Dual-Isotope SPECT Imaging with NIS Reporter Gene and Duramycin to Visualize Tumor Susceptibility to Oncolytic Virus Infection

Journal Pre-proof Dual Isotope SPECT imaging with NIS Reporter Gene and Duramycin To Visualize Tumor Susceptibility to Oncolytic Virus Infection Lianwen Zhang, Lukkana Suksanpaisan, Huailei Jiang, Timothy R. DeGrado, Stephen J. Russell, Ming Zhao, Kah-Whye Peng PII:

S2372-7705(19)30092-0

DOI:

https://doi.org/10.1016/j.omto.2019.10.002

Reference:

OMTO 148

To appear in:

Molecular Therapy: Oncolytics

Received Date: 22 August 2019 Accepted Date: 5 October 2019

Please cite this article as: Zhang L, Suksanpaisan L, Jiang H, DeGrado TR, Russell SJ, Zhao M, Peng K-W, Dual Isotope SPECT imaging with NIS Reporter Gene and Duramycin To Visualize Tumor Susceptibility to Oncolytic Virus Infection, Molecular Therapy: Oncolytics (2019), doi: https:// doi.org/10.1016/j.omto.2019.10.002. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 The Author(s).

1

Dual Isotope SPECT imaging with NIS Reporter Gene and Duramycin

2

To Visualize Tumor Susceptibility to Oncolytic Virus Infection

3 4

Lianwen Zhang1, Lukkana Suksanpaisan2, Huailei Jiang3, Timothy R DeGrado3, Stephen J Russell1,

5

Ming Zhao4, Kah-Whye Peng1

6

1

Department of Molecular Medicine, 3Department of Radiology, Mayo Clinic, Rochester, MN, 2Imanis Life

7

Sciences, Rochester, MN, 4Northwestern University, Chicago, IL

8

Co-first authors: Zhang and Suksanpaisan contributed equally to the study.

9 10 11

Corresponding author:

12

Kah Whye Peng

13

Molecular Medicine

14

Mayo Clinic

15

200 First Street SW

16

Rochester MN 55905

1

17

Abstract

18

Noninvasive dual imaging methods that provide an early readout on tumor permissiveness to virus infection

19

and tumor cell death could be valuable in optimizing development of oncolytic virotherapies. Here, we have

20

used the sodium iodide symporter (NIS) and

21

oncolytic Vesicular Stomatitis Virus (VSV) in VSV susceptible (MPC-11 tumor) versus resistant (CT26 tumor)

22

tumors in Balb/c mice. In conjunction, tumor cell death was imaged simultaneously using 99mTc-duramycin that

23

binds phosphatidylethanolamine in apoptotic and necrotic cells. Dual isotope SPECT/CT imaging showed

24

areas of virus infection (NIS and

25

imaging) in susceptible tumors. Multiple infectious foci arose early in MPC-11 tumors, which rapidly expanded

26

throughout the tumor parenchyma over time. There was a dose dependent increase in numbers of infectious

27

centers and

28

minimal in VSV resistant CT26 tumors. Combinatorial use of NIS and

29

simultaneous monitoring of OV spread, and the presence or absence of treatment associated cell death could

30

be useful to guide development of combination treatment strategies to enhance therapeutic outcome.

99m

125

I radiotracer to detect infection and replicative spread of an

125

I), which overlapped well with areas of tumor cell death (99mTc-duramycin

Tc-duramycin positive areas with viral dose. In contrast, NIS or duramycin signals were

2

99m

Tc-duramycin SPECT imaging for

31

Introduction

32

Oncolytic virotherapy (OV) is a promising new class of anticancer modality under development for a variety of

33

cancers1, 2. Oncolytic virotherapy uses recombinant replication competent viruses that have been genetically

34

engineered for enhanced immune activation and tumor selective replication3, 4. Viruses from diverse families

35

have demonstrated substantial efficacy in preclinical models, and are being assessed in various clinical

36

trials5. In addition to OV-mediated tumor killing by direct infection, the release of tumor-associated antigens

37

subsequent to viral-induced cell killing/death initiate a pro-inflammatory response, which promotes systemic

38

antitumor immunity

39

making it a highly attractive modality to combine in immuno-oncology drugs against a broad spectrum of

40

cancers8, 9.

3, 6, 7

. Accordingly, OV exposure may promote immediate and lasting anticancer effects,

41 42

Vesicular Stomatitis Virus (VSV), from the family Rhabdoviridae, is a promising oncolytic virus under

43

investigation in preclinical studies and in clinical trials10, 11. VSV is an effective anticancer vector platform due

44

to a variety of key features: 1) VSV has rapid replication kinetics and high burst size, 2) low sero-prevelance

45

allowing intravenous administration, and, importantly, 3) can be genetically engineered to encode transgenes

46

to improve specificity, and potency11. Previous strategies used in modulating VSV include the incorporation of

47

the IFNβ gene into the VSV genome to enhance tumor selectivity and potentiate immune mediated antitumor

48

activity12. In addition to genetic modifications used to achieve maximal tumor infection and oncolytic effects,

49

VSVs encoding reporter genes can provide an early readout during preclinical studies. The sodium iodide

50

symporter (NIS) is a self-protein expressed endogenously in the thyroid for the uptake of iodine for the

51

synthesis of thyroid hormones

52

imaging for the diagnosis of thyroid disorders, and treatment of metastatic thyroid cancer using beta-emitting

53

131

54

monitoring of pharmacokinetic activity and viral invasion of tumors when visualized via Single Photon

55

Emission Computed Tomography (SPECT/CT)9,

56

pharmacokinetic activity relating to viral infection and expression, target validation, and susceptibility of

57

specific tumor types

58

myeloma, we demonstrated the utility and significance of the NIS reporter gene in tracking measles virus

59

infection in plasmacytomas in patients over time

60

viral mediated cell killing and oncolytic potency by using

61

99m

62

probe which binds phosphatidylethanolamine (PTE) molecules17. Because PTE is translocated en masse from

13

. As such, NIS is used clinically in conjunction with

123

I gamma camera

I. Our group has previously engineered VSV to encode NIS which enables noninvasive, longitudinal

14, 15

, Furthermore, NIS can enable noninvasive tracking of

15

. In a Phase I clinical trial using a Measles Virus (MV)-NIS in patients with multiple

16 8

. SPECT/CT also can precisely measure the degree of 99m

Tc-duramycin as a radiotracer molecule. Indeed,

Tc-duramycin is well-characterized as a radiopharmaceutical and can serve as a highly sensitive molecular

3

99m

63

inner to outer plasma membranes during the early stages of apoptosis,

64

conjunction with SPECT imaging to detect apoptotic or necrotic cells18. Using this combinatorial approach of

65

VSV and duramycin, we wanted to determine if the methodology is sensitive enough for us to simultaneously

66

track virus infection, and monitor oncolytic potency while observing any off-target infection. If successful this

67

methodology could be particularly useful in combination studies whereby drugs and radioisotopes are added

68

in combination with virus to enhance efficacy 14, 19

69

4

Tc-duramycin could be used in

70

Results

71

Imaging kinetics of VSV infection in murine tumors

72

Here, we used a mouse myeloma MPC-11 tumor model that is known to be highly susceptible to VSV

73

infection for these studies20,

74

endogenous NIS expression in the thyroid, salivary glands and stomach, and no significant off-target infection

75

by VSV-IFNβ-NIS in Balb/c mice (data not shown)9. Representative fused SPECT/CT images of MPC-11 flank

76

tumors in mice given saline, 106 (low-dose group) 50% tissue culture infective dose (TCID50) or 108 TCID50

77

(high-dose group) VSV-mIFNβ-NIS are shown in Figure 1. Imaging scans were performed on days 1 (24h), 3,

78

and 5 post VSV administration. NIS positive signals were detectable in virus treated mice but not in saline

79

treated animals. No infectious foci were seen at day 1 in mice given 106 TCID50 virus, while numerous

80

infectious foci were detected in tumors from mice that received 108 TCID50 virus, indicating an initial dose-

81

dependent viral delivery, and spread. Mice treated with low dose virus showed increasing numbers of

82

positive foci over the course of 5 days. Signals were highest at day 3 in the 108 TCID50 group, followed by a

83

decrease by day 5, potentially due to death of the infected cells (Figure 1e). These imaging data reveals a

84

difference in initial viral infection and subsequent intratumoral spread between the low- and high-dose groups,

85

and underscores the importance of efficient virus delivery and infection in achieving maximal tumor infectivity.

86

In contrast, SPECT/CT imaging for NIS activity was negative in CT26 tumors, which are known to be resistant

87

to VSV-IFNβ-NIS therapy (Figure 2a, 2c) 22.

21

. Whole body imaging showed only physiologic uptake of

125

I due to

125

I NIS

88 89

Imaging tumor cell killing using [99mTc]Tc-duramycin

90

In addition to giving mice

91

phosphatidylethanolamine exposed on apoptotic cells, and performed dual isotope SPECT scans. As shown

92

in Figure 1b, the MPC-11 tumor was negative for duramycin uptake in saline treated mice although some

93

signal positive areas were seen, likely due to a central region of necrosis in the tumor. In both low and high

94

dose VSV groups, significant

95

well with areas of NIS expression in the VSV infected foci, confirming that in VSV responsive tumors, virus

96

infected cells rapidly dies from the oncolytic effect from day 1 (Figure 1e and 1f). As the NIS positive foci

97

increased intratumorally over several days, there was a corresponding progressive increase in duramycin

98

labeled areas in the tumors. The rapid increase of duramycin signals also indicates that VSV infection rapidly

99

induces apoptosis in the infected cells, and concurs with previous studies showing significant increase in

100

caspase-8 activity in infected cells at 8h post infection23. In contrast, minimal duramycin positive areas were

101

seen in the CT26 VSV treated tumors (Figure 2b, 2d).

125

I for NIS imaging, we co-administered

99m

99m

Tc-duramycin that binds to

Tc-duramycin signals were detected. These areas of cell killing overlap very

5

102 103

Immunofluorescence staining confirmed 125I SPECT imaging

104

To confirm that positive signals from 125I SPECT imaging correlate with VSV infection, we harvested tumors at

105

day 3 and day 5 immediately after SPECT imaging and stained the tumor tissues for VSV antigen, NIS protein

106

or TUNEL assay (Figure 3).

107

in coverage of tumor by NIS expressing cells over the 5 days (Figure 3a). Immunohistochemical staining

108

confirmed these areas expressed VSV and NIS antigens indicating that the

109

VSV-mIFNβ-NIS infection (Figure 3b and 3c). In parallel, we also focused on a specific slice from the SPECT

110

data to identify areas of

111

between duramycin labeled areas from the SPECT imaging with DNA damage detected by TUNEL assay

112

(Figure 3e). We also performed IHC staining of CT26 tumors which showed minimal NIS positive areas or

113

duramycin uptake. Indeed IHC staining did not show detectable levels of NIS antigen expression nor TUNEL

114

positivity (Figure 2e).

125

99m

I SPECT data at a specific slice of the tumor (radiohistology) showed increase

125

I signals were indeed due to

Tc-duramycin positivity (Figure 3d). IHC staining showed a good correlation

115 116

Space-filling models more accurately reflect the distribution of intratumoral infection

117

As shown in Figure 3, TUNEL staining overlaps well with VSV and NIS staining, indicating VSV amplification

118

is followed rapidly by tumor cell killing in these susceptible MPC-11 tumors. The SPECT imaging data can be

119

rendered into 3D model space-filling models to facilitate better visualization of the 3D tomographic data

120

(Figure 4). The 3D space-filling models allows us to combine all planar microSPECT/CT images into a

121

comprehensive volume rendering of intratumoral infection distribution to help us gain a better spatial context

122

and to visualize distributive relationships between these infectious centers (Figure 4, Supplementary Video

123

S1). Here, we can clearly see that there is a good concordance of the duramycin (red) and VSV activity

124

(yellow) in the MPC-11 tumor from mice given an IV dose of VSV-mIFNβ-NIS virus. As expected, the NIS

125

signals cover a larger volume of infection, and the apoptotic signals lag behind the VSV infection. The

126

significance of this technology is evident when comparing conclusions made from IHC stained tumor sections

127

or planar microSPECT/CT images with the space-filling models. While 2D slices do not provide contextual

128

information and can misrepresent the extent of virus infection through the entire tumor, 3D space-filling

129

models clearly demonstrate infection throughout the entire tumor.

130 131

Tumor dosimetry analysis show parallel trends between virus spread and tumor cell killing

132

Regions of interest (ROI) were drawn around the flank tumors in the SPECT images to determine the amount

133

of radiotracer uptake in the tumors in the respective animals (Figure 4). In MPC-11 xenografts of mice (n=4)

6

134

treated with low dose virus, there was gradual increase in 125I uptake over the course of five days (Figure 5a).

135

Mean percentage uptake of injected dose per cm3 of tissue (% ID/ cm3) is 1.08+0.56% ID/ cm3 on day 1,

136

increasing to 4.07+1.42% ID/ cm3 on day 3, and 5.15+2.76% ID/ cm3 on day 5. Mean uptake in mice (N=4) in

137

the high dose group was 2.07+0.36 % ID/ cm3 on day 1, 2.73+1.32% ID/ cm3 on day 3, followed by a steep

138

decline in signal by day 5 (1.20+0.16%ID/ cm3), Kinetics of tumor cell death, as measured by amount of

139

uptake, follows a similar trend to VSV infection in both low and high dose groups (Figure 5b). Conversely,

140

CT26 xenografts showed a mean of 0.37+0.37% ID/ cm3 over 5 days, confirming minimal viral infection in

141

these tumors (Figure 5c).

7

99m

Tc

142

Discussion

143

We demonstrated here for the first time, the feasibility of using

144

simultaneously evaluate the replication of an oncolytic VSV and the corresponding viral induced apoptotic cell

145

death.

146

140 keV, and a 6h physical half-life. For imaging dying infected cells, we used

147

short half-life, low background uptake by visceral organs and has been proven to bind efficiently to apoptotic

148

cells24-27. NIS reporter gene imaging is performed using

149

allows us to discriminate between the two profiles of the two isotopes to perform simultaneous imaging.

125

I and

99m

Tc dual isotope imaging to

125

I emits low energy photons of 35 keV with a 60 days physical half-life, and 99m

99m

Tc emits photons with

Tc-duramycin, that has a

125

I which has much lower energy at 35keV and

150 151

The areas of NIS positive signals, a surrogate of viral replication, correlated well with the areas of duramycin

152

signal that detects apoptotic/necrotic cells. Furthermore, the pharmacokinetics of viral gene expression and

153

subsequent death of virally infected cells can be quantitated and followed for each single animal as frequently

154

as desired by analysis of the SPECT data. Using longitudinal imaging, we detected increase in numbers of

155

infectious foci over several days after initial viral infusion in MPC tumors that are highly responsive to VSV

156

infection and killing. In MPC-11 mice given a low 106 TCID50 IV dose of VSV, viral infection gradually builds up

157

and peaked on day 5. There was a clear dose dependence increase in initial viral extravasation and infectivity

158

as NIS signals peaked more rapidly by day 3 in mice given 100-fold more virus, followed by decreasing

159

uptake over the next few days of surveillance. In conjunction,

160

mice treated with 108 TCID50 VSV than with 106 TCID50 VSV. From these dual imaging data, we were able to

161

conclude that response of MPC-11 tumor to VSV therapy is predominantly driven by initial oncolysis by the

162

virus, resulting in rapid death of the cell.

99m

Tc-duramycin uptake at day 2 was higher in

163 164

Dual isotope imaging that enables real time tracking of viral replication and cell killing could facilitate

165

understanding of mechanisms underlying therapy failure. While this approach is likely to be costly and not

166

easily adopted as standard of care in clinical practice, dual imaging could certainly help preclinical or early

167

phase clinical development of novel virus constructs and virus/drug combination studies. In contrast to MPC-

168

11 tumors, CT26 tumors are resistant to VSV therapy 28. Based on the imaging data, it is clear that treatment

169

failure is due predominantly to the lack of significant viral infection of the tumor cells after viral delivery. In vitro

170

studies showed that CT26 tumor cells are interferon responsive and are thus likely to have rapidly mounted a

171

robust antiviral response after initial infection through activation of the type I interferon response pathway28.

172

Studies have shown that addition of Jak1 inhibitors such as ruxolitinib could have a positive impact on

173

interferon responsive tumors by dampening the initial innate defense 29-31. In another scenario tumor cells that

8

174

are permissive to viral infection (thus NIS positive through SPECT imaging), but resistant to apoptosis would

175

show low/negligible duramycin binding. In these tumors with high NIS expression and limited cell death, a

176

timely dose of beta-emitting

177

shown using a NIS encoding VSV virus or measles virus respectively 14, 19.

131

I could potentially enhance virotherapy killing to boost cell killing as previously

178 179

The positive viral imaging data in this study was confirmed by IHC staining for VSV antigen and NIS

180

expression, confirming that the 125I uptake was indeed due to VSV infected cells expressing NIS. In contrast to

181

invasive and time-consuming protocols such as IHC staining, SPECT imaging is noninvasive and allows the

182

investigator to monitor the same living animal longitudinally over a desired time period. This not only reduces

183

animal waste but also provides a more refined and superior data set that includes tomographic data with

184

spatial information on relative distribution of the infectious foci and its changes over time. Instead of simply

185

obtaining a snapshot of the tumor in 2-dimensions, viral infection and cell death in the entire tumor can easily

186

be captured and rendered. The data is not only qualitative, but quantitative. Drawing regions of interest

187

around the flank tumor allows us to measure accurately the amount of radiotracer uptake over time per gram

188

of tumor. These measurements indicate that for high dose VSV, viral infection peaked at day 3 and declined

189

thereafter. Indeed, viral replication kinetics demonstrated in this study concurred with previous studies where

190

infectious virus recovery from tumor showed that VSV replication reached a peak by day 2-4, followed by

191

sharp decrease in activity by day 5, supporting our observations presented here20.

192 193

Reporter gene imaging for virus and cell tracking is often performed in rodents using bioluminescent

194

imaging32,

195

routine studies. However, it lacks resolution and for studies as described in this work where we are tracking

196

distribution of infectious foci, bioluminescent imaging would not be feasible as optical imaging lacks spatial

197

resolution, and is also negatively impacted by attenuation of photons from tissues more than a few mm

198

deep34. It is ironic that while there are abundant preclinical studies developing therapies use bioluminescent

199

imaging in vivo, investigators fail to incorporate these imaging studies during clinical development where it

200

perhaps matters most. In addition to NIS and duramycin imaging, there are other imaging modalities, e.g.

201

magnetic resonance reporter gene imaging, which could be incorporated and are compatible clinically

202

majority of oncolytic viruses in clinical trials do not encode a reporter gene, except for GL-ONC1 (encodes

203

Ruc-GFP, β-glucuronidase, and β-galactosidase genes), MV-NIS and VSV-IFNβ-NIS (symporter/transport

204

proteins (hNIS) Using NIS imaging, authors were able to demonstrate delivery of virus to metastatic tumor

205

deposits after intravenous administration of measles-NIS in myeloma patients8. Viral infection was seen

33

. Bioluminescent imaging offers high throughput screening and sensitivity and is acceptable for

9

35

. The

206

between days 8 to 15, followed by subsequent clearance of NIS infected cells by day 238. In another study,

207

Morris and colleagues have incorporated

208

killing of tumor cells by radiation therapy36. Timing is important in such radiovirotherapy studies; radiation

209

therapy should be applied during the peak of NIS expression as measured from 123I dosimetry measurements.

210

It is also envisaged, with better imaging equipment and more sensitive tracers37, 38, one would also be able to

211

track any off target infection by the oncolytic viruses in clinical trials to facilitate better understanding of any off

212

target toxicity in the future. In conclusion, we have shown, for the first time, that VSV-mediated tumor infection

213

and oncolytic activity can be monitored simultaneously with the combinatorial use of the NIS reporter gene

214

and 99mTc-duramycin via SPECT/CT. This technique can be readily adapted to provide an early prognosis and

215

reliable assessments of tumor responses to OV and is valuable in early phase clinical development studies.

123

I/NIS imaging with

131

I radiation therapy to induce cytoreductive

216 217

Methods

218

Cell culture and viruses

219

MPC-11 and CT26 cell lines (American Type Cell Culture) were cultured in DMEM supplemented with 10%

220

fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cell lines tested negative for

221

mycoplasma contamination. The generation and propagation of VSV-mIFNβ-NIS has been previously

222

described 9.

223 224

Animals

225

Animals were maintained and cared for in accordance with Mayo Clinic’s Institutional Animal Care and Use

226

Committee. Sixteen female BALB/c mice (Jackson Laboratory) aged 6–8 weeks old were used in this study.

227

5x106 MPC-11 cells or 5x106 CT26 cells were subcutaneously implanted in the right flank of the mice. When

228

tumors reached 0.5 cm in diameter, VSV-mIFNβ-NIS was administered intravenously via the tail vein at a

229

dose of 106, 107 or 108 TCID50 per mouse. Saline injections were used as controls in both xenograft models.

230

Mice were imaged under isoflurane inhalation anesthesia from day 2 post virus infusion.

231 232

Labeling Duramycin with 99mTc

233

Duramycin was labeled with technetium (99mTc) using tricine-phosphine coligands. The detailed procedure for

234

the kit preparation has been described previously

235

was added to the lyophilized vial. The labeling mixture was heated to 80°C in a lead-lined heating blo ck for 20

236

minutes. The vial seal was vented with a 25-G needle prior to removal from the heating block and was

237

equilibrated to room temperature before injection.

39

. About 20 mCi of 99mTc-pertechnetate in 500 µL of saline

10

238 239

SPECT/CT imaging

240

One hour prior to SPECT/CT imaging, all MPC11- and CT26-bearing BALB/c mice received 300 µCi

241

300 µCi

242

(MILabs, Utrecht, Netherlands). CT was performed for 5 min, followed by SPECT which simultaneously

243

captured both

244

window, creating two distinct data sets. Window modes are described as follows: 1)

245

keV (range 126 keV – 154 keV); 2) 125I: 100% window, 30 keV (range 15 keV – 45 keV). Data processing and

246

quantitation of regions of interest (ROI) were performed by Imanis Life Sciences (Rochester, MN), using

247

PMOD software (Zurich, Switzerland).

99m

125

I and

Tc-duramycin (99mTc-duramycin) intravenously. Mice were imaged in a USPECT-II/CT scanner

99m

Tc and

125

I signals. SPECT data was reconstructed using a standard

99m

99m

Tc window and

125

I

Tc: 20% window, 140

248 249

Immunofluorescence and TUNEL staining

250

Tumors were harvested at day 3 and day 5 after SPECT imaging, fixed in 10% buffered formalin followed by

251

transfer to saline solution 3 days later. When radioactive material was no longer detectable, tumor specimens

252

were embedded in paraffin and sectioned into 5 um-thick slices. Immunohistochemistry was performed using

253

an anti-hNIS antibody (1:1000, REA011; Imanis Life Sciences) or anti-VSV antibody (1:300, M2168; Mayo

254

VVPL). Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assays were

255

performed on the serial section slices as per the manufacturer’s instructions (G3250, Promega, Madison, WI).

256 257

Data analysis

258

SPECT data processing and quantitation of ROI were performed by Imanis Life Sciences (Rochester, MN),

259

using PMOD software (Zurich, Switzerland). GraphPad Prism (GraphPad Software, San Diego, CA) was used

260

for statistical calculations.

261 262

Author contributions

263

Conception and design, S.J.R. and K.W.P.; development of methodologies and performance of experiments,

264

L.Z, L.S, M.Z., H.J., T.D.; analysis and interpretation of data, L.Z and L.S.; statistical analysis of data, L.Z and

265

L.S.; writing, review, and/or revision of the manuscript, L.Z., L.S, M.Z., S.J.R, K.W.P.; and study supervision,

266

K.W.P

267 268 269

11

270

Disclosures

271

SJR, KWP and Mayo Clinic have a financial interest in the technology used in this research. VSV and MV are

272

licensed to Vyriad, and the NIS reporter gene technology is licensed to Imanis Life Sciences. LS is a full time

273

employee of Imanis.

274 275

Acknowledgements

276

We thank Teresa Decklever and Dianna Glynn of the Mayo Clinic Nuclear Medicine Molecular Imaging

277

Resource for their expert help in image acquisition.

12

278 279

Figure Legends

280

Figure 1. Intratumoral spread and dose dependent response after intravenous administration of VSV-

281

mIFNβ β-NIS virus in BALB/c mice with MPC-11 tumors

282

Representative fused images of tumors. Saline controls (a-b), and BALB/c mice administered with VSV-

283

mIFNβ-NIS at a dose of 106 TCID50 (c-d) or 108 TCID50 (e-f) were imaged by SPECT on day 1, 3, and 5

284

following virus treatment. Relative viral infectivity is measured as a function of VSV-mIFNβ-NIS -mediated

285

uptake as shown in the left column (a, c, e), and cell killing is measured by 99mTc-duramycin radiotracer activity

286

as shown in the right column (b, d, f). Percentage in figure indicates fractions of the entire tumor diameter.

125

I

287 288

Figure 2. Intratumoral spread of VSV-mIFNβ β -NIS virus after intravenous administration into BALB/c

289

mice with CT-26 tumors

290

Mice treated with saline (a-b), or VSV-mIFNβ-NIS at 107 TCID50 (c-d) were imaged by SPECT on days 1, 3,

291

and 5 following virus delivery. Relative viral infectivity is measured as a function of VSV-mIFNβ-NIS -mediated

292

125

293

activity as shown in the right column (b, d). Percentage in figure indicates fractions of the entire tumor

294

diameter. (e) immunohistochemical analysis of tumor sections staining for NIS protein or apoptotic cells

295

(TUNEL) showed minimal positivity.

I uptake as shown in the left column (a, c), and cell killing is measured by

99m

Tc-duramycin radiotracer

296 297

Figure 3. Correlating SPECT imaging data with protein expression (VSV and NIS staining) or cell

298

killing (TUNEL staining for DNA damage) in tumor sections

299

MPC-11 tumors from BALB/c mice that received saline or 106 TCID50 VSV-mIFNβ-NIS were harvested day 3

300

and 5. Representative images of tumor sections are shown.

301

confirmed via immunohistochemistry using anti-VSV antibody (b) and anti-hNIS antibody (c).

302

results at day 3 and 5 (d) are confirmed using TUNEL assay (e).

125

I SPECT results at day 3 and 5 (a) are 99m

Tc SPECT

303 304

Figure 4. Three-dimensional (3D) space-filled rendering of SPECT/CT tomographic data showing the

305

intratumoral distribution of viral infection

306

Maximal intensity projection images showing

307

areas of tumor cell death (b) in MPC-11 tumor at day 2. 3D space-filling models of saline treated mouse (c)

308

and VSV treated mouse (d), VSV infection (125I uptake) is colored yellow and apoptotic cells (99Tc-duramycin

309

uptake) are red.

125

I uptake in areas of VSV infection (a) and

13

99m

Tc uptake in

310 125

I uptake) and cell killing (99Tc-duramycin

311

Figure 5. Correlation of VSV infection (NIS-mediated

312

uptake) with the dose of VSV-mIFNβ β -NIS given intravenously to BALB/c mice

313

a)

314

mice) TCID50 VSV-mIFNβ-NIS. c)

315

TCID50 VSV-mIFNβ-NIS (blue, 4 mice) or saline (black, 2 mice). Data is measured as the collective mean

316

%ID/cm3 for each model. Error bars denote standard deviations associated with each data point.

125

I uptake or b)

99m

6

8

Tc uptake in MPC-11 tumors of mice treated with 10 (green, 4 mice ) or 10 (red, 4 125

I uptake or d)

99m

317

14

Tc uptake in CT26 tumors of mice treated with 10

7

318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381

Bilbliography 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

15. 16.

17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

Russell, S.J., K.W. Peng, and J.C. Bell, Oncolytic virotherapy. (2012) Nat Biotechnol. 30(7): p. 658-70. Lawler, S.E., et al., Oncolytic Viruses in Cancer Treatment: A Review. (2017) JAMA Oncol. 3(6): p. 841-849. Russell, L. and K.W. Peng, The emerging role of oncolytic virus therapy against cancer. (2018) Chin Clin Oncol. 7(2): p. 16. Seymour, L.W. and K.D. Fisher, Oncolytic viruses: finally delivering. (2016) British Journal of Cancer. 114(4): p. 357-361. J, G.P., et al., Trial Watch: Oncolytic viro-immunotherapy of hematologic and solid tumors. (2018) Oncoimmunology. 7(12): p. e1503032. de Graaf, J.F., et al., Armed oncolytic viruses: A kick-start for anti-tumor immunity. (2018) Cytokine Growth Factor Rev. 41: p. 28-39. Galanis, E., et al., Oncolytic measles virus expressing the sodium iodide symporter to treat drug-resistant ovarian cancer. (2015) Cancer Res. 75(1): p. 22-30. Russell, S.J., et al., Remission of Disseminated Cancer After Systemic Oncolytic Virotherapy. (2014) Mayo Clinic Proceedings. 89(7): p. 926-933. Naik, S., et al., Curative one-shot systemic virotherapy in murine myeloma. (2012) Leukemia. 26(8): p. 18701878. Melzer, M.K., A. Lopez-Martinez, and J. Altomonte, Oncolytic Vesicular Stomatitis Virus as a ViroImmunotherapy: Defeating Cancer with a "Hammer" and "Anvil". (2017) Biomedicines. 5(1). Hastie, E. and V.Z. Grdzelishvili, Vesicular stomatitis virus as a flexible platform for oncolytic virotherapy against cancer. (2012) Journal of General Virology. 93: p. 2529-2545. Westcott, M.M., et al., Interferon Beta and Interferon Alpha 2a Differentially Protect Head and Neck Cancer Cells from Vesicular Stomatitis Virus-Induced Oncolysis. (2015) J Virol. 89(15): p. 7944-54. Penheiter, A.R., S.J. Russell, and S.K. Carlson, The Sodium Iodide Symporter (NIS) as an Imaging Reporter for Gene, Viral, and Cell-based Therapies. (2012) Current Gene Therapy. 12(1): p. 33-47. Goel, A., et al., Radioiodide imaging and radiovirotherapy of multiple myeloma using VSV(Delta 51)-NIS, an attenuated vesicular stomatitis virus encoding the sodium iodide symporter gene. (2007) Blood. 110(7): p. 23422350. Miller, A., et al., Reporter gene imaging identifies intratumoral infection voids as a critical barrier to systemic oncolytic virus efficacy. (2014) Mol Ther Oncolytics. 1: p. 14005. Dispenzieri, A., et al., Phase I trial of systemic administration of Edmonston strain of measles virus genetically engineered to express the sodium iodide symporter in patients with recurrent or refractory multiple myeloma. (2017) Leukemia. 31(12): p. 2791-2798. Zhao, M., Z.X. Li, and S. Bugenhagen, Tc-99m-labeled duramycin as a novel phosphatidylethanolamine-binding molecular probe. (2008) Journal of Nuclear Medicine. 49(8): p. 1345-1352. Emoto, K., et al., Exposure of phosphatidylethanolamine on the surface of apoptotic cells. (1997) Experimental Cell Research. 232(2): p. 430-434. Dingli, D., et al., Image-guided radiovirotherapy for multiple myeloma using a recombinant measles virus expressing the thyroidal sodium iodide symporter. (2004) Blood. 103(5): p. 1641-1646. Zhang, L.W., et al., Robust Oncolytic Virotherapy Induces Tumor Lysis Syndrome and Associated Toxicities in the MPC-11 Plasmacytoma Model. (2016) Molecular Therapy. 24(12): p. 2109-2117. Ammayappan, A., K.W. Peng, and S.J. Russell, Characteristics of oncolytic vesicular stomatitis virus displaying tumor-targeting ligands. (2013) J Virol. 87(24): p. 13543-55. Ammayappan, A., S.J. Russell, and M.J. Federspiel, Recombinant mumps virus as a cancer therapeutic agent. (2016) Molecular Therapy-Oncolytics. 3. Gaddy, D.F. and D.S. Lyles, Vesicular stomatitis viruses expressing wild-type or mutant M proteins activate apoptosis through distinct pathways. (2005) Journal of Virology. 79(7): p. 4170-4179. Delvaeye, T., et al., Noninvasive Whole-Body Imaging of Phosphatidylethanolamine as a Cell Death Marker Using Tc-99m-Duramycin During TNF-Induced SIRS. (2018) Journal of Nuclear Medicine. 59(7): p. 1140-1145. Elvas, F., et al., (99m)Tc-Duramycin SPECT Imaging of Early Tumor Response to Targeted Therapy: A Comparison with (18)F-FDG PET. (2017) J Nucl Med. 58(4): p. 665-670. Elvas, F., et al., Characterization of [(99m)Tc]Duramycin as a SPECT Imaging Agent for Early Assessment of Tumor Apoptosis. (2015) Mol Imaging Biol. 17(6): p. 838-47. Hu, Y., et al., A Comparison of [Tc-99m]Duramycin and [Tc-99m]Annexin V in SPECT/CT Imaging Atherosclerotic Plaques. (2018) Molecular Imaging and Biology. 20(2): p. 249-259. Ruotsalainen, J.J., et al., Clonal variation in interferon response determines the outcome of oncolytic virotherapy in mouse CT26 colon carcinoma model. (2015) Gene Ther. 22(1): p. 65-75. Ghonime, M.G. and K.A. Cassady, Combination Therapy Using Ruxolitinib and Oncolytic HSV Renders Resistant MPNSTs Susceptible to Virotherapy. (2018) Cancer Immunology Research. 6(12): p. 1499-1510. Escobar-Zarate, D., et al., Overcoming cancer cell resistance to VSV oncolysis with JAK1/2 inhibitors. (2013) Cancer Gene Therapy. 20(10): p. 582-589. Dold, C., et al., Application of interferon modulators to overcome partial resistance of human ovarian cancers to VSV-GP oncolytic viral therapy. (2016) Molecular Therapy-Oncolytics. 3.

15

382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398

32. 33. 34.

35. 36. 37. 38. 39.

Rameix-Welti, M.A., et al., Visualizing the replication of respiratory syncytial virus in cells and in living mice. (2014) Nature Communications. 5. Mehle, A., Fiat Luc: Bioluminescence Imaging Reveals In Vivo Viral Replication Dynamics. (2015) Plos Pathogens. 11(9). Vandergaast, R., K.W. Peng, and L. Suksanpaisan, Precise Anatomical Localization and Deep Tissue Imaging of Cells Expressing Luciferase and the Sodium Iodide Symporter (NIS). (2018) Molecular Therapy. 26(5): p. 208209. Haddad, D. and Y. Fong, Molecular imaging of oncolytic viral therapy. (2015) Molecular Therapy-Oncolytics. 2. Eidenschink, B., et al., NIS-Mediated Radioiodine Therapy and Imaging for Pancreatic Cancer with Oncolytic Adenovirus. (2014) Molecular Therapy. 22: p. S65-S65. Jiang, H., et al., Synthesis and evaluation of (18)F-hexafluorophosphate as a novel PET probe for imaging of sodium/iodide symporter in a murine C6-glioma tumor model. (2018) Bioorg Med Chem. 26(1): p. 225-231. Jiang, H. and T.R. DeGrado, [(18)F]Tetrafluoroborate ([(18)F]TFB) and its analogs for PET imaging of the sodium/iodide symporter. (2018) Theranostics. 8(14): p. 3918-3931. Zhao, M. and Z.X. Li, A single-step kit formulation for the Tc-99m-labeling of HYNIC-Duramycin. (2012) Nuclear Medicine and Biology. 39(7): p. 1006-1011.

399

16