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CD1d-expressing breast cancer cells modulate NKT cell-mediated antitumor immunity in a murine model of breast cancer metastasis.

Hix LM, Shi YH, Brutkiewicz RR, Stein PL, Wang CR, Zhang M - PLoS ONE (2011)

Bottom Line: Tumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer.We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression.The results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.

ABSTRACT

Background: Tumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer. A novel syngeneic mouse model of breast cancer metastasis was developed in our lab to investigate mechanisms of immune regulation of breast cancer. Comparative analysis of low-metastatic vs. highly metastatic tumor cells isolated from these mice revealed several important genetic alterations related to immune control of cancer, including a significant downregulation of cd1d1 in the highly metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells known as natural killer T (NKT) cells. We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression.

Methodology/principal findings: In this study, we demonstrate in a mouse model of breast cancer metastasis that tumor downregulation of CD1d inhibits iNKT-mediated antitumor immunity and promotes metastatic breast cancer progression in a CD1d-dependent manner in vitro and in vivo. Using NKT-deficient transgenic mouse models, we demonstrate important differences between type I and type II NKT cells in their ability to regulate antitumor immunity of CD1d-expressing breast tumors.

Conclusions/significance: The results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer.

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Decreased expression of CD1d in highly metastatic murine and human breast cancer cells.(A) Real-time RT-PCR assay confirming significant downregulation of the CD1d1 gene in TM40D-MB cells, as compared to parental TM40D (low metastatic) cells. L19 serves as an internal control. Experiments were performed in triplicate, and data are represented as the mean ± SEM, * P≤0.05. (B) Flow cytometry analysis of CD1d using a PE-conjugated anti-CD1d mAb (1B1) or isotype IgG2b control. Average ± SD mean fluorescence intensity (MFI) for three independent experiments: TM40D (blue)  = 928.7±21.2, TM40D-MB (red)  = 541.0±57, ** P≤0.001. (C) Decreased expression of CD1d in human mammary epithelial cells correlates with increasing metastatic potential by RT-PCR. 71N, 81N: normal transformed human mammary epithelial cells. 21PT, ZR75: primary breast adenocarcinoma cells. MCF-7: minimally invasive adenocarcinoma cells. MDA-MB-468, MDA-MB-231: highly metastatic human breast adenocarcinoma cells, from patients of African-American (MDA-MB-468) and Caucasian (MDA-MB-231) descent. GAPDH serves as an internal housekeeping gene control.
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pone-0020702-g001: Decreased expression of CD1d in highly metastatic murine and human breast cancer cells.(A) Real-time RT-PCR assay confirming significant downregulation of the CD1d1 gene in TM40D-MB cells, as compared to parental TM40D (low metastatic) cells. L19 serves as an internal control. Experiments were performed in triplicate, and data are represented as the mean ± SEM, * P≤0.05. (B) Flow cytometry analysis of CD1d using a PE-conjugated anti-CD1d mAb (1B1) or isotype IgG2b control. Average ± SD mean fluorescence intensity (MFI) for three independent experiments: TM40D (blue)  = 928.7±21.2, TM40D-MB (red)  = 541.0±57, ** P≤0.001. (C) Decreased expression of CD1d in human mammary epithelial cells correlates with increasing metastatic potential by RT-PCR. 71N, 81N: normal transformed human mammary epithelial cells. 21PT, ZR75: primary breast adenocarcinoma cells. MCF-7: minimally invasive adenocarcinoma cells. MDA-MB-468, MDA-MB-231: highly metastatic human breast adenocarcinoma cells, from patients of African-American (MDA-MB-468) and Caucasian (MDA-MB-231) descent. GAPDH serves as an internal housekeeping gene control.

Mentions: Several lines of evidence support the idea that a small population of primary tumor cells possess existing molecular signatures for metastasis [46], [47]. In order to identify potential metastatic signatures using our mouse model of breast cancer metastasis, TM40D breast cancer cells of low metastatic potential were compared to the highly metastatic TM40D-MB cells by microarray [8]. Among the genes that were found differentially expressed between these two cell lines, we found a 4.72-fold downregulation of the cd1d1 gene in the TM40D-MB cells. (Figure S1). This expression difference was validated by real-time RT-PCR, comparing amplified cDNA for the murine cd1d1 gene by RT-PCR between the TM40D and TM40D-MB cells (Fig. 1A). Downregulation of surface CD1d expression in the TM40D-MB cells was also confirmed by fluorescence-activated cell sorting (FACS) (Fig. 1B). CD1d is known to be expressed in human breast tissue, in both ductal epithelial and vascular smooth muscle cells [48]. Downregulated CD1d expression has been correlated with decreased iNKT-mediated antitumor immunity in several human and murine hematopoietic malignancies [24], [25], [49]. In human solid tumors, a correlation of downregulated CD1d expression with increasing malignancy has been reported in malignant glioma, and most recently HPV-transformed cervical carcinoma cells [29], [50]. In order to assess the importance of CD1d downregulation in human breast cancer, we analyzed a panel of human mammary epithelial cell lines of increasing metastatic potential by RT-PCR for their expression of CD1d (Fig. 1C). CD1d was found to be expressed in normal human mammary epithelial cells, and with the exception of MDA-MB-468, is downregulated in the transition from normal to malignant breast cancer. These results suggest that downregulation of CD1d expression in both murine and human breast cancer may be an important mechanism for evading tumor immune surveillance and promoting metastatic cancer progression.


CD1d-expressing breast cancer cells modulate NKT cell-mediated antitumor immunity in a murine model of breast cancer metastasis.

Hix LM, Shi YH, Brutkiewicz RR, Stein PL, Wang CR, Zhang M - PLoS ONE (2011)

Decreased expression of CD1d in highly metastatic murine and human breast cancer cells.(A) Real-time RT-PCR assay confirming significant downregulation of the CD1d1 gene in TM40D-MB cells, as compared to parental TM40D (low metastatic) cells. L19 serves as an internal control. Experiments were performed in triplicate, and data are represented as the mean ± SEM, * P≤0.05. (B) Flow cytometry analysis of CD1d using a PE-conjugated anti-CD1d mAb (1B1) or isotype IgG2b control. Average ± SD mean fluorescence intensity (MFI) for three independent experiments: TM40D (blue)  = 928.7±21.2, TM40D-MB (red)  = 541.0±57, ** P≤0.001. (C) Decreased expression of CD1d in human mammary epithelial cells correlates with increasing metastatic potential by RT-PCR. 71N, 81N: normal transformed human mammary epithelial cells. 21PT, ZR75: primary breast adenocarcinoma cells. MCF-7: minimally invasive adenocarcinoma cells. MDA-MB-468, MDA-MB-231: highly metastatic human breast adenocarcinoma cells, from patients of African-American (MDA-MB-468) and Caucasian (MDA-MB-231) descent. GAPDH serves as an internal housekeeping gene control.
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pone-0020702-g001: Decreased expression of CD1d in highly metastatic murine and human breast cancer cells.(A) Real-time RT-PCR assay confirming significant downregulation of the CD1d1 gene in TM40D-MB cells, as compared to parental TM40D (low metastatic) cells. L19 serves as an internal control. Experiments were performed in triplicate, and data are represented as the mean ± SEM, * P≤0.05. (B) Flow cytometry analysis of CD1d using a PE-conjugated anti-CD1d mAb (1B1) or isotype IgG2b control. Average ± SD mean fluorescence intensity (MFI) for three independent experiments: TM40D (blue)  = 928.7±21.2, TM40D-MB (red)  = 541.0±57, ** P≤0.001. (C) Decreased expression of CD1d in human mammary epithelial cells correlates with increasing metastatic potential by RT-PCR. 71N, 81N: normal transformed human mammary epithelial cells. 21PT, ZR75: primary breast adenocarcinoma cells. MCF-7: minimally invasive adenocarcinoma cells. MDA-MB-468, MDA-MB-231: highly metastatic human breast adenocarcinoma cells, from patients of African-American (MDA-MB-468) and Caucasian (MDA-MB-231) descent. GAPDH serves as an internal housekeeping gene control.
Mentions: Several lines of evidence support the idea that a small population of primary tumor cells possess existing molecular signatures for metastasis [46], [47]. In order to identify potential metastatic signatures using our mouse model of breast cancer metastasis, TM40D breast cancer cells of low metastatic potential were compared to the highly metastatic TM40D-MB cells by microarray [8]. Among the genes that were found differentially expressed between these two cell lines, we found a 4.72-fold downregulation of the cd1d1 gene in the TM40D-MB cells. (Figure S1). This expression difference was validated by real-time RT-PCR, comparing amplified cDNA for the murine cd1d1 gene by RT-PCR between the TM40D and TM40D-MB cells (Fig. 1A). Downregulation of surface CD1d expression in the TM40D-MB cells was also confirmed by fluorescence-activated cell sorting (FACS) (Fig. 1B). CD1d is known to be expressed in human breast tissue, in both ductal epithelial and vascular smooth muscle cells [48]. Downregulated CD1d expression has been correlated with decreased iNKT-mediated antitumor immunity in several human and murine hematopoietic malignancies [24], [25], [49]. In human solid tumors, a correlation of downregulated CD1d expression with increasing malignancy has been reported in malignant glioma, and most recently HPV-transformed cervical carcinoma cells [29], [50]. In order to assess the importance of CD1d downregulation in human breast cancer, we analyzed a panel of human mammary epithelial cell lines of increasing metastatic potential by RT-PCR for their expression of CD1d (Fig. 1C). CD1d was found to be expressed in normal human mammary epithelial cells, and with the exception of MDA-MB-468, is downregulated in the transition from normal to malignant breast cancer. These results suggest that downregulation of CD1d expression in both murine and human breast cancer may be an important mechanism for evading tumor immune surveillance and promoting metastatic cancer progression.

Bottom Line: Tumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer.We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression.The results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.

ABSTRACT

Background: Tumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer. A novel syngeneic mouse model of breast cancer metastasis was developed in our lab to investigate mechanisms of immune regulation of breast cancer. Comparative analysis of low-metastatic vs. highly metastatic tumor cells isolated from these mice revealed several important genetic alterations related to immune control of cancer, including a significant downregulation of cd1d1 in the highly metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells known as natural killer T (NKT) cells. We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression.

Methodology/principal findings: In this study, we demonstrate in a mouse model of breast cancer metastasis that tumor downregulation of CD1d inhibits iNKT-mediated antitumor immunity and promotes metastatic breast cancer progression in a CD1d-dependent manner in vitro and in vivo. Using NKT-deficient transgenic mouse models, we demonstrate important differences between type I and type II NKT cells in their ability to regulate antitumor immunity of CD1d-expressing breast tumors.

Conclusions/significance: The results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer.

Show MeSH
Related in: MedlinePlus