Could Infecting Patients with Viruses Help in the Fight Against Glioblastomas?

Among the ever-growing number of angles researchers are using in the fight against cancer is virotherapy, or the technique of introducing via infection therapeutic genes and other protective agents to a tumor (Motalleb et al., 2009). This technique of tumoral infection of a virus, around since the 20th century, has illuminated the existence of oncolytic viruses, or viruses that naturally have an affinity towards, selectively replicate in, and kill cancerous growths (Chiocca et al, 2014). Oncolytic virotherapy yields many advantages that other modes of cancer therapy do not; perhaps most importantly is their ability to kill cancer cells far more efficiently than other modes of resistance. First, by hijacking the cell’s suicide mechanisms, oncolytic viruses can exhaust a cell’s resources and redirect them towards the effort of the virus’ spread before allowing the cell to die. Second, their ability to destroy tumor vasculature and stimulate immune responses affords them the opportunity to preemptively kill uninfected cancer cells before the virus even reaches those remote tumoral locations (Russell et al., 2012). Therefore, the efficacy of an oncolytic virus is dependent on both its ability to ward off interference from the host’s innate immune functions (which recognize and attack the foreign virus). In a recent study based at Ohio State University, researchers looked to enhance the effectiveness of the oncolytic herpes simplex virus (oHSV) specifically with regard to high grade glial cell cancers called glioblastomas (GBMs). GBMs  are the most invasive and aggressive cancers of the brain and are relatively resistant to resection and drug therapies (Davis et al., 2016).

As shown in previous studies and despite its relative successes in treating GBMs, melanomas and other cancers, treatment using oHSV is hindered by both the host’s immune response (thus limiting viral replication) and the extracellular matrix which slows viral spread (Kolodkin-Gal et al., 2007). A vital component of this innate innate immune response is a family of lymphocytes called natural killer (NK) cells, which are large, short-lived cells that use granules to puncture and kill target cells. NK cells, despite their valuable role in antitumor immune responses, are also well-known for their hindrance of oHSV viral spread. In this study, Xu et al. study hypothesized that, since oHSV is more effective with both enhanced viral infectivity and with simultaneous impairment of NK cells, a genetically-altered oHSV strain accounting for these two hurdles would yield better prognosis for mice with GBMs.   

NK cells' killer function is dependent on receptors found on their membrane that bind ligands on target cells, with their activatory receptors stimulating a cascade that ultimately kills the target cell while inhibitory receptors will skew towards the opposite anti-apoptotic function (Long et al., 2014). In this study, the molecule of interest is E-cadherin , a calcium-dependent gene that serves as a ligand to KLRG1, an inhibitory receptor found on NK cells, thereby making E-cadherin-expressing cells immune to NK-induced lysis, or cell destruction (Pecina-Slaus et al., 2003). In addition, E-cadherin cooperates with nectin-1, a molecule that uses cell-cell adhesion formation to facilitate the spread of viruses. Consequently, the researchers hypothesized that the  hyperexpression of E-cadherin in oHSV would improve its cell-to-cell infection without risking activation of NK cells which previously necessitated the knock out of local NK subsets in tumor regions to prevent its antiviral function. If true, this hypothesis would maintain NK cells natural  anti-tumoral activity, a best-of-both-worlds scenario.

This hypothesis led the researchers to genetically incorporate the gene CDH1 -which encodes E-cadherin- into the oHSV virus, henceforth referred to as OV-CDH1, to examine its effects on glioblastomas in mice. The results of the study were largely threehold: they found that E-cadherin inhibited NK activity and enhanced viral infection and spread , while also illuminating that the manipulated virus dampened the effects of glioblastomas.

To see if the manipulated virus had an effect on the susceptibility of GBM cells to NK-mediated death, the researchers investigated whether normal or OV-CDH1 viruses showed increased death. They found that, while cells infected with either virus globally increased their vulnerability to NK killing, it is notable that OV-CDH1-infected cells were less vulnerable to NKs than their non-manipulated counterparts when in the presence of a normal set of NK cells (of which half express the aforementioned inhibitory ligand KLRG1). Notably, when NK cells without the KLRG1 receptor were used, the manipulated virus yielded no benefit; but for NK cells with the inhibitory receptor, the manipulated virus had the hypothesized positive benefit of resistance to NK killing (Figure B). Figure A shows that the percentage of cell lysis is higher for the parental strain than the manipulated strain in the presence of bulk cells. Figure B shows similar levels of death between the two viruses in the presence KLRG(-) NKs but less death in manipulated viruses in the presence of KLRG(+) NKs.   It is important to keep in mind that the intention of the manipulated virus is to avoid NK-mediated cell death so the virus can spread and permeate through more of the tumor for its therapeutic effect.

Figure 1: OV-CDH1 infection reduces the cytotoxicity of human NK cells against OV-infected GBM cells

Next, the researchers confirmed that there was increased viral spread for the OV-CDH1 version of the virus compared to the parental, non-manipulated strain. To quantify this, the researchers tagged GBM-cell plaques, or visible regions of cell destruction, to green fluorescent protein so they were able to be viewed under a fluorescent microscope. Figure 2a below shows that the average size of the viral plaque is larger in the OV-CDH1 strain than in the parental strain, showing greater viral infection and accumulation from 24-72 hours post inoculation of the virus. The data is quantified in Figure 2b which shows the maximum plaque diameter of the manipulated viral plaques to be significantly greater than that of normal oHSV.

Figure 2: Cell–cell fusion facilitates viral spread of OV-CDH1.

The researchers also found that OV-CDH1-infected cells produced significantly more progeny than the parental OV-Q1 strain following inoculation. Figure 4A shows that viral production is significantly higher later in infection, perhaps due to the faster rate of viral entry in the tumoral mass shown in Figure 4E.

 

Figure 4: E-cadherin may accelerate viral entry and virus production.

Finally, the researchers conducted experiments to investigate whether the introduction of OV-CDH1 helps with GBM prognosis in vivo in mice. Five days after injecting mice with cancerous cells, Xu et al. injected mice with either the parental viral strain, OV-CDH1 or a mock. They found that OV-CDH1 was significantly more effective than OV-Q1 (parental strain) at reducing GBM tumor size (Figure 5E) with remarkably little luciferase signal (Figure 5C) which indicates significant regression in tumor growth. OV-CDH1 infected mice also lived significantly longer, showing that the manipulated virus had its desired anti-cancer therapeutic effect (Figure 5D).

Figure 5 : OV-CDH1 improves the efficacy of GBM virotherapy in vivo.

These remarkably telling results proposing the effectiveness of manipulating oHSV to both resist NK killing and facilitate its entry time and spread can have long-reaching effects with regard to virology as a whole. The significance of E-cadherins is evident in its ability to both inhibit the antiviral activity of NKs but not jeopardize its anti-tumor activity, a desirable balance in virology. The paper does a great job in the conclusion of talking about the pitfalls of modern oHSV development and why the addition of E-cadherins bypasses two prominent hurdles in the field. First, the attenuation strategy, in which viruses are genetically altered to limit their replication ability, lose oncolytic activity and infectivity; second, researchers attempt to retarget the virus to tumor cells by altering certain proteins in the virus, which compromises the virus’ infectivity upon entry into the tumor.

The greater context of this paper cannot be ignored. Glioblastomas, as such a deadly and heterogenous cancer for adults, are remarkably difficult to treat with immunotherapy, leaving patients with compromised brain function and low 5-year survival rates. Especially for a therapy that maintains certain advantages and disposes of certain drawbacks of prior trials, the follow-ups to the research are of utmost importance. Perhaps deeper insight into ways that other lymphocytes aside from natural killer cells can be manipulated by genetic alteration of the virus should be explored. In addition, possible insights into whether E-cadherins have benefit outside the context of cell-cell adhesion formation in the brain could yield tremendous benefit to patients of other types of cancers. Overall, the idea of using what we hate about viruses -e.g. their ability to hijack our cells for the sake of their proliferation and spread- and using those properties to our advantage is a thrilling angle in the fight against cancer.

Works cited

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