Altered expression of antiviral cytokine mRNAs associated with cyclophosphamide's enhancement of viral oncolysis

Abstract

Oncolytic viruses (OVs) are being used as anticancer agents in preclinical and clinical trials. Propagation of OVs inside infected tumors is critical to their efficacy and is mediated by the productive generation of progeny OVs within infected tumor cells. In turn, this progeny can spread the infection to other tumor cells in successive rounds of oncolysis. Previously, we had found that, in rats, cyclophosphamide (CPA) pretreatment increased infection of brain tumors by an intra-arterially administered herpes simplex virus type 1 OV, because it inhibited activation of complement responses, mediated by innate IgM. We also have previously shown that other pharmacologic inhibitors of complement, such as cobra venom factor (CVF), allowed for increased infection. However, in these studies, further inhibition of complement responses by CVF did not result in additional infection of brain tumor cells or in propagation of OV to surrounding tumor cells. In this study, we sought to determine if CPA did lead to increased infection/propagation from initially infected tumor cells. Unlike our results with CVF, we find that CPA administration does result in a time-dependent increase in infection of tumor cells, suggestive of increased propagation, in both syngeneic and athymic models of brain tumors. This increase was due to increased survival of OV within infected tumors and brain surrounding tumors. CPA's effect was not due to a direct enhancement of viral replication in tumor cells, rather was associated with its immunosuppressive effects. RT-PCR analysis revealed that CPA administration resulted in impaired mRNA production by peripheral blood mononuclear cells (PBMCs) of several cytokines (interferons alpha/beta, interferon gamma, TNFalpha, IL-15, and IL-18) with anti-HSV function. These findings suggest that the CPA-mediated facilitation of OV intraneoplastic propagation is associated with a general decrease of antiviral cytokines mRNAs in PBMCs. These findings not only suggest a potential benefit for the addition of transient immunosuppression in clinical applications of oncolytic HSV therapy, but also suggest that innate immunomodulatory pathways may be amenable to manipulation, in order to increase OV propagation and survival within infected tumors.

Figure 1

CPA increases the anatomical area of intratumoral infection by oncolytic HSV. In panel a, Fischer 344 rats from each experimental group (n=3), harboring 7-day old D74/HveC brain tumors, were killed 3 days after the injection of hrR3. Brains were then stained for expression of β-galactosidase. CPA treatment (either administered simultaneously or in a pretreatment fashion) markedly increased the anatomic area of β-galactosidase expression. The scale bar represents 2 mm. In panels b and c, the percentage of lacZ-expressing area of tumor was quantified using computer-assisted analysis. In panel d, Fisher 344 rats harboring rat 9L/HveC glioma tumors were injected with hrR3. Vehicle (upper two microphotographs) or CPA (lower two microphotographs) was administered i.p. the same day of hrR3 injection. After 3 days, brain tumors were harvested and stained for lacZ transgene expression. The scale bar represents 400 μm. In panels a, b, and d, 2 × 10⁷ PFUs of hrR3 (ICP6-defective, KOS strain) were injected while in panel c, 1.8 × 10⁶ PFUs of MGH-1 (ICP6 and ICP34.5-defective, F strain) were inoculated. For panels b and c, three tumor sections (each from the anterior, middle, and posterior one-third of the tumor) were selected for computer-assisted analyses of lacZ gene expression. The bars represent the mean percentage of lacZ-expressing area per tumor. Error bars represent the s.d. *P <0.05.

Figure 2

Immunohistochemical detection of HSV capsid protein. In parallel to the experiment described in Figure 1, brain sections were also immunocytochemically processed for the expression of HSV1 capsid antigens. In panel a, a representative tumor section from animals treated only with hrR3 is shown with brown precipitates indicative of HSV capsid antigen expression. In panel b, a representative section is shown from tumors harvested from animals treated with hrR3 and CPA on the same day. In panel c, a representative section is shown from tumors harvested from animals treated with hrR3 and pretreated with CPA (2 days before). The scale bar represents 100 μm.

Figure 3

CPA treatment improves the survival of OV within infected brain tumors. Titers of hrR3 were assayed in D74/HveC syngeneic brain tumors implanted in Fisher 344 rats that were either untreated or pretreated with CPA for 2 days before direct OV injection. Time points represent time after injection of OV. After recovering the OV from rat brains as described in Materials and methods, it was titrated on Vero cell monolayers. *P <0.01.

Figure 4

Long-term survival analysis of immunocompetent rats harboring rat malignant glioma. Fischer 344 Rats harboring D74/HveC glioma were treated intraperitoneally with either saline or CPA, 5 days after tumor cell implantation. After 2 days, animals were anesthetized and either hrR3 or HBSS was stereotactically injected into the tumor mass. All animals were observed daily to assess the survival. The treatment with CPA alone or hrR3 alone produced a significant increase in survival over mock treatment (P< 0.01, Wilcoxon test). The prolongation of survival was also significant with CPA + hrR3 combined treatment compared to the treatment with CPA alone or hrR3 alone (P <0.01, Wilcoxon test).

Figure 5

The active metabolite of CPA inhibits OV propagation in rat glioma cells in vitro. In panel a, three concentrations of 4-hydroperoxyCPA (0, 3, or 30 μM, as indicated) were added to D74/HveC cells in culture as cells were being infected with hrR3 (moi=0.01). After 30 h, cells were fixed in 0.5% glutaraldehyde and stained with X-gal to visualize the formation of plaques. The concentration of 30 μM was selected as the maximum value, because slightly higher concentrations resulted into apparent toxicity to cells in vitro (data not shown). In panel b, hrR3 (moi of 0.1) or vehicle was added onto cells in the presence of increasing concentrations of CPA (from 0 to 300 μM). Staining for lacZ expression was performed as described above.

Figure 6

CPA treatment also increases the anatomic area of OV-mediated transgene expression in athymic rats. D74/HveC glioma-harboring athymic rats were injected with hrR3 intratumorally after CPA or saline treatment. After 3 days, brain tumors were harvested and lacZ gene expression was quantitated by staining with X-gal and then by computer-assisted measurements of percentage of lacZ-expressing tumor area.

Figure 7

PBMCs and antiviral cytokine gene expression is decreased by CPA treatment. In panel a, the number of PBMCs isolated from Fisher 344 rats with D74/HveC brain tumors is shown 0, 24, and 48 h after treatment with CPA. In panel b, Fisher 344 rats with D74/HveC brain tumors were treated with saline (row labeled ‘−’), were treated with CPA for 2 days (row labeled ‘pre’), or were treated with CPA the same day (row labeled ‘simul.’) that brain tumors were injected with hrR3 or HBSS. At 12 h after OV injection, total RNA was prepared from PBMC and used for RT-PCR analysis to investigate the expression of a variety of cytokines. Equal numbers of PBMCs from each group were used to prepare RNAs.

Figure 8

PCR analysis of HSV genomes in rat organs after intratumoral administration of hrR3. Fischer 344 rats harboring D74/HveC brain tumors that were treated with saline (lanes labeled ‘−’) or with CPA (lanes labeled ‘+’) were then treated with intratumoral injection of hrR3. After 12 h, animals were euthanized and the indicated organs were harvested. DNA was prepared as described in Materials and methods and was then analyzed by PCR using primers specific to the sequence for glycoprotein C. Southern analysis of the PCR products was performed using a glycoprotein C DNA fragment as a probe. TG, trigeminal ganglia.