Rare therapeutic success with current therapies for malignant pleural mesothelioma indicates a need for new therapeutic modalities. Our group and others have shown in preclinical studies that delivery of type I IFNs using an adenoviral vector induces strong antitumor immune responses and has been highly effective in eliminating tumors in mice (Ahmed et al.
; Odaka et al.
; Brin et al.
). This antitumor activity appears to be present in patients also. Intrapleural administration of a replication-incompetent adenoviral vector expressing IFN-β to patients with mesothelioma and malignant pleural effusions resulted in humoral and cellular antitumor immune responses and some significant clinical responses (Sterman et al.
). Given the potential challenges of giving intravenous injections of oncolytic vectors such as VSV (Lun et al.
) and the potential induction of neutralizing antibodies (Power et al.
), the ability to safely and effectively deliver local gene therapy vectors to patients with tumors of the pleural space make mesothelioma an excellent disease target (Sterman et al.
). In recently completed work (Willmon et al.
), we have shown the efficacy of a VSV vector expressing murine IFN in a mouse model of mesothelioma with increased safety features. Importantly, this cell line was resistant to effects of murine IFN-β, thus supporting replication. Given this potential advantage of a replicating vector expressing mouse IFN-β in our preclinical models, the purpose of this study was to evaluate the potential antitumor efficacy of a replicating oncolytic virus expressing human IFN-β, VSV.hIFN-β, in a large panel of human mesothelioma cell lines.
Taking the advantages of species-specific binding for IFN-β and its receptor, a panel of human mesothelioma cells were screened for their susceptibility to VSV vectors expressing either human IFN-β (which is species specific and only interacts with the human IFN type I receptor) or mouse IFN-β (which only interacts with the mouse IFN type I receptor). Nine of 12 human mesothelioma lines (including a number of primarily isolated lines) were rapidly lysed by our control vector, VSV.mIFN-β (or by another control vector, VSV.GFP), whereas the four nontransformed mesothelial lines showed more limited toxicity with this vector (). These data are consistent with previous findings showing that tumor cells, compared with their normal counterparts, are more susceptible to the lytic effects of VSV (Balachandran and Barber, 2000
; Stojdl et al.
), a result that suggests that majority of mesothelioma lines have some defects in the IFN pathway. In addition, the VSV-mediated cell lysis was completely abolished in our nontransformed mesothelial lines when the vector expressed human IFN-β, a finding that supports the initiative for inserting this antiviral cytokine gene to improve safety (Obuchi et al.
However, we observed that more than half of the mesothelioma cell lines were protected from VSV-induced lysis in the presence of the hIFN-β transgene, revealing substantial tumor heterogeneity in their IFN responsiveness. We noted three classes of tumor cells with respect to their responses to IFN. Group 1 cells (3 of 12 lines) appeared to be resistant to both control VSV vectors and to VSV.hIFN-β and thus resembled the nontransformed mesothelial lines. Group 2 cells (5 of 12 lines) were lysed by control VSV vectors, but were resistant to lysis by the VSV.hIFN-β vector. In contrast, Group 3 cells (4 of 12 lines) appeared to be completely resistant to the antiviral effect of IFN-β and were thus lysed by both control and VSV.hIFN-β. These in vitro findings were consistent with animal studies conducted with flank mesothelioma tumors in SCID mice. Using the IFN nonresponsive MSTO line, flank tumor growth was significantly inhibited and showed similar sensitivity to VSV.hIFN-β and VSV.mIFN-β (). In the IFN-responsive REN flank model in SCID mice, significant tumor inhibition was seen with the VSV.mIFN-β, whereas the VSV.hIFN-β vector did not induce a statistically significant difference in tumor size compared with the control group ().
The heterogeneity in response to type I IFNs seen in our mesothelioma cell lines has been reported in other tumor cell lines in the literature, including cells derived from melanomas (Linge et al.
; Wong et al.
), lymphomas (Sun et al.
), bladder cancers (Matin et al.
), renal cancers (Pfeffer et al.
), and prostate cancers (Ahmed et al.
). This issue was addressed comprehensively by Stojdl et al
) using the NCI 60 tumor cell panel. They found that 81% of lines tested were “nonresponsive” to either IFN-α or IFN-β pretreatment, defined as 5 units of IFN being unable to significantly affect (<10-fold) the effective concentration required to induce 50% effect (EC50) of cells infected with wild-type VSV for 48
hr. The degree of IFN responsiveness (partial vs. total) was not explored in detail, however. Our data in mesothelioma lines, however, show a higher percentage of IFN-sensitive tumor cells.
The mechanism of this heterogeneity was explored in more detail by comparing the LP9 nontransformed line, the partially sensitive REN tumor line (Group 2), and the completely resistant LRK tumor line (Group 3). The resistant line (LRK) had multiple defects. Unlike the normal LP9 cells, no IFN-β mRNA and protein were upregulated in LRK cells following VSV infection (), and ISG mRNA levels were also unchanged in this cell line after treatment with IFN-β () or after infection with VSV (). In contrast, the partially sensitive REN line appeared to be intermediate in these responses. Although the REN cells produced high amounts of IFN-β message after VSV infection (), their ability to upregulate the message levels of IFN-dependent antiviral proteins after treatment with IFN-β () and after VSV infection () was significantly less than that seen with the nontransformed LP9 cell line. These findings are similar to those described in a panel of glioblastoma cells (Wollmann et al.
). In that study, normal human astrocytes were resistant to VSV infection, whereas four of five lines tested were highly susceptible to VSV infection, even in the presence of IFN-α. All of the susceptible cell lines showed defects in their ability to upregulate IFN-regulated proteins, such as MxA, and half had defects in their ability to produce IFN-β message after VSV infection.
To further investigate the mechanisms of heterogeneity in IFN response, quantitative real-time PCR analyses were performed to define possible defects in the type-I IFN signaling pathway among the mesothelioma lines (). Group 1 tumor cells (resistant to all types of VSV) appeared to have highly elevated levels of mRNA for Type I IFN activation pathway genes compared with primary mesothelial cells (). Group 2 tumor cells (sensitive to control VSV but resistant to lysis by VSVh.IFN-β) tended to have similar levels of mRNA for Type I IFN activation pathway genes as the primary mesothelial cells (). Group 3 tumor cells (sensitive to control and VSV.hIFN-β) had markedly reduced or undetectable levels of mRNA of most of the Type I IFN activation pathway genes. The results (with the exception of the MSTO line) indicate that there are losses of multiple components of the IFN pathway which explains why these cells do not respond to IFN.
We also extended these observations to mesothelioma tumors from patients. When we used immunohistochemistry to screen a tissue array of 48 primary tumors for their basal levels of three key components of IFN pathway that were downregulated in cell lines (IFNAR1, p48, and PKR), we found that about half of the primary tumors tested had no defects in any of these components (), suggesting they would be responsive to IFN. It is interesting to note that p48 (which was uniformly markedly downregulated in our IFN-nonresponsive cell lines) is encoded by a gene on chromosome 14q. A recent “loss of heterozygosity” analysis done by De Rienzo et al
) showed that approximately 43% of mesotheliomas displayed allelic losses from chromosome 14q, perhaps explaining these findings.
Using specimens from a recently conducted clinical trial (Sterman et al.
) of intrapleural delivery of Ad.IFN-β in mesothelioma patients, we were also able to directly determine if p48 status might predict response to IFN. In the three cases with sufficient cells, we observed clear p48 expression at baseline and this was associated with strong upregulation of the IFN-dependent protein OAS after Ad.IFN-β gene transfer ( and Supplemental Fig. S3
). Although more studies are needed (e.g., in specimens with p48 absent), these data suggest that screening tissue samples using immunohistochemistry for basal expression of proteins such as p48 might be useful for predicting whether viral replication of an INF-containing vector will be likely.
A clinical trial proposing the use of VSV.hIFN-β in patients with liver tumors has already been submitted to the DNA Recombinant Advisory Council and others will likely soon follow. Our data support studies to examine the use of VSV mutant vectors in patients with mesothelioma; however, it is likely that there would be heterogeneity in susceptibility to tumor lysis by this vector and suggests that tests to evaluate the sensitivity of a given tumor to a specific VSV vector might be useful (“personalized medicine”). This could include testing tumor samples for their ability to support VSV replication (Wollmann et al.
; Nguyen et al.
); however, it would be much more practical to use immunohistochemistry or PCR analysis on tumor biopsies to search for loss of key components of the IFN pathway, such as IFNARs, TyK2, or p48 (Wong et al.
). Our data have demonstrated that it is feasible to prescreen patients' responsiveness to IFN prior to treatment. This sort of susceptibility analysis may also be important in predicting the efficacy of other oncolytic viruses (i.e., measles, Newcastle Disease Virus, or Reovirus) that depend on defects in tumor IFN responses for efficacy. In addition to predicting patients who might have optimal response, knowing the IFN status of a tumor might also have practical therapeutic applications. The recent report that histone deacetylase inhibitors blunt the IFN pathway in tumor cells (Nguyen et al.
) suggests that these agents might be useful adjuncts in patients who have tumors with some residual IFN sensitivity.