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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

Dynamic change of the expression level of CXCR4 responding to the change of tumor environment.(A) Microscopic examination of cells obtained from the lung and orthotopic tumor in the culture conditions. Images on the left show the bright field view and images on the right show the fluorescent view. Green cells in the fluorescence view indicate breast cancer cells. Cells with no fluorescence around the cancer cells are the non-cancer cells. Scale bar: 200 μm. Representative images are shown. (B) Flow cytometric analysis of CXCR4 of the breast cancer cells obtained from the lung tissues in the culture condition. The histogram with blue color indicates cancer cells in the lung in vivo, purple indicates ex vivo cultured cancer cells obtained from the lung, and red indicates cancer cells in the orthotopic tumor in vivo. Representative histograms are shown. Cells in the light green colored area are cells with a high expression level of cell surface CXCR4. (C) Percentage of the ex vivo cultured cancer cells with a high expression level of cell surface CXCR4 in the low (70–80% confluent) and high (100% confluent) cell densities. At least three analyses were performed at each time point. Asterisks indicate the significant difference between the two groups. (D) The schema and histograms of the xenotransplantation experiment of the ex vivo cultured cancer cells originally obtained from the orthotopic tumor and the lung. Histograms show the expression level of cell surface CXCR4 of the cancer cells obtained from the orthotopic tumor and the lung of the mice that were xenotransplanted with the ex vivo cultured cancer cells originally obtained from the orthotopic tumor or the lung. The red colored histograms indicate the cancer cells obtained from the orthotopic tumors, and blue colored histograms indicate the cancer cells obtained from the lung. Representative histograms are shown. (E) Summary of our findings. The change of the tumor environment causes the change of the expression level of CXCR4. This change facilitated the tumor growth.
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pone.0130032.g005: Dynamic change of the expression level of CXCR4 responding to the change of tumor environment.(A) Microscopic examination of cells obtained from the lung and orthotopic tumor in the culture conditions. Images on the left show the bright field view and images on the right show the fluorescent view. Green cells in the fluorescence view indicate breast cancer cells. Cells with no fluorescence around the cancer cells are the non-cancer cells. Scale bar: 200 μm. Representative images are shown. (B) Flow cytometric analysis of CXCR4 of the breast cancer cells obtained from the lung tissues in the culture condition. The histogram with blue color indicates cancer cells in the lung in vivo, purple indicates ex vivo cultured cancer cells obtained from the lung, and red indicates cancer cells in the orthotopic tumor in vivo. Representative histograms are shown. Cells in the light green colored area are cells with a high expression level of cell surface CXCR4. (C) Percentage of the ex vivo cultured cancer cells with a high expression level of cell surface CXCR4 in the low (70–80% confluent) and high (100% confluent) cell densities. At least three analyses were performed at each time point. Asterisks indicate the significant difference between the two groups. (D) The schema and histograms of the xenotransplantation experiment of the ex vivo cultured cancer cells originally obtained from the orthotopic tumor and the lung. Histograms show the expression level of cell surface CXCR4 of the cancer cells obtained from the orthotopic tumor and the lung of the mice that were xenotransplanted with the ex vivo cultured cancer cells originally obtained from the orthotopic tumor or the lung. The red colored histograms indicate the cancer cells obtained from the orthotopic tumors, and blue colored histograms indicate the cancer cells obtained from the lung. Representative histograms are shown. (E) Summary of our findings. The change of the tumor environment causes the change of the expression level of CXCR4. This change facilitated the tumor growth.

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)

Dynamic change of the expression level of CXCR4 responding to the change of tumor environment.(A) Microscopic examination of cells obtained from the lung and orthotopic tumor in the culture conditions. Images on the left show the bright field view and images on the right show the fluorescent view. Green cells in the fluorescence view indicate breast cancer cells. Cells with no fluorescence around the cancer cells are the non-cancer cells. Scale bar: 200 μm. Representative images are shown. (B) Flow cytometric analysis of CXCR4 of the breast cancer cells obtained from the lung tissues in the culture condition. The histogram with blue color indicates cancer cells in the lung in vivo, purple indicates ex vivo cultured cancer cells obtained from the lung, and red indicates cancer cells in the orthotopic tumor in vivo. Representative histograms are shown. Cells in the light green colored area are cells with a high expression level of cell surface CXCR4. (C) Percentage of the ex vivo cultured cancer cells with a high expression level of cell surface CXCR4 in the low (70–80% confluent) and high (100% confluent) cell densities. At least three analyses were performed at each time point. Asterisks indicate the significant difference between the two groups. (D) The schema and histograms of the xenotransplantation experiment of the ex vivo cultured cancer cells originally obtained from the orthotopic tumor and the lung. Histograms show the expression level of cell surface CXCR4 of the cancer cells obtained from the orthotopic tumor and the lung of the mice that were xenotransplanted with the ex vivo cultured cancer cells originally obtained from the orthotopic tumor or the lung. The red colored histograms indicate the cancer cells obtained from the orthotopic tumors, and blue colored histograms indicate the cancer cells obtained from the lung. Representative histograms are shown. (E) Summary of our findings. The change of the tumor environment causes the change of the expression level of CXCR4. This change facilitated the tumor growth.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4470829&req=5

pone.0130032.g005: Dynamic change of the expression level of CXCR4 responding to the change of tumor environment.(A) Microscopic examination of cells obtained from the lung and orthotopic tumor in the culture conditions. Images on the left show the bright field view and images on the right show the fluorescent view. Green cells in the fluorescence view indicate breast cancer cells. Cells with no fluorescence around the cancer cells are the non-cancer cells. Scale bar: 200 μm. Representative images are shown. (B) Flow cytometric analysis of CXCR4 of the breast cancer cells obtained from the lung tissues in the culture condition. The histogram with blue color indicates cancer cells in the lung in vivo, purple indicates ex vivo cultured cancer cells obtained from the lung, and red indicates cancer cells in the orthotopic tumor in vivo. Representative histograms are shown. Cells in the light green colored area are cells with a high expression level of cell surface CXCR4. (C) Percentage of the ex vivo cultured cancer cells with a high expression level of cell surface CXCR4 in the low (70–80% confluent) and high (100% confluent) cell densities. At least three analyses were performed at each time point. Asterisks indicate the significant difference between the two groups. (D) The schema and histograms of the xenotransplantation experiment of the ex vivo cultured cancer cells originally obtained from the orthotopic tumor and the lung. Histograms show the expression level of cell surface CXCR4 of the cancer cells obtained from the orthotopic tumor and the lung of the mice that were xenotransplanted with the ex vivo cultured cancer cells originally obtained from the orthotopic tumor or the lung. The red colored histograms indicate the cancer cells obtained from the orthotopic tumors, and blue colored histograms indicate the cancer cells obtained from the lung. Representative histograms are shown. (E) Summary of our findings. The change of the tumor environment causes the change of the expression level of CXCR4. This change facilitated the tumor growth.
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