Limits...
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.

Show MeSH

Related in: MedlinePlus

Comparison of Immunohistochemistry and Autoradiography for Identifying MV-NIS-Infected Regions of Tumors. Nine mice with MV-NIS-infected BxPC-3 flank tumors (6 intratumorally-injected and 3 intravenously-injected) and two control mice (no MV-NIS) were injected with 99mTc04 (37 MBq). Tumors were excised, frozen, sectioned, and mounted on slides. Images were taken from a representative tumor, removed 14 days after intravenous injection of MV-NIS. Upper panel, Autoradiographic image of a tumor section. Lower panel, The same section processed for immunohistochemical detection of the measles virus N protein. All MV-NIS–infected syncytia were positive by autoradiography. Arrows denote alignment of MV-NIS–infected regions. Note that areas of high cell density (lower right corner) correspond to low levels of background 99mTcO4 localization; areas of necrosis (holes) in the tumor have higher levels of background localization due to radionuclide pooling. MV-NIS denotes measles virus expressing sodium iodide symporter.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198860&req=5

Figure 6: Comparison of Immunohistochemistry and Autoradiography for Identifying MV-NIS-Infected Regions of Tumors. Nine mice with MV-NIS-infected BxPC-3 flank tumors (6 intratumorally-injected and 3 intravenously-injected) and two control mice (no MV-NIS) were injected with 99mTc04 (37 MBq). Tumors were excised, frozen, sectioned, and mounted on slides. Images were taken from a representative tumor, removed 14 days after intravenous injection of MV-NIS. Upper panel, Autoradiographic image of a tumor section. Lower panel, The same section processed for immunohistochemical detection of the measles virus N protein. All MV-NIS–infected syncytia were positive by autoradiography. Arrows denote alignment of MV-NIS–infected regions. Note that areas of high cell density (lower right corner) correspond to low levels of background 99mTcO4 localization; areas of necrosis (holes) in the tumor have higher levels of background localization due to radionuclide pooling. MV-NIS denotes measles virus expressing sodium iodide symporter.

Mentions: Although the comparison between ex vivo radionuclide quantitation, IHC staining, or NIS RNA quantitation and micro-SPECT/CT analysis demonstrated the quantitative and predictive value of imaging, it was not clear whether the signal was coming from individual infected cells or multinucleated syncytia, nor was it clear whether all syncytia accumulated a similar concentration of imaging radionuclide. To address these questions, we compared IHC and autoradiography of the same tumor sections. Mice (n=11) with BxPC-3 flank tumors underwent intratumoral or intravenous injection with MV-NIS (3.5×106 50% tissue culture infective dose [TCID50]). Day 3 or day 14 after MV-NIS injection, mice were injected intraperitoneally with 99mTcO4 (37 MBq) and imaged 1 hour later with pinhole micro-SPECT/CT. Mice were euthanized immediately after imaging and tumors were removed for analysis. Figure 6 shows a high resolution image of a tumor section from the extreme of the group (14 days post IV injection of MV-NIS). These results, along with those of 10 other tumors (data not shown), demonstrated that 1) multinucleated syncytia retained adequate integrity to support 99mTcO4 transport across the multinucleated cell plasma membrane; 2) the majority of MV N-protein reactivity was associated with intact syncytia rather that lytic debris, even as late as 14 days after the initial infection; and 3) loss of NIS activity due to accumulation of random mutations in the viral NIS transgene did not appear to occur, at least not during this time frame of infection.


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)

Comparison of Immunohistochemistry and Autoradiography for Identifying MV-NIS-Infected Regions of Tumors. Nine mice with MV-NIS-infected BxPC-3 flank tumors (6 intratumorally-injected and 3 intravenously-injected) and two control mice (no MV-NIS) were injected with 99mTc04 (37 MBq). Tumors were excised, frozen, sectioned, and mounted on slides. Images were taken from a representative tumor, removed 14 days after intravenous injection of MV-NIS. Upper panel, Autoradiographic image of a tumor section. Lower panel, The same section processed for immunohistochemical detection of the measles virus N protein. All MV-NIS–infected syncytia were positive by autoradiography. Arrows denote alignment of MV-NIS–infected regions. Note that areas of high cell density (lower right corner) correspond to low levels of background 99mTcO4 localization; areas of necrosis (holes) in the tumor have higher levels of background localization due to radionuclide pooling. MV-NIS denotes measles virus expressing sodium iodide symporter.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198860&req=5

Figure 6: Comparison of Immunohistochemistry and Autoradiography for Identifying MV-NIS-Infected Regions of Tumors. Nine mice with MV-NIS-infected BxPC-3 flank tumors (6 intratumorally-injected and 3 intravenously-injected) and two control mice (no MV-NIS) were injected with 99mTc04 (37 MBq). Tumors were excised, frozen, sectioned, and mounted on slides. Images were taken from a representative tumor, removed 14 days after intravenous injection of MV-NIS. Upper panel, Autoradiographic image of a tumor section. Lower panel, The same section processed for immunohistochemical detection of the measles virus N protein. All MV-NIS–infected syncytia were positive by autoradiography. Arrows denote alignment of MV-NIS–infected regions. Note that areas of high cell density (lower right corner) correspond to low levels of background 99mTcO4 localization; areas of necrosis (holes) in the tumor have higher levels of background localization due to radionuclide pooling. MV-NIS denotes measles virus expressing sodium iodide symporter.
Mentions: Although the comparison between ex vivo radionuclide quantitation, IHC staining, or NIS RNA quantitation and micro-SPECT/CT analysis demonstrated the quantitative and predictive value of imaging, it was not clear whether the signal was coming from individual infected cells or multinucleated syncytia, nor was it clear whether all syncytia accumulated a similar concentration of imaging radionuclide. To address these questions, we compared IHC and autoradiography of the same tumor sections. Mice (n=11) with BxPC-3 flank tumors underwent intratumoral or intravenous injection with MV-NIS (3.5×106 50% tissue culture infective dose [TCID50]). Day 3 or day 14 after MV-NIS injection, mice were injected intraperitoneally with 99mTcO4 (37 MBq) and imaged 1 hour later with pinhole micro-SPECT/CT. Mice were euthanized immediately after imaging and tumors were removed for analysis. Figure 6 shows a high resolution image of a tumor section from the extreme of the group (14 days post IV injection of MV-NIS). These results, along with those of 10 other tumors (data not shown), demonstrated that 1) multinucleated syncytia retained adequate integrity to support 99mTcO4 transport across the multinucleated cell plasma membrane; 2) the majority of MV N-protein reactivity was associated with intact syncytia rather that lytic debris, even as late as 14 days after the initial infection; and 3) loss of NIS activity due to accumulation of random mutations in the viral NIS transgene did not appear to occur, at least not during this time frame of infection.

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