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Real-time monitoring of tumorigenesis, dissemination, & drug response in a preclinical model of lymphangioleiomyomatosis/tuberous sclerosis complex.

Liu F, Lunsford EP, Tong J, Ashitate Y, Gibbs SL, Yu J, Choi HS, Henske EP, Frangioni JV - PLoS ONE (2012)

Bottom Line: Surprisingly, TSC2-deficient cells administered intratracheally resulted in rapid dissemination to lymph node basins throughout the body, and histopathological changes in the lung consistent with LAM.Estrogen was found to be permissive for tumor growth and dissemination.We generated homogeneous NIS/GFP co-expressing TSC2-deficient, patient-derived cells that can proliferate and migrate in vivo after intratracheal instillation.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: TSC2-deficient cells can proliferate in the lungs, kidneys, and other organs causing devastating progressive multisystem disorders such as lymphangioleiomyomatosis (LAM) and tuberous sclerosis complex (TSC). Preclinical models utilizing LAM patient-derived cells have been difficult to establish. We developed a novel animal model system to study the molecular mechanisms of TSC/LAM pathogenesis and tumorigenesis and provide a platform for drug testing.

Methods and findings: TSC2-deficient human cells, derived from the angiomyolipoma of a LAM patient, were engineered to co-express both sodium-iodide symporter (NIS) and green fluorescent protein (GFP). Cells were inoculated intraparenchymally, intravenously, or intratracheally into athymic NCr nu/nu mice and cells were tracked and quantified using single photon emission computed tomography (SPECT) and computed tomography (CT). Surprisingly, TSC2-deficient cells administered intratracheally resulted in rapid dissemination to lymph node basins throughout the body, and histopathological changes in the lung consistent with LAM. Estrogen was found to be permissive for tumor growth and dissemination. Rapamycin inhibited tumor growth, but tumors regrew after the drug treatment was withdrawn.

Conclusions: We generated homogeneous NIS/GFP co-expressing TSC2-deficient, patient-derived cells that can proliferate and migrate in vivo after intratracheal instillation. Although the animal model we describe has some limitations, we demonstrate that systemic tumors formed from TSC2-deficient cells can be monitored and quantified noninvasively over time using SPECT/CT, thus providing a much needed model system for in vivo drug testing and mechanistic studies of TSC2-deficient cells and their related clinical syndromes.

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Lymph node metastasis and invasion by TSC2-deficient cells.A. Normal lymph node (LN; top row) and lymph node identified by GFP and NIS expression (bottom row) 4 weeks after intratracheal administration of 621-327 cells. Left column shows in vivo color video image. Right columns show same nodes ex vivo after resection, placement on black paper, and imaging using color video, GFP fluorescence, and SPECT/CT, respectively. Scale bars = 1 mm. B. Hematoxylin and eosin (H&E) staining of frozen sections from normal (top row) and tumor-infiltrated (bottom row) lymph nodes 2 weeks after intratracheal administration of 621-327 cells. Dotted rectangle inset  =  higher magnification. Consecutive tissue sections were also stained with anti-GFP antibody. Scale bars = 50 µm. C. H&E staining of paraffin-embedded, tumor-infiltrated lymph nodes at 15 weeks post-administration of 621-327 cells. Dotted rectangle inset  =  higher magnification. Scale bars = 50 µm.
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pone-0038589-g003: Lymph node metastasis and invasion by TSC2-deficient cells.A. Normal lymph node (LN; top row) and lymph node identified by GFP and NIS expression (bottom row) 4 weeks after intratracheal administration of 621-327 cells. Left column shows in vivo color video image. Right columns show same nodes ex vivo after resection, placement on black paper, and imaging using color video, GFP fluorescence, and SPECT/CT, respectively. Scale bars = 1 mm. B. Hematoxylin and eosin (H&E) staining of frozen sections from normal (top row) and tumor-infiltrated (bottom row) lymph nodes 2 weeks after intratracheal administration of 621-327 cells. Dotted rectangle inset  =  higher magnification. Consecutive tissue sections were also stained with anti-GFP antibody. Scale bars = 50 µm. C. H&E staining of paraffin-embedded, tumor-infiltrated lymph nodes at 15 weeks post-administration of 621-327 cells. Dotted rectangle inset  =  higher magnification. Scale bars = 50 µm.

Mentions: Resection of tissue with high radiotracer uptake (Figure S2) revealed a pattern consistent with lymphatic or hematogenous spread from the lung to lymph node basins. Indeed, histopathological analysis of lymph nodes at early time points after intratracheal administration revealed NIS−/GFP-expressing tumor deposits within lymph nodes (Figure 3A). Early tumor nodules were often seen within semi-encapsulated structures in lymph nodes (Figure 3B). By weeks 3 to 4, most lymph nodes analyzed were completely obliterated by the tumor cells with abundant or medium cytoplasm (arrow head). Interestingly, by 15 weeks postinoculation, some tumor cells developed a smooth muscle cell morphology, and some lymph nodes developed cystic or fluid-filled structures, although these cysts are not histologically identical to those observed in human LAM (Figure 3C). The weak SPECT signal measured in living mouse lungs (Figure 2A), and the strong signal seen after sacrifice (Figure 4A) suggest that the signal-to-noise ratio in the lungs was significantly degraded by respiratory motion.


Real-time monitoring of tumorigenesis, dissemination, & drug response in a preclinical model of lymphangioleiomyomatosis/tuberous sclerosis complex.

Liu F, Lunsford EP, Tong J, Ashitate Y, Gibbs SL, Yu J, Choi HS, Henske EP, Frangioni JV - PLoS ONE (2012)

Lymph node metastasis and invasion by TSC2-deficient cells.A. Normal lymph node (LN; top row) and lymph node identified by GFP and NIS expression (bottom row) 4 weeks after intratracheal administration of 621-327 cells. Left column shows in vivo color video image. Right columns show same nodes ex vivo after resection, placement on black paper, and imaging using color video, GFP fluorescence, and SPECT/CT, respectively. Scale bars = 1 mm. B. Hematoxylin and eosin (H&E) staining of frozen sections from normal (top row) and tumor-infiltrated (bottom row) lymph nodes 2 weeks after intratracheal administration of 621-327 cells. Dotted rectangle inset  =  higher magnification. Consecutive tissue sections were also stained with anti-GFP antibody. Scale bars = 50 µm. C. H&E staining of paraffin-embedded, tumor-infiltrated lymph nodes at 15 weeks post-administration of 621-327 cells. Dotted rectangle inset  =  higher magnification. Scale bars = 50 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376142&req=5

pone-0038589-g003: Lymph node metastasis and invasion by TSC2-deficient cells.A. Normal lymph node (LN; top row) and lymph node identified by GFP and NIS expression (bottom row) 4 weeks after intratracheal administration of 621-327 cells. Left column shows in vivo color video image. Right columns show same nodes ex vivo after resection, placement on black paper, and imaging using color video, GFP fluorescence, and SPECT/CT, respectively. Scale bars = 1 mm. B. Hematoxylin and eosin (H&E) staining of frozen sections from normal (top row) and tumor-infiltrated (bottom row) lymph nodes 2 weeks after intratracheal administration of 621-327 cells. Dotted rectangle inset  =  higher magnification. Consecutive tissue sections were also stained with anti-GFP antibody. Scale bars = 50 µm. C. H&E staining of paraffin-embedded, tumor-infiltrated lymph nodes at 15 weeks post-administration of 621-327 cells. Dotted rectangle inset  =  higher magnification. Scale bars = 50 µm.
Mentions: Resection of tissue with high radiotracer uptake (Figure S2) revealed a pattern consistent with lymphatic or hematogenous spread from the lung to lymph node basins. Indeed, histopathological analysis of lymph nodes at early time points after intratracheal administration revealed NIS−/GFP-expressing tumor deposits within lymph nodes (Figure 3A). Early tumor nodules were often seen within semi-encapsulated structures in lymph nodes (Figure 3B). By weeks 3 to 4, most lymph nodes analyzed were completely obliterated by the tumor cells with abundant or medium cytoplasm (arrow head). Interestingly, by 15 weeks postinoculation, some tumor cells developed a smooth muscle cell morphology, and some lymph nodes developed cystic or fluid-filled structures, although these cysts are not histologically identical to those observed in human LAM (Figure 3C). The weak SPECT signal measured in living mouse lungs (Figure 2A), and the strong signal seen after sacrifice (Figure 4A) suggest that the signal-to-noise ratio in the lungs was significantly degraded by respiratory motion.

Bottom Line: Surprisingly, TSC2-deficient cells administered intratracheally resulted in rapid dissemination to lymph node basins throughout the body, and histopathological changes in the lung consistent with LAM.Estrogen was found to be permissive for tumor growth and dissemination.We generated homogeneous NIS/GFP co-expressing TSC2-deficient, patient-derived cells that can proliferate and migrate in vivo after intratracheal instillation.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: TSC2-deficient cells can proliferate in the lungs, kidneys, and other organs causing devastating progressive multisystem disorders such as lymphangioleiomyomatosis (LAM) and tuberous sclerosis complex (TSC). Preclinical models utilizing LAM patient-derived cells have been difficult to establish. We developed a novel animal model system to study the molecular mechanisms of TSC/LAM pathogenesis and tumorigenesis and provide a platform for drug testing.

Methods and findings: TSC2-deficient human cells, derived from the angiomyolipoma of a LAM patient, were engineered to co-express both sodium-iodide symporter (NIS) and green fluorescent protein (GFP). Cells were inoculated intraparenchymally, intravenously, or intratracheally into athymic NCr nu/nu mice and cells were tracked and quantified using single photon emission computed tomography (SPECT) and computed tomography (CT). Surprisingly, TSC2-deficient cells administered intratracheally resulted in rapid dissemination to lymph node basins throughout the body, and histopathological changes in the lung consistent with LAM. Estrogen was found to be permissive for tumor growth and dissemination. Rapamycin inhibited tumor growth, but tumors regrew after the drug treatment was withdrawn.

Conclusions: We generated homogeneous NIS/GFP co-expressing TSC2-deficient, patient-derived cells that can proliferate and migrate in vivo after intratracheal instillation. Although the animal model we describe has some limitations, we demonstrate that systemic tumors formed from TSC2-deficient cells can be monitored and quantified noninvasively over time using SPECT/CT, thus providing a much needed model system for in vivo drug testing and mechanistic studies of TSC2-deficient cells and their related clinical syndromes.

Show MeSH
Related in: MedlinePlus