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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 Rapamycin inhibited tumor growth, but tumors regrew after the drug treatment was withdrawn.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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Generation and characterization of in vivo trackable TSC2-deficient cells.A. Expression cassettes of adenoviral (Ad-NIS/GFP) and retroviral (Retro-NIS/GFP) vectors. CMV  =  cytomegalovirus promoter, NIS  =  sodium-iodide symporter, LITR  =  left-handed inverted terminal repeat, RITR  =  right-handed inverted terminal repeat, GFP  =  green fluorescent protein, ΔE1, ΔE3 =  E1 and E3 deletions of adenovirus type 5 (Ad5) backbone sequence, 5′ LTR = 5′ long terminal repeat, φ signal  =  virus packing signal, IRES  =  internal ribosome entry site, 3′ LTR  = 3′ long terminal repeat. B. In vitro uptake of 99mTc-pertechnetate and GFP fluorescence in stable Retro-NIS−/GFP-expressing 621-327 cells (top row) and control 621-101 cells (bottom row). Scale bar = 6 mm. C. Immunofluorescent detection of GFP, NIS, and tuberin in TSC2-deficient 621-327 cells (top row), control 621-101 cells (middle row), and HeLa cells (bottom row). GFP fluorescence (2nd column), staining with primary anti-NIS specific antibody (3rd column) or anti-tuberin antibody (right column) with secondary Cy3 antibody conjugates is shown along with phase contrast (left column). Scale bar = 50 µm. D. Western blot analysis of TSC2-expressing HEK293T control cells, and TSC2-deficient 621-101 cells and 621-327 cells, using antibodies to key signaling proteins, NIS, and GFP. A beta actin loading control is also shown, as are long exposure times (long exp.) for tuberin and hamartin.
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pone-0038589-g001: Generation and characterization of in vivo trackable TSC2-deficient cells.A. Expression cassettes of adenoviral (Ad-NIS/GFP) and retroviral (Retro-NIS/GFP) vectors. CMV  =  cytomegalovirus promoter, NIS  =  sodium-iodide symporter, LITR  =  left-handed inverted terminal repeat, RITR  =  right-handed inverted terminal repeat, GFP  =  green fluorescent protein, ΔE1, ΔE3 =  E1 and E3 deletions of adenovirus type 5 (Ad5) backbone sequence, 5′ LTR = 5′ long terminal repeat, φ signal  =  virus packing signal, IRES  =  internal ribosome entry site, 3′ LTR  = 3′ long terminal repeat. B. In vitro uptake of 99mTc-pertechnetate and GFP fluorescence in stable Retro-NIS−/GFP-expressing 621-327 cells (top row) and control 621-101 cells (bottom row). Scale bar = 6 mm. C. Immunofluorescent detection of GFP, NIS, and tuberin in TSC2-deficient 621-327 cells (top row), control 621-101 cells (middle row), and HeLa cells (bottom row). GFP fluorescence (2nd column), staining with primary anti-NIS specific antibody (3rd column) or anti-tuberin antibody (right column) with secondary Cy3 antibody conjugates is shown along with phase contrast (left column). Scale bar = 50 µm. D. Western blot analysis of TSC2-expressing HEK293T control cells, and TSC2-deficient 621-101 cells and 621-327 cells, using antibodies to key signaling proteins, NIS, and GFP. A beta actin loading control is also shown, as are long exposure times (long exp.) for tuberin and hamartin.

Mentions: Adenoviral and retroviral gene delivery vectors (Figure 1A) were used to deliver NIS and GFP genes into human cell line 621-101, which was derived from a LAM-associated angiomyolipoma. After initial proof of principle using adenovirus expression, retrovirus expression was used exclusively. Retrovirus-transduced NIS/GFP cells were selected and subcloned. A stable cell line designated 621-327 was obtained. Clonal 621-327 cells were adapted to grow in DMEM/F12 medium supplemented with 15% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL). No EGF or other additives were added into the cell culture medium. 621-327 cells characterized by simultaneous expression of GFP and NIS are capable of mediating radiotracer 99mTcO4- uptake, while the control 621-101 cells were not (Figure 1B). After continuous subculturing of a vial of these frozen cells for over 6 months, the tested 621-327 cells showed stable high-level expression of functional NIS and GFP, but low-level expression of tuberin (Figure 1C), and retained homogeneous morphology.


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)

Generation and characterization of in vivo trackable TSC2-deficient cells.A. Expression cassettes of adenoviral (Ad-NIS/GFP) and retroviral (Retro-NIS/GFP) vectors. CMV  =  cytomegalovirus promoter, NIS  =  sodium-iodide symporter, LITR  =  left-handed inverted terminal repeat, RITR  =  right-handed inverted terminal repeat, GFP  =  green fluorescent protein, ΔE1, ΔE3 =  E1 and E3 deletions of adenovirus type 5 (Ad5) backbone sequence, 5′ LTR = 5′ long terminal repeat, φ signal  =  virus packing signal, IRES  =  internal ribosome entry site, 3′ LTR  = 3′ long terminal repeat. B. In vitro uptake of 99mTc-pertechnetate and GFP fluorescence in stable Retro-NIS−/GFP-expressing 621-327 cells (top row) and control 621-101 cells (bottom row). Scale bar = 6 mm. C. Immunofluorescent detection of GFP, NIS, and tuberin in TSC2-deficient 621-327 cells (top row), control 621-101 cells (middle row), and HeLa cells (bottom row). GFP fluorescence (2nd column), staining with primary anti-NIS specific antibody (3rd column) or anti-tuberin antibody (right column) with secondary Cy3 antibody conjugates is shown along with phase contrast (left column). Scale bar = 50 µm. D. Western blot analysis of TSC2-expressing HEK293T control cells, and TSC2-deficient 621-101 cells and 621-327 cells, using antibodies to key signaling proteins, NIS, and GFP. A beta actin loading control is also shown, as are long exposure times (long exp.) for tuberin and hamartin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376142&req=5

pone-0038589-g001: Generation and characterization of in vivo trackable TSC2-deficient cells.A. Expression cassettes of adenoviral (Ad-NIS/GFP) and retroviral (Retro-NIS/GFP) vectors. CMV  =  cytomegalovirus promoter, NIS  =  sodium-iodide symporter, LITR  =  left-handed inverted terminal repeat, RITR  =  right-handed inverted terminal repeat, GFP  =  green fluorescent protein, ΔE1, ΔE3 =  E1 and E3 deletions of adenovirus type 5 (Ad5) backbone sequence, 5′ LTR = 5′ long terminal repeat, φ signal  =  virus packing signal, IRES  =  internal ribosome entry site, 3′ LTR  = 3′ long terminal repeat. B. In vitro uptake of 99mTc-pertechnetate and GFP fluorescence in stable Retro-NIS−/GFP-expressing 621-327 cells (top row) and control 621-101 cells (bottom row). Scale bar = 6 mm. C. Immunofluorescent detection of GFP, NIS, and tuberin in TSC2-deficient 621-327 cells (top row), control 621-101 cells (middle row), and HeLa cells (bottom row). GFP fluorescence (2nd column), staining with primary anti-NIS specific antibody (3rd column) or anti-tuberin antibody (right column) with secondary Cy3 antibody conjugates is shown along with phase contrast (left column). Scale bar = 50 µm. D. Western blot analysis of TSC2-expressing HEK293T control cells, and TSC2-deficient 621-101 cells and 621-327 cells, using antibodies to key signaling proteins, NIS, and GFP. A beta actin loading control is also shown, as are long exposure times (long exp.) for tuberin and hamartin.
Mentions: Adenoviral and retroviral gene delivery vectors (Figure 1A) were used to deliver NIS and GFP genes into human cell line 621-101, which was derived from a LAM-associated angiomyolipoma. After initial proof of principle using adenovirus expression, retrovirus expression was used exclusively. Retrovirus-transduced NIS/GFP cells were selected and subcloned. A stable cell line designated 621-327 was obtained. Clonal 621-327 cells were adapted to grow in DMEM/F12 medium supplemented with 15% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL). No EGF or other additives were added into the cell culture medium. 621-327 cells characterized by simultaneous expression of GFP and NIS are capable of mediating radiotracer 99mTcO4- uptake, while the control 621-101 cells were not (Figure 1B). After continuous subculturing of a vial of these frozen cells for over 6 months, the tested 621-327 cells showed stable high-level expression of functional NIS and GFP, but low-level expression of tuberin (Figure 1C), and retained homogeneous morphology.

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