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Pinhole micro-SPECT/CT for noninvasive monitoring and quantitation of oncolytic virus dispersion and percent infection in solid tumors.

Penheiter AR, Griesmann GE, Federspiel MJ, Dingli D, Russell SJ, Carlson SK - Gene Ther. (2011)

Bottom Line: The purpose of our study was to validate the ability of pinhole micro-single-photon emission computed tomography/computed tomography (SPECT/CT) to: 1) accurately resolve the intratumoral dispersion pattern and 2) quantify the infection percentage in solid tumors of an oncolytic measles virus encoding the human sodium iodide symporter (MV-NIS).We reliably resolved multiple distinct intratumoral zones of infection from non-infected regions.Pinhole micro-SPECT/CT imaging using the NIS reporter demonstrated precise localization and quantitation of oncolytic MV-NIS infection, and can replace more time-consuming and expensive analyses (for example, autoradiography and IHC) that require animal killing.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905, USA.

ABSTRACT
The purpose of our study was to validate the ability of pinhole micro-single-photon emission computed tomography/computed tomography (SPECT/CT) to: 1) accurately resolve the intratumoral dispersion pattern and 2) quantify the infection percentage in solid tumors of an oncolytic measles virus encoding the human sodium iodide symporter (MV-NIS). Sodium iodide symporter (NIS) RNA level and dispersion pattern were determined in control and MV-NIS-infected BxPC-3 pancreatic tumor cells and mouse xenografts using quantitative, real-time, reverse transcriptase, polymerase chain reaction, autoradiography and immunohistochemistry (IHC). Mice with BxPC-3 xenografts were imaged with (123)I or (99)TcO(4) micro-SPECT/CT. Tumor dimensions and radionuclide localization were determined with imaging software. Linear regression and correlation analyses were performed to determine the relationship between tumor infection percentage and radionuclide uptake (% injected dose per gram) above background and a highly significant correlation was observed (r(2)=0.947). A detection threshold of 1.5-fold above the control tumor uptake (background) yielded a sensitivity of 2.7% MV-NIS-infected tumor cells. We reliably resolved multiple distinct intratumoral zones of infection from non-infected regions. Pinhole micro-SPECT/CT imaging using the NIS reporter demonstrated precise localization and quantitation of oncolytic MV-NIS infection, and can replace more time-consuming and expensive analyses (for example, autoradiography and IHC) that require animal killing.

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A, Human NIS RNA Levels after Infection with MV-NIS. Tumor homogenates were created from subcutaneous tumors in nude mice (7 MV-NIS–injected tumors and 7 noninjected tumors from the opposite flank). Cultured BxPC-3 cells were optimally infected with MV-NIS. RNA levels were measured by qRT-PCR. The level of NIS RNA/μg in opposite flank tumors and uninfected BxPC-3 cells in vitro was not significantly different. NIS RNA/μg in MV-NIS injected tumors was significantly different than the level in opposite flank tumors (p < 0.01 by t-test). B, Linear regression of 123I uptake (%ID/g) in infected and uninfected BxPC-3 subcutaneous dual flank tumors (n = 7 mice) versus percent infected BxPC-3 cells (as calculated by RNA yield). The dotted line shows the detection sensitivity threshold value (3.55 %ID/g, 1.5 times y-intercept of the regression line) (r2 of linear regression = 0.872, p < 0.001). This corresponded to 2.7% infected tumor cells. %ID/g denotes percent injected dose per gram; MV-NIS, measles virus expressing human NIS; NIS, sodium iodide symporter; qRT-PCR, quantitative, real-time, reverse transcriptase, polymerase chain reaction.
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Figure 1: A, Human NIS RNA Levels after Infection with MV-NIS. Tumor homogenates were created from subcutaneous tumors in nude mice (7 MV-NIS–injected tumors and 7 noninjected tumors from the opposite flank). Cultured BxPC-3 cells were optimally infected with MV-NIS. RNA levels were measured by qRT-PCR. The level of NIS RNA/μg in opposite flank tumors and uninfected BxPC-3 cells in vitro was not significantly different. NIS RNA/μg in MV-NIS injected tumors was significantly different than the level in opposite flank tumors (p < 0.01 by t-test). B, Linear regression of 123I uptake (%ID/g) in infected and uninfected BxPC-3 subcutaneous dual flank tumors (n = 7 mice) versus percent infected BxPC-3 cells (as calculated by RNA yield). The dotted line shows the detection sensitivity threshold value (3.55 %ID/g, 1.5 times y-intercept of the regression line) (r2 of linear regression = 0.872, p < 0.001). This corresponded to 2.7% infected tumor cells. %ID/g denotes percent injected dose per gram; MV-NIS, measles virus expressing human NIS; NIS, sodium iodide symporter; qRT-PCR, quantitative, real-time, reverse transcriptase, polymerase chain reaction.

Mentions: By using quantitative, real-time, reverse transcriptase, polymerase chain reaction (qRT-PCR), we determined the total number of NIS RNA copies per cell in control and maximally MV-NIS-infected BxPC-3 human pancreatic cancer cells in vitro. Control (uninfected) BxPC-3 cells contained 8.28 × 103 ± 1.01 × 103 NIS RNA copies per μg of total RNA. BxPC-3 cells infected under conditions that yielded maximal radioiodide uptake (multiplicity of infection, 0.1 at 48 h) contained 5.29 × 107 ± 2.18 × 107 NIS RNA copies/ μg (Figure 1A). The total yield of NIS RNA from control BxPC-3 cells was 19.33 pg/cell. From this number, we calculated that uninfected BxPC-3 cells expressed 0.16 copies of NIS RNA per cell and optimally MV-NIS-infected BxPC-3 cells expressed 1,023 copies of NIS RNA/cell.


Pinhole micro-SPECT/CT for noninvasive monitoring and quantitation of oncolytic virus dispersion and percent infection in solid tumors.

Penheiter AR, Griesmann GE, Federspiel MJ, Dingli D, Russell SJ, Carlson SK - Gene Ther. (2011)

A, Human NIS RNA Levels after Infection with MV-NIS. Tumor homogenates were created from subcutaneous tumors in nude mice (7 MV-NIS–injected tumors and 7 noninjected tumors from the opposite flank). Cultured BxPC-3 cells were optimally infected with MV-NIS. RNA levels were measured by qRT-PCR. The level of NIS RNA/μg in opposite flank tumors and uninfected BxPC-3 cells in vitro was not significantly different. NIS RNA/μg in MV-NIS injected tumors was significantly different than the level in opposite flank tumors (p < 0.01 by t-test). B, Linear regression of 123I uptake (%ID/g) in infected and uninfected BxPC-3 subcutaneous dual flank tumors (n = 7 mice) versus percent infected BxPC-3 cells (as calculated by RNA yield). The dotted line shows the detection sensitivity threshold value (3.55 %ID/g, 1.5 times y-intercept of the regression line) (r2 of linear regression = 0.872, p < 0.001). This corresponded to 2.7% infected tumor cells. %ID/g denotes percent injected dose per gram; MV-NIS, measles virus expressing human NIS; NIS, sodium iodide symporter; qRT-PCR, quantitative, real-time, reverse transcriptase, polymerase chain reaction.
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Related In: Results  -  Collection

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Figure 1: A, Human NIS RNA Levels after Infection with MV-NIS. Tumor homogenates were created from subcutaneous tumors in nude mice (7 MV-NIS–injected tumors and 7 noninjected tumors from the opposite flank). Cultured BxPC-3 cells were optimally infected with MV-NIS. RNA levels were measured by qRT-PCR. The level of NIS RNA/μg in opposite flank tumors and uninfected BxPC-3 cells in vitro was not significantly different. NIS RNA/μg in MV-NIS injected tumors was significantly different than the level in opposite flank tumors (p < 0.01 by t-test). B, Linear regression of 123I uptake (%ID/g) in infected and uninfected BxPC-3 subcutaneous dual flank tumors (n = 7 mice) versus percent infected BxPC-3 cells (as calculated by RNA yield). The dotted line shows the detection sensitivity threshold value (3.55 %ID/g, 1.5 times y-intercept of the regression line) (r2 of linear regression = 0.872, p < 0.001). This corresponded to 2.7% infected tumor cells. %ID/g denotes percent injected dose per gram; MV-NIS, measles virus expressing human NIS; NIS, sodium iodide symporter; qRT-PCR, quantitative, real-time, reverse transcriptase, polymerase chain reaction.
Mentions: By using quantitative, real-time, reverse transcriptase, polymerase chain reaction (qRT-PCR), we determined the total number of NIS RNA copies per cell in control and maximally MV-NIS-infected BxPC-3 human pancreatic cancer cells in vitro. Control (uninfected) BxPC-3 cells contained 8.28 × 103 ± 1.01 × 103 NIS RNA copies per μg of total RNA. BxPC-3 cells infected under conditions that yielded maximal radioiodide uptake (multiplicity of infection, 0.1 at 48 h) contained 5.29 × 107 ± 2.18 × 107 NIS RNA copies/ μg (Figure 1A). The total yield of NIS RNA from control BxPC-3 cells was 19.33 pg/cell. From this number, we calculated that uninfected BxPC-3 cells expressed 0.16 copies of NIS RNA per cell and optimally MV-NIS-infected BxPC-3 cells expressed 1,023 copies of NIS RNA/cell.

Bottom Line: The purpose of our study was to validate the ability of pinhole micro-single-photon emission computed tomography/computed tomography (SPECT/CT) to: 1) accurately resolve the intratumoral dispersion pattern and 2) quantify the infection percentage in solid tumors of an oncolytic measles virus encoding the human sodium iodide symporter (MV-NIS).We reliably resolved multiple distinct intratumoral zones of infection from non-infected regions.Pinhole micro-SPECT/CT imaging using the NIS reporter demonstrated precise localization and quantitation of oncolytic MV-NIS infection, and can replace more time-consuming and expensive analyses (for example, autoradiography and IHC) that require animal killing.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905, USA.

ABSTRACT
The purpose of our study was to validate the ability of pinhole micro-single-photon emission computed tomography/computed tomography (SPECT/CT) to: 1) accurately resolve the intratumoral dispersion pattern and 2) quantify the infection percentage in solid tumors of an oncolytic measles virus encoding the human sodium iodide symporter (MV-NIS). Sodium iodide symporter (NIS) RNA level and dispersion pattern were determined in control and MV-NIS-infected BxPC-3 pancreatic tumor cells and mouse xenografts using quantitative, real-time, reverse transcriptase, polymerase chain reaction, autoradiography and immunohistochemistry (IHC). Mice with BxPC-3 xenografts were imaged with (123)I or (99)TcO(4) micro-SPECT/CT. Tumor dimensions and radionuclide localization were determined with imaging software. Linear regression and correlation analyses were performed to determine the relationship between tumor infection percentage and radionuclide uptake (% injected dose per gram) above background and a highly significant correlation was observed (r(2)=0.947). A detection threshold of 1.5-fold above the control tumor uptake (background) yielded a sensitivity of 2.7% MV-NIS-infected tumor cells. We reliably resolved multiple distinct intratumoral zones of infection from non-infected regions. Pinhole micro-SPECT/CT imaging using the NIS reporter demonstrated precise localization and quantitation of oncolytic MV-NIS infection, and can replace more time-consuming and expensive analyses (for example, autoradiography and IHC) that require animal killing.

Show MeSH
Related in: MedlinePlus