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Imagable 4T1 model for the study of late stage breast cancer.

Tao K, Fang M, Alroy J, Sahagian GG - BMC Cancer (2008)

Bottom Line: Biophotonic imaging was used to characterize growth and metastasis of the lines in vivo and an improved gene expression approach was used to characterize the basis for the metastatic phenotype that was observed.Regression of growth, which occurred in weeks 3-4, was associated with extensive necrosis and infiltration of leukocytes.The new cell lines will greatly facilitate the study of late stage breast and preclinical assessment of cancer drugs and other therapeutics particularly those targeting immune system effects on tumor metastasis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA. kai.tao@tufts.edu

ABSTRACT

Background: The 4T1 mouse mammary tumor cell line is one of only a few breast cancer models with the capacity to metastasize efficiently to sites affected in human breast cancer. Here we describe two 4T1 cell lines modified to facilitate analysis of tumor growth and metastasis and evaluation of gene function in vivo. New information regarding the involvement of innate and acquired immunity in metastasis and other characteristics of the model relevant to its use in the study of late stage breast cancer are reported.

Methods: The lines were engineered for stable expression of firefly luciferase to allow tracking and quantitation of the cells in vivo. Biophotonic imaging was used to characterize growth and metastasis of the lines in vivo and an improved gene expression approach was used to characterize the basis for the metastatic phenotype that was observed.

Results: Growth of cells at the primary site was biphasic with metastasis detected during the second growth phase 5-6 weeks after introduction of the cells. Regression of growth, which occurred in weeks 3-4, was associated with extensive necrosis and infiltration of leukocytes. Biphasic tumor growth did not occur in BALB/c SCID mice indicating involvement of an acquired immune response in the effect. Hematopoiesis in spleen and liver and elevated levels of circulating leukocytes were observed at week 2 and increased progressively until death at week 6-8. Gene expression analysis revealed an association of several secreted factors including colony stimulatory factors, cytokines and chemokines, acute phase proteins, angiogenesis factors and ECM modifying proteins with the 4T1 metastatic phenotype. Signaling pathways likely to be responsible for production of these factors were also identified.

Conclusion: The production of factors that stimulate angiogenesis and ECM modification and induce hematopoiesis, recruitment and activation of leukocytes suggest that 4T1 tumor cells play a more direct role than previously appreciated in orchestrating changes in the tumor environment conducive to tumor cell dissemination and metastasis. The new cell lines will greatly facilitate the study of late stage breast and preclinical assessment of cancer drugs and other therapeutics particularly those targeting immune system effects on tumor metastasis.

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4T1 genes categorized by cellular location and function. Genes associated with the 4T1 metastatic phenotype that fall into the categories shown are listed in the figure. Genes shown in red are elevated in 4T1 and genes shown in blue reduced in 4T1. The blue line to the right of middle represents the plasma membrane with genes falling to the right of it representing secreted genes. The blue rectangle to the left of middle represents intracellular membranes and genes falling inside the rectangle are genes located within intracellular organelles.
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Figure 10: 4T1 genes categorized by cellular location and function. Genes associated with the 4T1 metastatic phenotype that fall into the categories shown are listed in the figure. Genes shown in red are elevated in 4T1 and genes shown in blue reduced in 4T1. The blue line to the right of middle represents the plasma membrane with genes falling to the right of it representing secreted genes. The blue rectangle to the left of middle represents intracellular membranes and genes falling inside the rectangle are genes located within intracellular organelles.

Mentions: Genes differentially expressed in 4T1 were categorized with respect to cellular location and function (Fig. 10). Among the genes are substantial numbers involved in cell adhesion, migration, angiogenesis, and extracellular matrix modification; cytoskeleton function; cell proliferation, apoptosis and survival; cellular metabolism; and inflammation and immune response. Altered expression of several transcription factors and genes involved in chromatin modification that regulate these processes were also observed. Elevated expression of genes associated with tight junctions (Cldn3, Cldn4, Cldn7 and Tjp2), adherins junctions (Cdh1 and Vil1) focal adhesions (Itga3, Itga6 and Lama5), and intermediate filaments (Krt1-18 and Krt2-7) indicate that the 4T1 line has greater epithelial character than the non-metastatic lines. An increased propensity for extracellular matrix (ECM) remodeling is suggested by elevated expression of matrix metalloproteinases (Mmp3, Mmp9 and Mmp13), urokinase-type plasminogen activator (Plau) and secreted protease inhibitors (Serpina3g, Serpin2 and Lcn2).


Imagable 4T1 model for the study of late stage breast cancer.

Tao K, Fang M, Alroy J, Sahagian GG - BMC Cancer (2008)

4T1 genes categorized by cellular location and function. Genes associated with the 4T1 metastatic phenotype that fall into the categories shown are listed in the figure. Genes shown in red are elevated in 4T1 and genes shown in blue reduced in 4T1. The blue line to the right of middle represents the plasma membrane with genes falling to the right of it representing secreted genes. The blue rectangle to the left of middle represents intracellular membranes and genes falling inside the rectangle are genes located within intracellular organelles.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2529338&req=5

Figure 10: 4T1 genes categorized by cellular location and function. Genes associated with the 4T1 metastatic phenotype that fall into the categories shown are listed in the figure. Genes shown in red are elevated in 4T1 and genes shown in blue reduced in 4T1. The blue line to the right of middle represents the plasma membrane with genes falling to the right of it representing secreted genes. The blue rectangle to the left of middle represents intracellular membranes and genes falling inside the rectangle are genes located within intracellular organelles.
Mentions: Genes differentially expressed in 4T1 were categorized with respect to cellular location and function (Fig. 10). Among the genes are substantial numbers involved in cell adhesion, migration, angiogenesis, and extracellular matrix modification; cytoskeleton function; cell proliferation, apoptosis and survival; cellular metabolism; and inflammation and immune response. Altered expression of several transcription factors and genes involved in chromatin modification that regulate these processes were also observed. Elevated expression of genes associated with tight junctions (Cldn3, Cldn4, Cldn7 and Tjp2), adherins junctions (Cdh1 and Vil1) focal adhesions (Itga3, Itga6 and Lama5), and intermediate filaments (Krt1-18 and Krt2-7) indicate that the 4T1 line has greater epithelial character than the non-metastatic lines. An increased propensity for extracellular matrix (ECM) remodeling is suggested by elevated expression of matrix metalloproteinases (Mmp3, Mmp9 and Mmp13), urokinase-type plasminogen activator (Plau) and secreted protease inhibitors (Serpina3g, Serpin2 and Lcn2).

Bottom Line: Biophotonic imaging was used to characterize growth and metastasis of the lines in vivo and an improved gene expression approach was used to characterize the basis for the metastatic phenotype that was observed.Regression of growth, which occurred in weeks 3-4, was associated with extensive necrosis and infiltration of leukocytes.The new cell lines will greatly facilitate the study of late stage breast and preclinical assessment of cancer drugs and other therapeutics particularly those targeting immune system effects on tumor metastasis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA. kai.tao@tufts.edu

ABSTRACT

Background: The 4T1 mouse mammary tumor cell line is one of only a few breast cancer models with the capacity to metastasize efficiently to sites affected in human breast cancer. Here we describe two 4T1 cell lines modified to facilitate analysis of tumor growth and metastasis and evaluation of gene function in vivo. New information regarding the involvement of innate and acquired immunity in metastasis and other characteristics of the model relevant to its use in the study of late stage breast cancer are reported.

Methods: The lines were engineered for stable expression of firefly luciferase to allow tracking and quantitation of the cells in vivo. Biophotonic imaging was used to characterize growth and metastasis of the lines in vivo and an improved gene expression approach was used to characterize the basis for the metastatic phenotype that was observed.

Results: Growth of cells at the primary site was biphasic with metastasis detected during the second growth phase 5-6 weeks after introduction of the cells. Regression of growth, which occurred in weeks 3-4, was associated with extensive necrosis and infiltration of leukocytes. Biphasic tumor growth did not occur in BALB/c SCID mice indicating involvement of an acquired immune response in the effect. Hematopoiesis in spleen and liver and elevated levels of circulating leukocytes were observed at week 2 and increased progressively until death at week 6-8. Gene expression analysis revealed an association of several secreted factors including colony stimulatory factors, cytokines and chemokines, acute phase proteins, angiogenesis factors and ECM modifying proteins with the 4T1 metastatic phenotype. Signaling pathways likely to be responsible for production of these factors were also identified.

Conclusion: The production of factors that stimulate angiogenesis and ECM modification and induce hematopoiesis, recruitment and activation of leukocytes suggest that 4T1 tumor cells play a more direct role than previously appreciated in orchestrating changes in the tumor environment conducive to tumor cell dissemination and metastasis. The new cell lines will greatly facilitate the study of late stage breast and preclinical assessment of cancer drugs and other therapeutics particularly those targeting immune system effects on tumor metastasis.

Show MeSH
Related in: MedlinePlus