi K, Azuma T, Takai Y - PLoS ONE (2015)

Dormancy of breast cancer cells in the xenograft tumor-bearing mice.(A) Immunofluorescent images for the endothelial cell marker, CD31, and MDA-MB-231 breast cancer cells in the orthotopic tumor and metastatic lesions in the lung. The arrowhead indicates the metastatic tumor. Green: ZsGreen1; red: CD31; blue: nucleus. Scale bars: 100 μm for the low power field; 10 μm for the high power field. Representative images are shown. (B) Immunofluorescent images for Ki-67 in the MDA-MB-231 breast cancer cells in the orthotopic tumor and metastatic lesions in the lung. The arrowheads indicate the metastatic tumor lesions in the lung. Green: human leukocyte antigen (HLA)-A, B, C; red: Ki-67; blue: nucleus. Scale bars: 100 μm for the low power field; 10 μm for the high power field. Representative images are shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4470829&req=5

pone.0130032.g001: Dormancy of breast cancer cells in the xenograft tumor-bearing mice.(A) Immunofluorescent images for the endothelial cell marker, CD31, and MDA-MB-231 breast cancer cells in the orthotopic tumor and metastatic lesions in the lung. The arrowhead indicates the metastatic tumor. Green: ZsGreen1; red: CD31; blue: nucleus. Scale bars: 100 μm for the low power field; 10 μm for the high power field. Representative images are shown. (B) Immunofluorescent images for Ki-67 in the MDA-MB-231 breast cancer cells in the orthotopic tumor and metastatic lesions in the lung. The arrowheads indicate the metastatic tumor lesions in the lung. Green: human leukocyte antigen (HLA)-A, B, C; red: Ki-67; blue: nucleus. Scale bars: 100 μm for the low power field; 10 μm for the high power field. Representative images are shown.
Mentions: To examine the characteristics of dormant breast cancer cells, we analyzed xenograft tumors in mice that we generated by orthotopic xenotransplantation of human breast cancer tissues obtained from breast cancer patients and the human breast cancer cell line MDA-MB-231 [9]. In the following experiments, we used one of the PDX lines and one from the cell line that allowed us to detect and isolate the cancer cells by the green fluorescence protein ZsGreen1 that was transduced to the cell line or the expression of the HLA-A, B, C (Figs 1, 3, 4 and 5; S1, S2 and S3 Figs). The human breast cancer PDX was generated from the ER (+)/PR (+)/HER2 (-) breast cancer and the MDA-MB-231 cells showed triple-negative breast cancer phenotype [9,22]. The growth rate of the PDX was much lower than that of the cell line-derived xenografts: the PDX and cell line-derived xenografts reached approximately 1–2 cm in about 3 months and 5–6 weeks, respectively. In the cell line-derived tumor model, when the tumors grew to these sizes, a small number of the cancer cells spontaneously metastasized to the lung and located at the perivascular area (Fig 1A and 1B). The metastatic cancer cells in the lung were also detected by flow cytometry (S1 Fig). Essentially the same results were obtained in the human PDX model (S2A and S2B Fig). In the cell line-derived tumor model, the metastatic cancer cells in the lung were present solely or in small clusters in contrast to the cancer cells in the orthotopic tumors, in which a large number of the cancer cells were densely packed in the tumors (Fig 1A and 1B). More than 80% of the cancer cells in the orthotopic tumors excluding those in the necrotizing tissue were positively stained for anti-Ki-67 pAb, although there was a small fraction of Ki-67-negative cancer cells (Fig 1B and Table 1). In contrast, consistent with a previous report [5], cancer cells not stained with the anti-Ki-67 pAb were dominant in the lung, and less than 20% of the metastasized cancer cells in the lung were Ki-67-positive (Fig 1B and Table 1). The Ki-67-positive normal lung cells were rarely observed (data not shown). Essentially the same results were obtained in the PDX model (S2B Fig and Table 1). Therefore, cancer cells in the orthotopic tumor are prone to be in the proliferating state, whereas the metastasized cancer cells in the lung tend to be in the dormant state. These findings suggest that the proliferating activity of the cancer cells is heterogeneous, and that the number of the proliferating cancer cells changes depending on the change of the tumor environment.

Bottom Line: In the gene expression analysis of the orthotopic cancer cells by a single-cell multiplex real-time quantitative reverse transcription PCR followed by flow cytometric analysis, restrained cellular proliferation was associated with downregulation of the chemokine receptor CXCR4.In the immunohistological and flow cytometric analyses, the expression level of CXCR4 in the metastasized cancer cells was decreased compared with that in the cancer cells in orthotopic tumors, although the expression level of the CXCR4 ligand CXCL12 was not reduced in the lung.These findings indicate that CXCR4 is downregulated in metastasized breast cancer cells and implicated in their dormancy.

View Article: PubMed Central - PubMed

Affiliation: Division of Pathogenetic Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan; Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan; Division of Molecular and Cellular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan.

ABSTRACT
Our understanding of the mechanism of cancer dormancy is emerging, but the underlying mechanisms are not fully understood. Here we analyzed mouse xenograft tumors derived from human breast cancer tissue and the human breast cancer cell line MDA-MB-231 to identify the molecules associated with cancer dormancy. In immunohistological examination using the proliferation marker Ki-67, the tumors included both proliferating and dormant cancer cells, but the number of dormant cells was remarkably increased when they metastasized to the lung. In the gene expression analysis of the orthotopic cancer cells by a single-cell multiplex real-time quantitative reverse transcription PCR followed by flow cytometric analysis, restrained cellular proliferation was associated with downregulation of the chemokine receptor CXCR4. In the immunohistological and flow cytometric analyses, the expression level of CXCR4 in the metastasized cancer cells was decreased compared with that in the cancer cells in orthotopic tumors, although the expression level of the CXCR4 ligand CXCL12 was not reduced in the lung. In addition, the proliferation of the metastasized cancer cells was further decreased by the CXCR4 antagonist administration. In the ex vivo culture of the metastasized cancer cells, the expression level of CXCR4 was increased, and in the xenotransplantation of ex vivo cultured cancer cells, the expression level of CXCR4 was again decreased in the metastasized cancer cells in the lung. These findings indicate that CXCR4 is downregulated in metastasized breast cancer cells and implicated in their dormancy.

No MeSH data available.


Related in: MedlinePlus