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 (). 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 (). Staining with the luminal-specific epithelial anti-cytokeratin-8/18 antibody, further showed that both β-gal+
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 2 Analysis of G47Δ infection of prostate surgical samples. (a) Tissues were cut into 2–4 mm3 pieces, incubated with G47Δ (1.5 × 106 p.f.u.) for 1.5 h in 250 µl of POC medium and thereafter, placed on a semi-submersed (more ...)
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 (). 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 (). 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 (). 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 (). 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. summarizes these results. Although, as anticipated, both engineered oHSV replicated less well than did wild-type, G47Δ (1.2 × 106
± 4.5 × 105
) was consistently superior to G207 (4.3 × 104
± 1.15 × 104
) achieving a 28-fold increase in viral titer. We estimate that on average, the input for each infection was 6 × 104
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 3 A comparative analysis of oncolytic herpes simplex virus (oHSV) and wild-type HSV-1 infectivity and replication. (a) Tissue distribution of G47Δ, G207 and strain F 3 days post infection. Prostate cancer tissues were infected as detailed above (more ...)
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.