Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus

Abstract

Introduction: Oncolytic viruses show promise for treating cancer. However, to assess therapeutic efficacy and potential toxicity, a noninvasive imaging modality is needed. This study aimed to determine if insertion of the human sodium iodide symporter (hNIS) cDNA as a marker for non-invasive imaging of virotherapy alters the replication and oncolytic capability of a novel vaccinia virus, GLV-1h153.

Methods: GLV-1h153 was modified from parental vaccinia virus GLV-1h68 to carry hNIS via homologous recombination. GLV-1h153 was tested against human pancreatic cancer cell line PANC-1 for replication via viral plaque assays and flow cytometry. Expression and transportation of hNIS in infected cells was evaluated using Westernblot and immunofluorescence. Intracellular uptake of radioiodide was assessed using radiouptake assays. Viral cytotoxicity and tumor regression of treated PANC-1tumor xenografts in nude mice was also determined. Finally, tumor radiouptake in xenografts was assessed via positron emission tomography (PET) utilizing carrier-free 124I radiotracer.

Results: GLV-1h153 infected, replicated within, and killed PANC-1 cells as efficiently as GLV-1h68. GLV-1h153 provided dose-dependent levels of hNIS expression in infected cells. Immunofluorescence detected transport of the protein to the cell membrane prior to cell lysis, enhancing hNIS-specific radiouptake (P < 0.001). In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts (P < 0.001). Finally, intratumoral injection of GLV-1h153 facilitated imaging of virus replication in tumors via 124I-PET.

Conclusion: Insertion of the hNIS gene does not hinder replication or oncolytic capability of GLV-1h153, rendering this novel virus a promising new candidate for the noninvasive imaging and tracking of oncolytic viral therapy.

Figure 1

GLV-1h153 construct. a. GLV-1h153 was derived from GLV-1h68 by replacing the gus A expression cassette at the A56R locus with the hNIS expression cassette through in vivo homologous recombination. Both viruses contain RUC-GFP and lacZ expression cassettes at the F14.5L and J2R loci, respectively. PE, PE/L, P11, and P7.5 are VACV synthetic early, synthetic early/late, 11K, and 7.5K promoters, respectively. TFR is human transferrin receptor inserted in the reverse orientation with respect to the promoter PE/L.b. Confirmation of GFP, LacZ, and lack of gus A marker gene expression in GLV-1h153 infected CV-1 cells. While the gus A gene cassette is expressed in cells infected with parent virus GLV-1h68, this has been replaced by the hNIS gene cassette in GLV-1h153, leading to loss of gus A expression.

Figure 2

Viral proliferation assay of GLV-1h153-in PANC-1 cells. a. PANC-1 cells were grown in 6-well plates and infected with GLV-1h153 or GLV-1h68 at an MOI of 0.01 and 1.0. Three wells of each virus were harvested at 1, 24, 48, and 72 hours postinfection. GLV-1h153 replicated in a similar manner to GLV-1h68, with a 4-log increase in viral load at an MOI of 0.01 by 72 hours, reaching similar levels as that in cells infected with an MOI of 1.0. This demonstrates that GLV-1h153 is able to replicate efficiently within PANC-1 cells in vitro as well as parental virus GLV-1h68. b. GFP expression was quantified via flow cytometry in PANC-1 cells infected with GLV-1h153 at MOIs of 1.0 and 0.01 and was shown to be MOI dependent. GFP expression mimicked the viral replication growth curve, with GFP expression in the MOI 0.01 infected cells reaching similar levels as the MOI of 1.0 by 72 hours after infection. c. GFP expression was quantified via flow cytometry in PANC-1 cells infected with an MOI of 0.01, 0.1, 0.5, 1.0 2.0, and 5.0 at 24 hours after infection, and was shown to be MOI-dependent.

Figure 3

Assessment of hNIS expression in GLV-1h153-infected PANC-1 cells. a. Microarray analysis of cells infected with an MOI of 5.0 of GLV-1h153 yielded an almost 2000-fold increase by 6 hours and an almost 5000-fold increase by 24 hours in hNIS mRNA production as compared to noninfected control. b. PANC-1 cells were either mock infected or infected with GLV-1h68 at an MOI of 1.0 or infected with GLV-1h153 at an MOI of 1.0 or 5.0 for 24 hours. The hNIS protein was detected by Western blot analysis using monoclonal anti-hNIS antibody. Only GLV-1h153-infected cells expressed the hNIS protein, but cells either mock infected or infected with GLV-1h68 did not. The molecular weight marker bands (in kiloDaltons) are shown on the left. c. PANC-1 cells were mock infected or infected with GLV-1h68 or GLV-1h153 at a MOI of 1.0 for 24 hours. The hNIS protein was detected by immunofluorescence microscopy using monoclonal anti-hNIS antibody, which recognizes the intracellular domain of the protein. Mock- or GLV-1h68-infected cells (as demonstrated by GFP expression) did not express the hNIS protein, whereas the hNIS protein on the cell membrane of PANC-1 cells infected with GLV-1h153 was readily detectable.

Figure 4

Assessment of in vitro ¹³¹I radiouptake of GLV-1h153-infected PANC-1 cells. a. PANC-1 cells were infected with an MOI of 0, 0.01, 0.1, and 1.0 of GLV-1h153 and MOI of 1.0 of GLV-1h68. PCCL3 was used as a positive control. Twenty-four hours after infection, there is a >70-fold enhanced radiouptake at an MOI of 1.0 as compared to an MOI of 0 in GLV-1h153, and radiouptake is shown to be MOI dependent and hNIS specific (as shown with blocking with competitive inhibitor of hNIS, NaClO4). b. Maximum radiouptake with an MOI of 1.0 24 hrs after infection corresponded to maximum GFP expression.

Figure 5

GLV-1h153 infection and killing in cell culture and in vivo. a. PANC-1 cells were infected by various GLV-1h153at MOIs of 0.01, 0.1, and 1.0. Cell viability was determined via lactate dehydrogenase assays, and was set at 100% before infection. GLV-1h153 infected and was cytotxic at various MOIs, with less than 20% survival of cells as compared to control at an MOI of 1.0 by day 9. The values are the mean of triplicate samples, and bars indicate SD. b. GFP expression is shown to be time-dependent, with abundant GFP expression by day 3. Phase overlay pictures shows gradual cell death and thus decline of GFP expression by day 7. Closer examination of infected cells reveals loss of normal morphology and cell progressive cell detachment. c. 2 × 10⁶ PFUs of GLV-1h153 or GLV-1h68, or PBS were injected IVly or ITly into nude mice bearing s.c. PANC-1 tumors on the hindleg (~100 mm³). GLV-1h153 was able to regress pancreatic tumor xenograft both ITly and IVly starting at day 13. The values are a mean of 4-5 mice, with bars indicating SEM. d. GLV-1h153 infection of pancreatic tumor xenografts did not have adverse effects on body weight at 5 weeks post injection, with the IT group even gaining weight compared to control.

Figure 6

PET imaging of enhanced radiouptake in GLV-1h153-infected PANC-1 xenografts. Two × 10⁷ PFU of GLV-1h153, GLV-1h68, or PBS was injected intratumorally into PANC-1 hindleg tumor-bearing mice. ¹²⁴I-PET scanning was obtained 48 hours after infection and 1 hour after radiotracer administration. GLV-1h153-infected PANC-1 tumors were easily visualized, while no enhanced signal was seen in the PBS- or GLV-1h68 injected tumors. The stomach and thyroid were also imaged due to native NIS expression, and the bladder due to tracer excretion.