ARTICLE Oncolytic herpes simplex virus kills stem-like tumor-initiating colon cancer cells

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1 Citation: Moleular Therapy Onolytis (216) 3, 1613; doi:1.138/mto ARLE Onolyti herpes simplex virus kills stem-like tumor-initiating olon aner ells Susanne G Warner 1, Dana Haddad 2, Joye Au 3, Joshua S Carson 4, Mihael P O Leary 1, Christina Lewis 5, Seastien Monette 6 and Yuman Fong 1 Stem-like tumor-initiating ells (s) are impliated in aner progression and reurrene, and an e identified y sphere-formation and tumorigeniity assays. Onolyti viruses infet, repliate in, and kill a variety of aner ells. In this study, we seek proof of priniple that s are suseptile to viral infetion. HCT8 human olon aner ells were sujeted to serum-free ulture to generate tumorspheres. ells and s were infeted with HSV-1 sutype NV166. Cytotoxiity, viral repliation, and Akt1 expression were assessed. tumorigeniity was onfirmed and NV166 effiay was assessed in vivo. NV166 infetion was highly ytotoxi to oth parent HCT8 ells and s. In oth populations, ell-kill of >8% was ahieved within 3 days of infetion at a multipliity of infetion (MOI) of 1.. However, the parent ells required 2-log greater viral repliation to ahieve the same ytotoxiity. s overexpressed Akt1 in vitro and formed flank tumors from as little as 1 ells, growing earlier, faster, larger, and with greater histologi atypia than tumors from parent ells. Treatment of -indued tumors with NV166 yielded tumor regression and slowed tumor growth. We onlude that olon s are seleted for y serum-free ulture, overexpress Akt1, and are suseptile to onolyti viral infetion. Moleular Therapy Onolytis (216) 3, 1613; doi:1.138/mto ; pulished online 15 June 216 INTRODUCTION Caner stem ell (CSC) theory represents a reent paradigm shift that has hallenged previously aepted aner development proesses. In theory, a CSC is a ell within a tumor that possesses the apaity to self-renew and to ause the heterogeneous lineages of aner ells that omprise the tumor. 1 CSC theory offers a logial explanation as to why traditional hemoradiotherapies ommonly result in transient regression followed y disappointing reurrene. Within the framework of CSC theory, traditional therapy, whih targets rapidly dividing aner ells, spares quiesent CSCs therey failitating tumor reurrene months to years after initial therapy. 2,3 In addition to demonstrating the need for novel therapies, this theory implies that one annot ure a aner without eradiating CSCs. 2 Controversy aounds regarding the optimal method for CSC identifiation. 4 7 Authors have used various means, suh as ell surfae markers, intraellular funtional proteins like aldehyde dehydrogenase (ALDH), expulsion of Hoesht dye, formation of tumor spheres in serum-free or soft agar ulture, and ATP-inding assette (ABC) transporters that efflux hemotherapeuti drugs. 1,7 12 All of these methods, however, are measured against the funtional assay and gold standard of in vivo tumorigeniity from a very small amount of ells. 3,8,13 With variations of the aove tehniques, CSCs have een identified among many different solid tumor types inluding rain, 14 head and nek, 15,16 reast, 17 lung, 18,19 esophageal, 2 panreati, 21 olon, 22,23 and ovarian aners, 24 as well as melanoma. 25 Several investigators have even estalished that a single CSC an initiate tumor formation. 11,26 The PI3/Akt pathway has een impliated in tumorigeniity and the CSC phenotype. Akt is the primary effetor kinase of the lipid kinase PI3K and is onstitutively ative in many malignant tissues, with promotion of ell survival, proliferation, angiogenesis, and invasion. 27 Several authors have shown that upregulated memers of the PI3/Akt pathway are ruial to stemness in various solid tumors. 11,28,29 Onolyti viral therapy has reently emerged as a forerunner in the rae to develop novel adjuvant therapies to treat aner. Onolyti viruses infet, repliate in, and kill aner ells, with speifiity that leaves normal ells unharmed. 3,31 In terms of CSC treatment, the very aerrations that failitate CSC resistane to traditional therapies atually make them ideal targets for onolyti viruses. 32 For instane, defetive apoptoti signaling has een shown to failitate p53-independent death in response to viral infetion of hemotherapy-resistant ells. This implies that CSCs with strengthened traditional apoptoti pathways would still e suseptile to viral therapy. 33 Several authors have demonstrated the potential of various onolyti viruses against CSCs or stem-like ells using gliolastoma, neurolastoma, esophageal, and reast aner models. 3,33,34 HSV-1-derived attenuated viruses are unique and potent onolyti virus that has proven suessful in prelinial 1 Department of Surgery, City of Hope, Duarte, California, USA; 2 Department of Radiology, Memorial Sloan Kettering Caner Center, New York, New York, USA; 3 Department of Surgery Fox Chase Caner Center, Philadelphia, Pennsylvania, USA; 4 Department of Surgery University of Texas Medial Branh, Galveston, Texas, USA; 5 Thomas Jefferson College of Mediine, Philadelphia, Pennsylvania, USA; 6 Laoratory of Comparative Pathology and the Genetially Engineered Mouse Phenotyping Servie, Memorial Sloan Kettering Caner Center, New York, New York, USA. Correspondene: SG Warner (suwarner@oh.org) Reeived 14 Deemer 215; aepted 1 Marh 216

2 2 and linial models NV166 is an engineered HSV-1 that has shown effiay against more than 11 ell lines derived from at least 16 aner ell types, inluding oloretal aner. 35 We hypothesized that antiapoptoti pathways are upregulated in oloretal CSCs, making them ideal targets for viral therapy. We further theorized that CSCs are suseptile to onolyti viral infetion. As we use a funtional tumorigeniity assay to define our stemlike supopulation of aner ells in this study, we favor the term tumor-initiating ells or s. 5 RESULTS Serum-free ulture selets for tumor spheres omposed of stem-like HCT8 ells that overexpress AKT1 (and underexpress BAD) Serum-free ulture relialy produed tumor spheres. Cells in tumor spheres appeared morphologially different from adherent-growing parent HCT8 ells with rounded shape, with sujetively lessonsistent dimensions (Figure 1). Tumorspheres also repliated at a slower rate than their adherent parental ounterparts (Tale 1). Cell douling time within a tumorsphere ould take up to 48 hours, whereas parent HCT8 ells douled at least one every 24 hours. Moreover, parent HCT8 ell ounts inreased 1-fold within 72 hours of plating, versus fivefold in the population (Tale 1). These differenes in repliation speed were onsistently oserved, and approahed ut did not ahieve statistial signifiane. upregulation of Akt1 was onfirmed with oth Western lot and RT-polymerase hain reation (PCR) (Figure 1). HCT8 s are highly tumorigeni ompared with parent ells Injetion of the same amount of s into the flanks of 7 9-weekold nude athymi mie onsistently yielded tumor growth from as few as 1 s as soon as 2 days following injetion (Figure 2a). Injetion of the same quantities of parent HCT8 ells rarely generated tumors. Tumors reated y injetion with s onsistently grew faster, larger, sooner, and with greater ellular atypia than those rare tumors formed from parent HCT8 injetions (Figure 2). After 6 days, no tumors resulted from injetion of 1 parent HCT8 ells. At 35 days postinjetion, tumors egan to appear in three of five mie injeted with 1, parent tumor ells. These tumors grew muh later, slower, and with dereased ellular atypia when ompared to tumors initiated with 1, s. Upon sarifie, no adominal or thorai metastases were visile in any of the animals, though asites was oasionally present. NV166 infets and expresses GFP in HCT8 parent ells and s In this experiment, NV166 suessfully infeted and expressed green fluoresent protein (GFP) in HCT8 parent ells and s at all MOIs of.1,.1, and 1.. GFP expression was deteted y oth fluoresent mirosopy and y flow ytometry. Tale 2 demonstrates sustained dose-dependent inreases in GFP expression in oth ell populations. Representative experiments at MOI of.1 are shown (Figure 3a). NV166 repliates within and is more effiiently ytotoxi to s versus parent HCT8 ells Both HCT8 parent ells and s support viral repliation. In the parent population, viral titers peaked on day 3 at MOI 1. and day 5 at MOI.1 and.1. In the population, viral titers peaked on day 3 at MOI 1. and.1, and peaked on day 5 at MOI.1. Expetedly, lower viral doses resulted in inreased viral repliation in order to ahieve the same ell kill as higher initial doses. Thus, in oth ell populations, an MOI of.1 resulted in the maximum oserved amplifiation of viral titers (Figure 3,). Interestingly, far less viral repliation was needed to ahieve similar ytotoxiity in the versus the parent HCT8 ells. For instane, at an MOI of.1, peak viral titer in s represented a 75-fold inrease over initial dose versus a greater-than 3,8-fold inrease in the parent HCT8 ells. As shown in Figure 3d, ytotoxiity was ahieved in a dose-dependent fashion over a 1-fold range of viral doses. In the population, an MOI of 1. resulted in greater than 75% ell kill after 3 versus 4 days in the parent ell population. Furthermore, greater than 8% ell kill was ahieved y day 7 at all 3 MOIs in the experiments ompared to day 9 in the parent ell experiments. A representative experiment is shown (Figure 3d,e). Relative mrna expression a AKT1 Tuulin Figure 1 HCT8 parent ells versus tumor-initiating ells (s). (a) ells appear morphologially different from their ounterparts forming tumorspheres (). The ells are further distinguishale y varied expression of Akt1 (). NV166 infetion ends in onolyti ell death, and may indue apoptosis in neighoring noninfeted ells Muh deate exists in the literature regarding preise mehanisms and pathways of onolyti ell death as a result of viral infetion. As previously estalished, viral infetion an indue apoptosis in neighoring noninfeted ells. 43 In order to further haraterize the nature and effets of virally-indued ell death in the population, tumorsphere infetion was oserved using real-time and timelapsed fluoresent onfoal mirosopy. Via time-lapsed mirosopy, Tale 1 Cell douling time HCT8 P value Cells/well hours hours hours

3 a Avg tumor volume (m 3 ) E3 1 E2 1 E1 1 E Days post ell injetion Figure 2 Tumor-initiating ells (s) are highly tumorigeni when ompared to equal numer of parent ells injeted. (a) Tumors initiated with 1E5 ells of eah tumor population were examined histologially. In addition to growing muh faster, tumors () exhiited muh higher levels of histologi atypia ompared to tumors initiated y parent ells () as noted y H&E staining. Tale 2 Perent green fluoresent protein-expressing ells Day 1 Day 3 Day 5 MOI MOI MOI MOI MOI MOI MOI, multipliity of infetion;, tumor-initiating ells. noninfeted ells were oserved oth separating from tumorspheres, and undergoing ell-death (Figure 4a). Infeted ells were stained with Cy5-tagged Annexin V and 4,6-diamidino-2-phenylindole (DAPI) DAPI and examined via fluoresent mirosopy. Annexin V is a protein that attahes to phosphatidylserine residues on pre-apoptoti ells. Many non-gfp-expressing, noninfeted ells adjaent to GFP-expressing infeted ells were positive for Annexin V. These noninfeted ells were frequently also positive for DAPI staining, indiating memrane permeaility and impending later-stage apoptosis (Figure 4). Interestingly, very few ells were doule-positive for either GFP and Annexin-V-Cy5 or GFP and DAPI, suggesting that viral lyti ell death is a proess that likely ours independent of ystander ell apoptoti pathways. However it should e pointed out that HSV-1 infetion an result in phosphatidlyserine transloation, thus there is a possiility that neighoring ells presumed to e noninfeted ould simply not yet e expressing GFP. Further investigations are needed to estalish reproduiility and validity of this possile asopal effet. NV166 arogates tumor growth of flank tumors In the animal model, injetion of virus into oth and parent HCT8-generate tumors resulted in ell-swelling, ytoplasmi vauolization, and intranulear inlusion odies with marginalization of hromatin (Figure 5). Furthermore, in virally-treated tumors, immunohistohemial staining for GFP was positive in areas of nerosis surrounded y areas of tumor regression. Figure 5 shows H&E appearane of treated tumor versus 5 days stained for GFP. On a marosopi level, among a ohort of 1 mie with indued tumors, 4 were treated with PBS and 6 were treated with NV166. Viral therapy arogated tumor growth and indued regression in some ases, as indiated y Figure 5a. In fat, phosphateuffered saline (PBS)-treated mie experiened fourfold greater tumor growth than their virus-treated ounterparts. DISCUSSION This study demonstrates a distint sensitivity of olon adenoarinoma s to the HSV-1-ased onolyti virus NV166 in vitro and shows effiay of this same vetor against -derived tumors in vivo. While other authors have shown that HSV-1 kills stem-like gliolastoma, neurolastoma, and reast ells; to our knowledge, ours is the first study to investigate the suseptiility of stem-like oloni adenoarinoma ells to HSV-1 onolyti viral therapy. Several investigators have previously assoiated poor oloretal aner outomes and inreased metastases with expression of ell-surfae markers assoiated with stemness Thus the sensitivity of oloretal s disovered in this study proves a priniple that may pave the way for further fous of targeted therapies aimed at s. To identify and haraterize s, this study utilized a serum-free tumorsphere ulture of a oloretal aner ell line, the presene of AKT overexpression, as well as generation of tumors from as few as 1 ells. Despite many studies advoating use of ell surfae markers as CSC identifiers, it remains unlear whether stemness and differentiation are mutually exlusive in CSCs. The more

4 4 a 12 % GFP-expressing ells Days after infetion E+6 d 12 Viral plaque forming units (PFU) 2.5E+6 2.E+6 1.5E+6 1.E+6 5.E+5 Initial dose Maximum titer Cell survival (% ontrol) E+ MOI.1 MOI.1 Viral dose MOI Days post-infetion 1.6E+4 e 12 Viral plaque forming units (PFU) 1.4E+4 1.2E+4 1.E+4 8.E+3 6.E+3 4.E+3 2.E+3.E+ MOI.1 MOI.1 Viral dose MOI 1. Initial dose Maximum titer Cell survival (% ontrol) Days post-infetion Figure 3 Green fluoresent protein (GFP) expression inreases over time after infetion. GFP expression reahed and sustained lose to 1% expression in oth HCT8 parent (dashed line, irles) and tumor-initiating ells () (solid line, squares) populations. A representative experiment at MOI.1 is shown (a). HCT8 parent () and () ells support viral repliation at MOIs of.1,.1, and 1., with exponentially inreased viral PFUs on day of maximal urst (lak ars) ompared to initial doses (white ars). However, viral repliation does appear to our on a different sale etween populations. In oth HCT8 parent (d) and (e) populations, NV166 was ytotoxi at MOI of.1 (irles),.1 (squares), and 1. (triangles). Cell kill is represented as perentages of live ells ompared to ontrol untreated ells grown under idential onditions. MOI: multipliity of infetion, ratio of viral partiles to ells; PFU: plaque-forming units, numer of viral partiles. likely truth is that ells exist in varying degrees of stemness within a tumor, and that stemness is a flexile harateristi. Thus, several authors advoate a move away from surfae markers, or at least the orrelation of surfae markers with funtional ellular assays. 51 Most likely, the population indued y our tumorsphere ulture was heterogeneous. Inaility to produe lonally idential ells is perhaps the foremost limitation of the study. However, we estalished onsistent upregulation of AKT1 in the s and y doing so further related them to putative CSCs. Similarly, Vermeulen et al. 11 were ale to isolate a population of CSCs whih ould form tumorspheres from a single ell. They then noted that CSC markers like CD133 + and CD24 + are oexpressed on tumorigeni ells, and further demonstrated that inhiition of PI3K pathway atually results in differentiation of stem-like ells, indiating the ruial role of the PI3K/Akt pathway in maintaining stem-like harateristis. 11 The suseptiility of human oloretal aner ell lines to HSV onstruts and speifially to NV166 has een previously estalished. 35,52 This study also estalished that oloretal s are highly sensitive to onolyti HSV-1. Interestingly, while viral repliation ourred effiiently in oth parent and populations, several

5 a 5 hours 12 hours 24 hours d 36 hours 48 hours 6 hours Figure 4 Fluoresene mirosopy demonstrates virus-indued green fluoresent protein (GFP) expression and presumaly noninfeted ystander ell apoptosis. (a) Images aquired y fluoresent and differential interferene ontrast onfoal mirosopy show a tumorsphere on post-infetion day 3 following infetion with an MOI of 1.. Cells expressing GFP (green) are infeted with NV166. () A merged image of ontrol, uninfeted ells expressing only small amounts of Annexin V and DAPI. () The ontrol DIC image of the stained tumorsphere. (d) Cy-5 tagged Annexin V-positive ells appear magenta, whih indiates phosphatidylserine residues of preapoptoti ells. Cells stained lue have taken up DAPI, indiating inreased ellular permeaility and a later stage of apoptosis. thousand more PFUs were neessary to ahieve parent HCT8 ell kill. This finding implies that s are more sensitive to viral infetion than their parent ell ounterparts. It is important to note that infetions of parent and populations were performed in parallel, with ells seated and infeted at the same time, using the same methods aside from serum-free nonadherent ulture required to reate the tumor spheres, and same viral stok and repeated in tripliate. Viral titers were also performed in parallel and repeated in tripliate. Thus, we do not feel this differene in repliation was merely an issue of ell-plating or differene in viral stok. While a small portion of this differene ould e aounted for y the oserved inreased douling time of the population, a several thousand fold disparity more likely indiates a fundamental differene in ellular mahinery. The finding of distint effiay of NV166 against a ell population holds great linial promise in that CSC-targeted viral therapy ould e administered without generating exessive daughter viral partiles. In light of the aforementioned theories, there is reason to suspet that suh -targeted therapy may represent a means of preventing the reurrenes that plague ontemporary treatment regimens. This study also showed that s exhiit upregulated onstitutive expression of AKT1 ompared to their parent ells. As previously disussed, AKT1 plays a entral role in protein synthesis and antiapoptosis proesses known to e dysregulated in oloretal aner. Apoptosis is a protetive mehanism employed y infeted ells to disale ellular mahinery neessary for viral repliation. Host ells infeted with HSV-1 an undergo phosphorylation of eukaryoti initiation fator-2α whih terminates the viral lyti yle y inhiiting protein synthesis. 53 To avert apoptosis, HSV-1 enodes genes like γ , whih enodes the ICP45 protein that in turn prevents phosphorylation of eukaryoti initiation fator- 2α therey failitating ontinued protein synthesis However, many engineered onolyti HSV-1 strains are attenuated y single deletions of γ , whih failitates onotropism given that aner ells usurp antiapoptoti pathways. Thus, even with defiient γ , onolyti HSV-1 has time and mahinery to repliate and propagate. Interestingly, in addition to assoiation with stemness, PI3/Akt has een impliated in the upregulation of genes that alter the intraellular environment to failitate more effiient HSV-1 infetion. 11,57 Thus, this study opens the door for future researh onerning the importane of Akt to viral onolysis and sensitivity to HSV-1. This study also oserved that while infeted ells serve viral purposes y prolonging ell life, neighoring, noninfeted ells experiene a ystander effet and undergo apoptosis. This has een previously demonstrated with HSV-1 infetion of gastri aner ells, theorizing that future inhiition of apoptosis ould enhane lateral viral spread y making more ells availale to potentially produe viral progeny. 43 Using annexin-v and DAPI staining in this study, we were ale to visualize a similar phenomenon among infeted s. Moreover, we found that very few infeted ells (e.g., GFP-expressing ells) were also positive for annexin-v or DAPI, indiating that infeted ells do not present phosphatidylserine residues, whih are thought to result from aspase asade ativation. This is aligned with previous studies indiating that engineered HSV-1 sutypes indue aspaseindependent ell death. 58 This has several important impliations. For instane, inhiition of the protetive apoptosis of ells adjaent to infeted ells ould enhane lateral spread of virus, and therey failitate tumor destrution at dereased doses. Even more intriguing is that previous authors have orrelated

6 6 a P =.3 Tumor volume (m 3 ) PBS NV Days postinjetion d Figure 5 Histologi examination of viral treated tumors and in vivo effiay of viral infetion. (a) Viral injetion of tumor-initiating ells ()-initiated tumors aates tumor growth and indues regression in some ases. Tumors initiated y 1, s were treated with two onseutive doses of NV166 using 5E6 PFU, with the seond dose ourring at day 3 on the graph. () Mirosopi hanges in infeted tumors show lear signs of tumor ell death. V: viale area of tumor, N: neroti area, T: transition zone in whih ells are undergoing degeneration and nerosis. In this zone note ell swelling, ytoplasmi vauolation, and numerous ells with intranulear inlusion odies with margination of hromatin (arrows) typially seen after viral infetion. () In virally-treated tumors, areas of nerosis orrespond with (d) green fluoresent protein expression. onstitutive Akt1 overexpression with ellular protetion from apoptosis and memrane phosphatidylserine exposure. 59 This again demonstrates the need for further examination of the effets of onolyti virus on the PI3K/Akt pathway. Conlusion Stem-like tumor-initiating oloretal aner ells overexpress AKT1, and are suseptile to onolyti HSV-1. Further researh is needed to determine if memers of the PI3/Akt pathway ould yield targets for onolyti viral therapies aimed at CSC eradiation. MATERIALS AND METHODS Cell lines The human oloretal aner ell line HCT8 (Amerian Type Culture Colletion (ATCC), Rokville, MD) was studied. HCT8s were grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 1 U/ml peniillin, 1 mg/ml streptomyin, and 1% fetal alf serum (FCS). Vero ells (ATCC) were grown in minimum essential medium (MEM) supplemented with 1 U/ml peniillin, 1 mg/ml streptomyin, and 1% FCS. Cells were maintained in a 5% CO 2 humidified inuator at 37 C. Sphere isolation and haraterization After a minimum of three passages, HCT8 (ATCC, Manassas, VA) ells were washed in PBS and harvested with.25% trypsin in.2% ethylenediaminetetraaeti aid and entrifuged for 5 minutes at 8 rpm. They were then resuspended in PBS, reentrifuged, and finally resuspended in serum-free Roswell Park Memorial Institute medium (RPMI) supplemented with 2 μg/l human reominant epidermal growth fator (hregf), 1 μg/l firolast growth fator (FGF), and 5 ml/l insulin-transferrin-selenium (ITS) liquid media supplement 1 (Sigma-Aldrih, St. Louis, MO). Cells were then plaed overnight in a regular adherene flask. The next day, ells growing in suspension were removed from the flask, entrifuged, resuspended in serum-free RPMI, and ultured in ultra-low attahment flasks (Corning Inorporated, Corning, NY). Spheres were haraterized using Western lot and PCR for Akt1 expression (tehniques desried elow). Hemoytometer standard ounting methods were employed to disern ell quantities. To determine douling times, s and parent HCT8 ells were seated in six-well plates and ounted at 24-hour intervals. Six measurements from eah well were taken and the total alulated ells per well of at least three wells were averaged to yield daily values. Virus NV166 is a repliation-ompetent, attenuated herpes simplex type-1 virus (HSV-1) whose onstrution has een desried in detail previously. 6 This virus is derived from the wild-type HSV-1 F-strain akone, and has single opy deletions of ICP-4, ICP-, and γ , whih inrease tumor seletivity and derease virulene (Figure 6). NV166 also ontains a transgene for enhaned GFP under the ontrol of a onstitutively ative ytomegalovirus promoter. Infeted ells thus express GFP that an e deteted in vitro y oth fluoresent mirosopy and flow ytometry. 35 Viral stoks were propagated on Vero ells and titered y standard plaque assay. 52 Cytotoxiity assay HCT8 ells ( ) were plated and grown overnight in standard 24-well flatottom plates, while suspended HCT8 tumor spheres were grown in Corning

7 TR L (NV166) pa pa TRS pa oril oris IR L IR S oris Deleted α & γ34.5 Deleted α4 Figure 6 NV166 viral onstrut. Herpes simplex virus type 1 is a doule-stranded DNA virus with a 152 k DNA genome omprised of unique long and short segments, eah flanked y inverted (IR L and IR S ) and terminal repeats (TR L and TR S ). The genome further ontains three DNA pakaging (pa) signals, whih enale onstrution of virions. There are two different origins of repliation, one in the unique long segment (oril), and one in the unique short segment (oris). Several genes are dupliated as a result of the inverted repeats. These inlude oris, γ34.5, α, and α4. Deletion of these genes enhanes viral speifiity and dereased neurovirulene. An enhaned green fluoresent protein (egfp) is enoded on a onstitutively ative ytomegalovirus (CMV) promoter. Infeted ells thus express egfp. ultra-low attahment 24-well plates. Cells were infeted with NV166 at multipliities of infetion (MOIs) of 1.,.1, and.1, and inuated at 37 C in a 5% CO 2 humidified inuator for 1 to 9 days. Eah day after infetion, adherent HCT8 supernatant was removed and ells were lysed in 24-well plates with a 1.5% Triton-X solution (% volume/pbs). HCT8 tumorsphere suspensions were olleted and entrifuged at 1,2 rpm for 5 minutes and then lysed with the same Triton-X solution to release intraellular latate dehydrogenase. The ytotoxi effet was determined y omparing release of intraellular latate dehydrogenase from virally infeted tumor ells to untreated ontrol ells grown under idential onditions. Latate dehydrogenase was quantified using a Cytotox 96 nonradioative ytotoxiity assay (Promega, Madison, WI) that measures the onversion of a tetrazolium salt into a formazan produt. Asorane was measured at 49 nm using a SpetraMAX 19 miroplate reader with the assistane of SoftMax Pro software v5..1 (Moleular Devies, Sunnyvale, CA). Results are expressed as the surviving fration of treated ells ompared to untreated ontrol ells. All samples were tested in tripliate and the experiment was repeated at least three times. Mirosopi examination of infeted ells All infeted ells were visualized under fluoresent mirosopy. All samples were evaluated with an inverted mirosope (Nikon Elipse TE3, Nikon, Tokyo, Japan) using phase-ontrast and fluoresene mirosopy. A GFP emission filter was used to detet green fluoresene, and images were otained using NIS software. Infeted tumorspheres were further examined with a Leia TCS AOBS SP2 spetral onfoal mirosope on an upright stand (Leia Mirosystems, Wetzlar, Germany) following staining with Annexin V- Cy5 Apoptosis Detetion Kit (BioVision, Mountain View, CA) and 4, 6-diamidino-2-phenylindole dihydrohloride DAPI for measures of early and late apoptosis. Time-lapse images of sphere formation and ellular infetion were otained using a Zeiss LSM 5 Live onfoal laser sanning mirosope (Carl Zeiss, Oerkohen, Germany). Viral repliation assay Standard plaque assays were performed to quantify viral repliation in HCT8 ells following infetion with NV166. Cells (1 1 3 ) were plated in six-well flat-ottom assay plates in 2 ml media. After 12 to 24 hours, ells were treated with NV166 at MOIs of.1,.1, and 1. in 1 μl media. Cells maintained at 37 C served as ontrols. Supernatants and ells were olleted from ulture wells eah day after infetion for 7 days. Serial dilutions of supernatants and ell lysates were ultured on onfluent layers of Vero ells and viral titers were determined y ounting viral plaques after 72 hours. All samples were tested, and experiments were repliated in tripliate. Flow ytometry for GFP HCT8 ells were grown in standard six-well plates and the same numer of ells from HCT8 tumorspheres were grown in ultra-low attahment six-well plates (Corning Inorporated). Cells were infeted at MOIs of 1.,.1, and.1. Adherent HCT8 ells were harvested with.25% trypsin in.2% ethylenediaminetetraaeti aid, washed in PBS, and then resuspended in 2 μl PBS. 5 μl of 7-amino-atinomyin (7-AAD, BD Pharmingen, San Diego, CA) was added as an exlusion dye for ell viaility. Data for GFP expression was aquired on a FACSCaliur mahine (BD Biosienes, San Jose, CA) and analyzed with FlowJo software (Tree Star, Ashland, OR). Flank tumor model Eight-to-ten-week-old athymi nude female mie otained from Harlan Sprague Dawley laoratories were provided with food and water ad liitum. All animal work ourred with the approval and strit guidane of the Memorial Sloan-Kettering Institutional Animal Care and Use Committee. Mouse flanks were injeted with to HCT8 human oloretal aner ells or an equal numer of ells from tumor spheres produed y serum-free ulture. Cells were injeted as a 1:1 mixture with Growth-Fator- Redued BD Matrigel (BD Biosienes). Animals were weighed and tumors measured every 3 5 days. Tumor development ourred relialy 2 5 weeks following injetion depending upon amount and type of ells injeted. Tumor volume was alulated using the following formula: Volume (m 3 ) = (length width2) (π 6). Animals in whih either exessive weight loss or tumor growth ourred were sarified prior to experimental ompletion. In one ohort of ten indued tumor-earing mie, when visile tumor growth was ahieved, five mie were treated with PFU NV166 in 1 μl PBS tumor injetion whereas five reeived 1 μl PBS alone for two onseutive doses administered on days 1 and 3. Histologi analysis Following sarifie, flank tumors were removed en lo. Tumor and arass weights were reorded. Tumors were fixed in 4% paraformaldehyde overnight, then rinsed with PBS, and plaed in 7% ethanol. Samples were then emedded in paraffin loks, routinely setioned at 4 μm, and stained for hematoxylin and eosin, as well as GFP. All pathology slides were reviewed y a veterinary histopathologist, linded to tumor treatment modalities. Western lot Protein from total ell lysates were resolved y sodium dodeyl sulfate -polyarylamide gel eletrophoresis (SDS-PAGE) SDS-PAGE, transferred onto Immuno-Blot polyvinylidene difluoride memranes (Bio-Rad, Herules, CA), and proed with antiodies inluding AKT1, β-atin, and tuulin (Cell Signaling Tehnology, Danvers, MA). Detetion was performed with enhaned hemiluminesene reagents (Amersham Biosienes, England). Real-time PCR (RT-PCR) RNA was isolated using an RNeasy Mini-Kit (Qiagen, Valenia, CA) as desried y the manufaturer. Reverse transription was performed using random hexamer priming and TaqMan Reverse Transription Reagents (Applied Biosystems, Foster City, CA). RNA was quantified y spetrophotometry with the Nanodrop 1 spetrophotometer (Thermo Sientifi, Waltham, MA). RT-PCR was performed on a ThermoHyrid thermoyler (Thermo Sientifi). For eah reation, 2 μg of RNA was amplified in a 1 μl reation ontaining 2 μl of deoxynuleotide triphosphate (2.5 mmol/l eah dntp), 22 μl MgCL 2 (25 mmol/l), 1 μl 1 -RT uffer, 5 μl random hexamers (5 μmol/l), 2 μl RNase inhiitor (2 U/μl), and a variale amount of free water to total 1 μl depending on RNA sample onentration. Samples were transferred to the Thermo Hyrid thermoyler at 25 C for 1 minutes, 48 C for 3 minutes, and 95 C for 5 minutes. The resulting DNA samples were stored at 2 C. Quantitative realtime PCR (RT-PCR) using TaqMan Assay-on-Demand Gene Expression Assays (Applied Biosystems) was performed on the ABI Prism 79HT Sequene Detetion System. DNA was amplified in a 2 μl reation ontaining 1 μmol/l of the desired primer, 2 μl DNA, and TaqMan Universal PCR Master Mix (Applied Biosystems). Eah PCR reation was sujeted to 3 minutes at 48 C and 1 minutes at 95 C followed y 4 yles at 95 C for 15 seonds and 6 C for 6 seonds. Eah sample was assayed at least in tripliate and experiments were repeated at least three times. Results were analyzed using SDS version 2.3 software (Applied Biosystems). The relative expressions of desried genes were determined relative to the endogenous ontrol 18s rrna using ΔCT analysis. Statistial analysis All data were expressed as mean ± standard error of the mean. A two-tailed Student t-test was used to determine signifiane etween treatment groups in oth in vitro and in vivo experiments. A P value of <.5 was onsidered statistially signifiant. 7

8 8 CONFLICT OF INTEREST The authors delare no onflit of interest. ACKNOWLEDGMENTS The authors would like to thank Tony Riley, of the Medial Graphis and Photography Department at the Memorial Sloan-Kettering Caner Center for his invaluale illustration of Figure 1. The authors further thank Jakie Candelier of the laoratory of omparative pathology for assisting in the proessing of tumor samples. The authors thank Viola Weeda of Bhuvanesh Singh s laoratory for her invaluale assistane with various laoratory tehniques. The authors thank Sho Fujisawa, Yevgeniy Romin, and Tao Tong of the Moleular Cytology Core Faility at Memorial Sloan-Kettering Caner Center for assistane with the mirosopi aspets of this projet. Finally, the authors espeially thank Meryl Greenlatt for her editorial assistane, and also Sott Tuorto of the Department of Surgery for his researh assistane. REFERENCES 1. Clarke, MF, Dik, JE, Dirks, PB, Eaves, CJ, Jamieson, CH, Jones, DL et al. (26). 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