Oncolytic herpes simplex viruses type 1 (oHSVs) such as G47Δ and G207 are genetically engineered for selective replication competence in cancer cells. Several factors can influence the overall effectiveness of oHSV tropism, including HSV-1 receptor expression, extracellular matrix milieu and cellular permissiveness. We have taken advantage of human prostate organ cultures derived from radical prostatectomies to investigate oHSV tropism. In this study, we show that both G47Δ and G207 specifically replicate in epithelial cells of the prostatic glands but not in the surrounding stroma. In contrast, both the epithelial and stromal cell compartments were readily infected by wild-type HSV-1. Analysis of oHSV replication in prostate surgical specimens 3 days post infection showed that G47Δ generated ∼30-fold more viral progeny than did G207. This correlated with the enhanced expression of G47Δ-derived glycoprotein gB protein levels as compared with G207. In benign prostate tissues, G207 and G47Δ titers were notably reduced, whereas strain F titers were maintained at similar levels compared with prostate cancer specimens. Overall, our results show that these oncolytic herpes vectors show both target specificity and replication competence in human prostate cancer specimens and point to the utility of using human prostate organ cultures in assessing oHSV tropism and cellular specificity.
Oncolytic virotherapy is based on the selective killing of tumor cells while sparing the surrounding normal cells. Selectively replication-competent viral vectors, such as oncolytic herpes simplex viruses type 1 (oHSVs) which are genetically engineered from herpes-simplex virus (HSV) type I, represent an attractive strategy for tumor-based therapies because these viruses can specifically replicate and spread in cancer cells in situ, exhibiting oncolytic activity through direct cytopathic effects while sparing normal cells. G207 is a previously described oHSV that has been used in clinical trials.1 It contains deletions of both copies of gamma34.5 and a LacZ insertion within the ICP6 gene causing its inactivation.2 G47Δ is a replication-competent HSV-1 vector derived from G207 that also contains an additional deletion within the nonessential α47 gene.3 A deletion of α47 places the late US11 gene under the control of the immediate early α47 promoter. As a consequence, US11 expression immediately after infection results in the enhanced growth of G47Δ by precluding the shutoff of protein synthesis.4, 5 oHSV vectors have undergone extensive preclinical and clinical testing. Our laboratory has previously shown that G47Δ is more effective than G207 in several tumor models.3, 6
Importantly for prostate cancer, oHSV vectors have been shown to be nontoxic to normal prostate and the surrounding nerves after intraprostatic inoculation in mice and in non-human primates.7, 8 In addition, oHSVs are effective against human prostate cancer irrespective of hormone status or radiosensitivity,6, 9, 10 a major advantage in its application for advanced forms of the disease, in which hormone and radiation refractory tumor is a common cause of progression. Moreover, preexisting immunity does not preclude the efficacy of oHSV vectors in mice.11, 12, 13 This is critical for translational purposes, as many humans are seropositive for HSV.
Although the use of oHSV for tumor therapy has shown promise, several issues that have hampered their further development for use in clinics still remain. Some of the factors that may limit the overall effectiveness of oHSV as a cancer therapeutic modality include expression of HSV-1 entry receptors, extracellular matrix barrier and intracellular resistance mechanisms.14 Strategies developed to assess oHSV tropism in monolayer tissue culture do not recapitulate the three-dimensional tumor physiology of multiple cell types as observed in patient and in-patient specimens.
Both human and murine prostate organ cultures have been used extensively to study a wide variety of biological processes ex vivo.15, 16, 17, 18 We have taken advantage of a prostate organ culture system derived from radical prostatectomies for cancer and from surgeries removing benign prostatic tissue (BPT) to assess oHSV tropism. The advantages of prostate organ cultures are the following: (1) the use of primary human prostate cancer material (as opposed to passaged prostate cancer cell cultures or animal models); (2) the three-dimensional structure remains intact (in contrast to typical monolayer cultures; (3) tumor foci exist at different stages of tumor progression; and (4) the genetic heterogeneity of cancer cells can be studied within and between patient samples. Therefore, the factors that may affect viral entry and replication including cell–cell interactions and cell–matrix interactions remain preserved in this three-dimensional milieu and are amenable to exploration.
Assessment of prostate organ cultures for viability and HSV-1 entry receptor expression
Human prostate specimens derived from radical prostatectomies were assessed for both tissue integrity and viability either immediately after resection or 72 h after incubation ex vivo on a semi-submersed collagen sponge (Ultrafoam, Davol, Cranston, RI, USA). A histological analysis showed that the morphological appearance of prostatic glandular regions as well as the surrounding stroma remained intact over the 72-h incubation period. It is noted that, epithelial cell–cell contacts within these glandular regions appeared to be preserved as well as epithelial cell contacts contiguous with the basement membrane (Figure 1a). The cell viability of prostate tissues was also assessed using MTS assays (Promega, Madison, WI, USA). As shown in Figure 1b, MTS enzymatic activity was similar for freshly isolated tissues as compared with those cultured over a 3-day period. Nectin-1 is a major cell-surface receptor for HSV-1 entry, the expression of which correlates with oHSV susceptibility and has been shown to be ubiquitously expressed in multiple tissues, including in the prostate.19, 20 Therefore, we assessed the tissue distribution of nectin-1 in prostate surgical specimens by immunohistochemistry. Anti-nectin-1 (R166) antibody21, 22 strongly stained epithelial cells within glandular regions of the prostate while a faint staining pattern appears in the surrounding stroma. The staining of the epithelia appeared enhanced along their edges and at epithelial cell–cell contacts (Figure 1c). Overall, organ cultures derived from prostate surgical specimens represent a viable target for assessing oHSV tropism.
oHSV preferentially targets epithelial cells of the prostatic ducts but not of stromal cells
We initially assessed the target specificity of G47Δ within prostate surgical specimens by X-gal staining. G47Δ contains a LacZ insertion in the ICP6 gene, which allows for viral tracking. Organ culture conditions were optimized to detect G47Δ infectivity while minimizing incubation times to preserve tissue integrity. G47Δ (1.5 × 106 p.f.u. (plaque forming units)) was placed in a medium with prostate tissue specimens for 1.5 h, and the specimens were subsequently transferred into a semi-submersed collagen-matrix for an additional 2–3 days resulting in the detection of β-gal+ cells (Figure 2a). It is noted that, X-gal staining appeared to be confined to epithelial cells within the prostatic glands with negligible staining detected in the surrounding stroma. Senescence-induced β-gal expression in prostate tissues has been detected by X-gal staining;23 however, β-gal expression was not detected in uninfected tissues (data not shown). Immunostaining of serial sections with anti-HSV gB, which recognizes the cell-surface glycoprotein B, a late gene product of HSV-1, confirmed the replication of G47Δ within these glandular regions but not in the stroma (Figure 2a). Staining with the luminal-specific epithelial anti-cytokeratin-8/18 antibody, further showed that both β-gal+ and gB+ regions likely represented luminal epithelial cells. LacZ+ and gB+ staining were frequently observed toward the center of the tissue section, suggesting viral penetration. The nuclear protein p63 is expressed in normal basal epithelial cells of the prostate. Furthermore, anti-p63 staining is frequently used as a diagnostic aid the expression of which is either decreased or absent in prostate adenocarcinomas but is present in BPT.24 Staining with anti-p63 on serial sections derived from these organ cultures revealed minimal detection in the prostate cancer specimens, whereas the nuclear expression of p63 staining was indeed observed in an organ culture derived from a BPT specimen used as a control (Figure 2b).
Next, G47Δ infectivity was compared with G207 and the nonengineered parental HSV-1 (strain F). Anti-gB and anti-cytokeratin-8/18 staining indicated that G207 infectivity was confined to the epithelium of the prostatic glands similar to that of G47Δ (Figure 3a). This was in striking contrast to the pattern of infectivity observed for strain F as previously observed in mice.7 By day 3 post infection with wild-type strain F, anti-HSV gB-positive staining appeared not only within the epithelial-containing glandular regions but was also prevalent in the stroma as verified by anti-cytokeratin 8/18 immunostaining (Figure 3a). That is, HSV-1 gB expression was noted in regions that lacked anti-cytokeratin 8/18 staining. Thus, these data show that although G47Δ and G207 infection appears confined to the epithelia within the prostatic glands, wild-type HSV-1 is more promiscuous in nature, spreading throughout both the epithelia as well as the stroma. In addition, these data also suggest that although G47Δ has the capacity to infect both epithelial and stroma cells similar to strain F, its genetic alterations restrict its ability to spread in the epithelium. In BPT specimens, G47Δ infectivity resulted in limited distribution to glands at the periphery of tissue, whereas strain F infectivity was frequently observed in both glandular and non-glandular areas throughout the tissue (Figure 3c). In parallel, prostate cancer tissue lysates derived from G47Δ and G207-infected organ cultures were monitored by immunoblot analysis over a 3-day period for HSV-1 gB expression levels, an indicator of viral replication (Figure 3b). Although gB expression was undetected for either vector by D1 post infection, G47Δ-expressed gB was clearly visible by D3 and moreover, was substantially more prominent than for G207. Finally, we assessed oHSV replication by quantifying viral progeny in these prostate surgical specimens. Tissue samples (n=6) were infected as described above with G207, G47Δ or strain F, and infectious viral titers were subsequently determined 3 days post infection. Figure 3c summarizes these results. Although, as anticipated, both engineered oHSV replicated less well than did wild-type, G47Δ (1.2 × 106 p.f.u. mg−1±4.5 × 105) was consistently superior to G207 (4.3 × 104 p.f.u. mg−1±1.15 × 104) achieving a 28-fold increase in viral titer. We estimate that on average, the input for each infection was 6 × 104 p.f.u. mg−1 of protein from ∼25 mg of total tissue weight. The increase in strain F viral titers likely reflects, at least in part, its non-selectivity in its ability to spread and replicate in both epithelial and stromal cell compartments. Finally, oHSV infection of BPT specimens resulted in notably reduced viral titers for both G207, G47Δ, whereas viral titers for strain F were maintained at comparable levels relative to prostate cancer samples (Figure 3d).
Collectively, our studies show the suitability of using prostate surgical specimens to assess oHSV tropism. In accordance with previously published studies, prostate tissues can be maintained on a matrix support system.15, 16 The heterogeneous nature of prostate cancer specimens as well as the presence of Nectin-1, a major HSV-1 entry receptor, makes their use in organ cultures particularly attractive to evaluate oHSV tropism. Although both G47Δ and G207 were observed to preferentially target cytokeratin-8/18-positive epithelial cells, wild-type HSV-1 was also prevalent throughout the stroma. Moreover, the replication efficacy of G47Δ was consistently superior to that of G207 in these surgical samples. The molecular basis for these differences in replication is due to the immediate-early expression of Us11 after G47Δ infection that compensates for the loss of gamma34.5. Us11 protein has been shown to counteract the activities of eIF2α kinase PKR, PACT and 2′-5′-oligoadenylate synthetase, which are all critical for host defense.25 Finally, although we did not observe a correlation between tumor grade and viral infectivity more samples may need to be assessed. Thus, the unique genetic modifications built into these oHSVs confer cell-type specific replication competence in prostate surgical specimens. When these data are considered in conjunction with the equivalent lack of neurotoxicity of G47Δ and G207 after direct injection in the brains of HSV susceptible mice,3, 26 these data suggest that G47Δ should be considered for further development for prostate cancer therapy. The specificity of this model system should also allow assessment of the efficacy of oHSV virotherapy in combination with chemotherapeutics and appropriate small molecule pharmaceuticals.
Conflict of interest
The authors declare no conflict of interest.
We would like to thank Drs T. Kasic and A. Viola (University of Padova; Padova, Italy) for their helpful advice on setting up conditions for prostate organ cultures. We are grateful to Dr CA Krummenacher (University of Pennsylvania School of Dental Medicine; Philadelphia, PA, USA) for supplying the R166 antibody. We thank Dr G Fulci (Massachusetts General Hospital) for expertise on immunohistochemistry and Ms. Melissa Marinelli for laboratory assistance. RLM an SDR are consultants to MediGene Ag, which has a license from Georgetown University for G207. Support for this study was in part obtained from a grant to RLM (RO1 CA102139).
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International Journal of Urology (2017)