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High neuropeptide Y release associates with Ewing sarcoma bone dissemination - in vivo model of site-specific metastases.

Hong SH, Tilan JU, Galli S, Izycka-Swieszewska E, Polk T, Horton M, Mahajan A, Christian D, Jenkins S, Acree R, Connors K, Ledo P, Lu C, Lee YC, Rodriguez O, Toretsky JA, Albanese C, Kitlinska J - Oncotarget (2015)

Bottom Line: Hypoxia up-regulates NPY and activates its pro-metastatic functions.To test the impact of NPY on ES metastatic pattern, ES cell lines, SK-ES1 and TC71, with high and low peptide release, respectively, were used in an orthotopic xenograft model.SK-ES1 xenografts resulted in thoracic extra-osseous metastases (67%) and dissemination to bone (50%) and brain (25%), while TC71 tumors metastasized to the lungs (70%).

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Georgetown University, Washington DC, USA.

ABSTRACT
Ewing sarcoma (ES) develops in bones or soft tissues of children and adolescents. The presence of bone metastases is one of the most adverse prognostic factors, yet the mechanisms governing their formation remain unclear. As a transcriptional target of EWS-FLI1, the fusion protein driving ES transformation, neuropeptide Y (NPY) is highly expressed and released from ES tumors. Hypoxia up-regulates NPY and activates its pro-metastatic functions. To test the impact of NPY on ES metastatic pattern, ES cell lines, SK-ES1 and TC71, with high and low peptide release, respectively, were used in an orthotopic xenograft model. ES cells were injected into gastrocnemius muscles of SCID/beige mice, the primary tumors excised, and mice monitored for the presence of metastases. SK-ES1 xenografts resulted in thoracic extra-osseous metastases (67%) and dissemination to bone (50%) and brain (25%), while TC71 tumors metastasized to the lungs (70%). Bone dissemination in SK-ES1 xenografts associated with increased NPY expression in bone metastases and its accumulation in bone invasion areas. The genetic silencing of NPY in SK-ES1 cells reduced bone degradation. Our study supports the role for NPY in ES bone invasion and provides new models for identifying pathways driving ES metastases to specific niches and testing anti-metastatic therapeutics.

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TC71 and SK-ES1 ES cells varying in NPY expression and release give rise to invasive primary tumorsA. NPY mRNA levels measured by real-time RT-PCR in SK-ES1 and TC71 cells cultured in vitro. B. NPY concentrations in conditioned media from SK-ES1 and TC71 cells measured by ELISA. C. TC71 and SK-ES1 cells were injected into gastrocnemius muscles of SCID/bg mice. NPY mRNA levels in primary tumors were measured by real-time RT-PCR. D. The growth of TC71 and SK-ES1 primary tumors was monitored by periodical measurements of the tumor-bearing hindlimb. E. Primary tumor mass of SK-ES1 xenograft. F. Positive staining of SK-ES1 primary tumor for the ES marker, CD99. G. Muscle invasion within SK-ES1 primary tumor. H. Bone degradation within SK-ES1 primary tumor. Panels E-H contain representative photographs of the SK-ES1 primary tumors. However, tumor morphology was similar in SK-ES1 and TC71 xenografts. T - tumor; H&E – hematoxylin and eosin staining.
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Figure 1: TC71 and SK-ES1 ES cells varying in NPY expression and release give rise to invasive primary tumorsA. NPY mRNA levels measured by real-time RT-PCR in SK-ES1 and TC71 cells cultured in vitro. B. NPY concentrations in conditioned media from SK-ES1 and TC71 cells measured by ELISA. C. TC71 and SK-ES1 cells were injected into gastrocnemius muscles of SCID/bg mice. NPY mRNA levels in primary tumors were measured by real-time RT-PCR. D. The growth of TC71 and SK-ES1 primary tumors was monitored by periodical measurements of the tumor-bearing hindlimb. E. Primary tumor mass of SK-ES1 xenograft. F. Positive staining of SK-ES1 primary tumor for the ES marker, CD99. G. Muscle invasion within SK-ES1 primary tumor. H. Bone degradation within SK-ES1 primary tumor. Panels E-H contain representative photographs of the SK-ES1 primary tumors. However, tumor morphology was similar in SK-ES1 and TC71 xenografts. T - tumor; H&E – hematoxylin and eosin staining.

Mentions: As an EWS-FLI1 target, NPY is universally expressed in ES [18-20]. However, ES cell lines vary significantly in their levels of NPY expression and release. To determine if high NPY secretion influences the pattern of metastases, we used two ES cell lines, SK-ES1 and TC71, which express high and low NPY levels, respectively (Fig. 1A). These differences in expression of the peptide translated to a variability in its release. Conditioned media from SK-ES1 cells contained high levels of NPY (average of 0.6 ng/ml/106 cells), while no secretion to the media was observed in TC71 cells (Fig. 1B).


High neuropeptide Y release associates with Ewing sarcoma bone dissemination - in vivo model of site-specific metastases.

Hong SH, Tilan JU, Galli S, Izycka-Swieszewska E, Polk T, Horton M, Mahajan A, Christian D, Jenkins S, Acree R, Connors K, Ledo P, Lu C, Lee YC, Rodriguez O, Toretsky JA, Albanese C, Kitlinska J - Oncotarget (2015)

TC71 and SK-ES1 ES cells varying in NPY expression and release give rise to invasive primary tumorsA. NPY mRNA levels measured by real-time RT-PCR in SK-ES1 and TC71 cells cultured in vitro. B. NPY concentrations in conditioned media from SK-ES1 and TC71 cells measured by ELISA. C. TC71 and SK-ES1 cells were injected into gastrocnemius muscles of SCID/bg mice. NPY mRNA levels in primary tumors were measured by real-time RT-PCR. D. The growth of TC71 and SK-ES1 primary tumors was monitored by periodical measurements of the tumor-bearing hindlimb. E. Primary tumor mass of SK-ES1 xenograft. F. Positive staining of SK-ES1 primary tumor for the ES marker, CD99. G. Muscle invasion within SK-ES1 primary tumor. H. Bone degradation within SK-ES1 primary tumor. Panels E-H contain representative photographs of the SK-ES1 primary tumors. However, tumor morphology was similar in SK-ES1 and TC71 xenografts. T - tumor; H&E – hematoxylin and eosin staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4466675&req=5

Figure 1: TC71 and SK-ES1 ES cells varying in NPY expression and release give rise to invasive primary tumorsA. NPY mRNA levels measured by real-time RT-PCR in SK-ES1 and TC71 cells cultured in vitro. B. NPY concentrations in conditioned media from SK-ES1 and TC71 cells measured by ELISA. C. TC71 and SK-ES1 cells were injected into gastrocnemius muscles of SCID/bg mice. NPY mRNA levels in primary tumors were measured by real-time RT-PCR. D. The growth of TC71 and SK-ES1 primary tumors was monitored by periodical measurements of the tumor-bearing hindlimb. E. Primary tumor mass of SK-ES1 xenograft. F. Positive staining of SK-ES1 primary tumor for the ES marker, CD99. G. Muscle invasion within SK-ES1 primary tumor. H. Bone degradation within SK-ES1 primary tumor. Panels E-H contain representative photographs of the SK-ES1 primary tumors. However, tumor morphology was similar in SK-ES1 and TC71 xenografts. T - tumor; H&E – hematoxylin and eosin staining.
Mentions: As an EWS-FLI1 target, NPY is universally expressed in ES [18-20]. However, ES cell lines vary significantly in their levels of NPY expression and release. To determine if high NPY secretion influences the pattern of metastases, we used two ES cell lines, SK-ES1 and TC71, which express high and low NPY levels, respectively (Fig. 1A). These differences in expression of the peptide translated to a variability in its release. Conditioned media from SK-ES1 cells contained high levels of NPY (average of 0.6 ng/ml/106 cells), while no secretion to the media was observed in TC71 cells (Fig. 1B).

Bottom Line: Hypoxia up-regulates NPY and activates its pro-metastatic functions.To test the impact of NPY on ES metastatic pattern, ES cell lines, SK-ES1 and TC71, with high and low peptide release, respectively, were used in an orthotopic xenograft model.SK-ES1 xenografts resulted in thoracic extra-osseous metastases (67%) and dissemination to bone (50%) and brain (25%), while TC71 tumors metastasized to the lungs (70%).

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Georgetown University, Washington DC, USA.

ABSTRACT
Ewing sarcoma (ES) develops in bones or soft tissues of children and adolescents. The presence of bone metastases is one of the most adverse prognostic factors, yet the mechanisms governing their formation remain unclear. As a transcriptional target of EWS-FLI1, the fusion protein driving ES transformation, neuropeptide Y (NPY) is highly expressed and released from ES tumors. Hypoxia up-regulates NPY and activates its pro-metastatic functions. To test the impact of NPY on ES metastatic pattern, ES cell lines, SK-ES1 and TC71, with high and low peptide release, respectively, were used in an orthotopic xenograft model. ES cells were injected into gastrocnemius muscles of SCID/beige mice, the primary tumors excised, and mice monitored for the presence of metastases. SK-ES1 xenografts resulted in thoracic extra-osseous metastases (67%) and dissemination to bone (50%) and brain (25%), while TC71 tumors metastasized to the lungs (70%). Bone dissemination in SK-ES1 xenografts associated with increased NPY expression in bone metastases and its accumulation in bone invasion areas. The genetic silencing of NPY in SK-ES1 cells reduced bone degradation. Our study supports the role for NPY in ES bone invasion and provides new models for identifying pathways driving ES metastases to specific niches and testing anti-metastatic therapeutics.

Show MeSH
Related in: MedlinePlus