Stephen H. Thorne

Consensus guidelines for the detection of immunogenic cell death

Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named “immunogenic cell death” (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.

Oncolytic virotherapy : Molecular targets in tumor-selective replication and carrier cell-mediated delivery of oncolytic viruses

Tremendous advances have been made in developing oncolytic viruses (OVs) in the last few years. By taking advantage of current knowledge in cancer biology and virology, specific OVs have been genetically engineered to target specific molecules or signal transduction pathways in cancer cells in order to achieve efficient and selective replication. The viral infection and amplification eventually induce cancer cells into cell death pathways and elicit host antitumor immune responses to further help eliminate cancer cells. Specifically targeted molecules or signaling pathways (such as RB/E2F/p16, p53, IFN, PKR, EGFR, Ras, Wnt, anti-apoptosis or hypoxia) in cancer cells or tumor microenvironment have been studied and dissected with a variety of OVs such as adenovirus, herpes simplex virus, poxvirus, vesicular stomatitis virus, measles virus, Newcastle disease virus, influenza virus and reovirus, setting the molecular basis for further improvements in the near future. Another exciting new area of research has been the harnessing of naturally tumor-homing cells as carrier cells (or cellular vehicles) to deliver OVs to tumors. The trafficking of these tumor-homing cells (stem cells, immune cells and cancer cells), which support proliferation of the viruses, is mediated by specific chemokines and cell adhesion molecules and we are just beginning to understand the roles of these molecules. Finally, we will highlight some avenues deserving further study in order to achieve the ultimate goals of utilizing various OVs for effective cancer treatment.

High‐mobility group box 1 activates caspase‐1 and promotes hepatocellular carcinoma invasiveness and metastases

Hypoxia is often found in solid tumors and is associated with tumor progression and poor clinical outcomes. The exact mechanisms related to hypoxia‐induced invasion and metastasis remain unclear. We elucidated the mechanism by which the nuclear‐damage–associated molecular pattern molecule, high‐mobility group box 1 (HMGB1), released under hypoxic stress, can induce an inflammatory response to promote invasion and metastasis in hepatocellular carcinoma (HCC) cells. Caspase‐1 activation was found to occur in hypoxic HCC cells in a process that was dependent on the extracellular release of HMGB1 and subsequent activation of both Toll‐like receptor 4 (TLR4)‐ and receptor for advanced glycation endproducts (RAGE)‐signaling pathways. Downstream from hypoxia‐induced caspase‐1 activation, cleavage and release of proinflammatory cytokines interleukin (IL)‐1β and ‐18 occurred. We further demonstrate that overexpression of HMGB1 or treatment with recombinant HMGB1 enhanced the invasiveness of HCC cells, whereas stable knockdown of HMGB1 remarkably reduced HCC invasion. Moreover, in a murine model of HCC pulmonary metastasis, stable knockdown of HMGB1 suppressed HCC invasion and metastasis. Conclusion: These results suggest that in hypoxic HCC cells, HMGB1 activates TLR4‐ and RAGE‐signaling pathways to induce caspase‐1 activation with the subsequent production of multiple inflammatory mediators, which, in turn, promote cancer invasion and metastasis. (HEPATOLOGY 2012;55:1866–1875)

Importance of glycolysis and oxidative phosphorylation in advanced melanoma

Serum lactate dehydrogenase (LDH) is a prognostic factor for patients with stage IV melanoma. To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma. Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2. Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS), we subsequently determined using tissue microarray (TMA) analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes. To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT) 1 and 4. Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma. Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS. Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy.

Enhancing Poxvirus Oncolytic Effects through Increased Spread and Immune Evasion

The antitumoral effects of oncolytic viruses have generally been limited by inefficient spread of the viruses within infected tumors and by inefficient systemic delivery, particularly in preimmunized hosts. Tumor-selective poxviruses have biological characteristics that may overcome these limitations. Nevertheless, physical barriers within the tumor microenvironment, including the extracellular matrix, can still limit intratumoral spread, and neutralizing antibodies can impede systemic delivery. To counter these limitations, we sought to take advantage of a naturally occurring poxvirus form known as extracellular enveloped virus (EEV). The EEV is shrouded by a host cell-derived lipid bilayer containing anticomplement proteins and is typically released from infected cells early during the infection cycle. Therefore, the EEV form evolved for rapid systemic spread within the host and for evasion of immune-mediated clearance. We compared the oncolytic potential of low versus high EEV-producing strains of vaccinia. EEV-enhanced vaccinia strains displayed improved spread within tumors after systemic delivery, resulting in significantly improved antitumor effects. The EEV-enhanced strains also displayed a greater ability to spread between injected and noninjected distant tumors through the blood and, importantly, displayed reduced clearance by neutralizing antibody. Safety was unaffected. The incorporation of EEV-enhancing mutations into next generation oncolytic vaccinia strains may improve the potency of these viruses without sacrificing safety.

The use of oncolytic vaccinia viruses in the treatment of cancer: a new role for an old ally?

The use of genetically engineered, tumor-targeting viruses as oncolytic agents has recently emerged as a promising new area for the development of novel cancer therapies. The first viruses to enter the clinic, such as ONYX-015 (an oncolytic adenovirus), provided evidence both for the safety and for the anti-tumor potential of this approach. The results of these early trials have also allowed investigators to examine the limitations of these viruses and to develop potentially far more effective approaches. In this review the development of such next generation viruses, in particular the potential use of strains of vaccinia virus, will be discussed. Vaccinia has an enormous history of use in humans and possesses many of the features felt to be beneficial for the creation of a successful virotherapy agent. It causes no known disease in humans, yet is capable of infecting almost all cell types with a subsequent rapid and lytic infection, which subsequently induces a vigorous local CTL immune response at the site of infection. Vaccinia also displays natural tumor tropism, and several approaches have been used to further limit viral replication to tumor cells and to optimize the immune response induced at the site of the tumor. Finally, the large cloning capacity of vaccinia allows for the addition of multiple foreign genes into the viral genome. This has been exploited to increase the bystander effect of the virus by immune modulation or by expression of pro-drug converting enzymes as well as to incorporate safety controls and reporters for in vivo molecular imaging. Initial clinical trials with these viruses further highlights their potential as the next generation of oncolytic agents and as highly effective future cancer therapies.

Ethyl pyruvate administration inhibits hepatic tumor growth

EP is a potent inhibitor of HMGB1 release that has significant anti–inflammatory activities and exerts a protective effect in animal models of inflammation. As inflammation is linked to cancer growth, we hypothesized that EP would have anti–tumor activity and explored its effects in a liver tumor model. Mice injected intraportally with MC38 colorectal cancer cells led to the growth of visible hepatic tumors within 2 weeks. Pretreatment with EP 30 min prior to infusion of tumor cells and continuing daily for 9 days inhibited tumor growth significantly in a dose–dependent manner, with 80 mg/kg EP achieving >70% reduction in the number of tumor nodules when compared with untreated animals. Delayed treatment with EP also suppressed tumor growth significantly, although to a lesser extent. Tumors had early, marked leukocytic infiltrates, and EP administration decreased innate (NK cells, monocytes) and adaptive (T and B cell lymphocytic) immune cell infiltrates acutely and significantly in the liver. Serum IL–6 and HMGB1 levels, which were elevated following tumor injection, were decreased significantly in EP–treated animals. Tumors showed an increase in apoptosis in EP–treated mice, and tumor cells treated in vitro with EP had marked increases in LC3–II and cleaved PARP, consistent with enhanced autophagic flux and apoptosis. Thus, EP inhibition of tumor growth in the liver was mediated by tumor (induction of apoptosis) and host (decreased inflammation) effects. EP administration may have a therapeutic role in the treatment of cancer in conjunction with other therapeutic agents.

Sustained inhibition of PKCα reduces intravasation and lung seeding during mammary tumor metastasis in an in vivo mouse model

Metastasis is the major reason for breast cancer-related deaths. Although there is a host of indirect evidence for a role of protein kinase C (PKC) α in primary breast cancer growth, its role in the molecular pathways leading to metastasis has not been studied comprehensively. By treating mice with αV5-3, a novel peptide inhibitor selective for PKCα, we were able to determine how PKCα regulates metastasis of mammary cancer cells using a syngeneic and orthotopic model. The primary tumor growth was not affected by αV5-3 treatment. However, the mortality rate was reduced and metastasis in the lung decreased by more than 90% in the αV5-3-treated mice relative to the control-treated mice. αV5-3 treatment reduced intravasation by reducing matrix metalloproteinase-9 activities. αV5-3 treatment also reduced lung seeding of tumor cells and decreased cell migration, effects that were accompanied by a reduction in nuclear factor kappa B activity and cell surface levels of the CXCL12 receptor, CXCR4. αV5-3 treatment caused no apparent toxicity in non-tumor-bearing naïve mice. Rather, inhibiting PKCα protected against liver damage and increased the number of immune cells in tumor-bearing mice. Importantly, αV5-3 showed superior efficacy relative to anti-CXCR4 antibody in reducing metastasis in vivo. Together, these data show that pharmacological inhibition of PKCα effectively reduces mammary cancer metastasis by targeting intravasation and lung seeding steps in the metastatic process and suggest that PKCα-specific inhibitors, such as αV5-3, can be used to study the mechanistic roles of PKCα specifically and may provide a safe and effective treatment for the prevention of lung metastasis of breast cancer patients.

New enzyme for reductive cancer chemotherapy, YieF, and its improvement by directed evolution

Reductive prodrugs, mitomycin C and 5-aziridinyl-2,4-dinitrobenzamide (CB 1954), are nontoxic in their native form but become highly toxic upon reduction. Their effectiveness in cancer chemotherapy can be enhanced by delivering to tumors enzymes with improved prodrug reduction kinetics. We report the discovery of a new prodrug-reducing enzyme, YieF, from Escherichia coli, and the improvement of its kinetics for reducing mitomycin C and CB 1954. A YieF-derived enzyme, Y6, killed HeLa spinner cells with ≥5-fold efficiency than the wild-type enzymes, YieF and NfsA, at a variety of drug and enzyme concentrations and incubation times. With adhered HeLa cells and Salmonella typhimurium SL 7838 bacteria as enzyme delivery vehicle, at least an order of magnitude less of Y6-producing bacteria were required to kill >90% of tumor cells compared with bacteria expressing the wild-type enzymes, which at a comparable level killed <5% of the cells. Thus, Y6 is a promising enzyme for use in cancer chemotherapy, and Salmonella strain SL 7838, which specifically targets tumors, may be used to deliver the prodrug-activating enzymes to tumors. [Mol Cancer Ther 2006;5(1):97–103]

Synergistic anti-tumor effects between oncolytic vaccinia virus and paclitaxel are mediated by the IFN response and HMGB1

Recent developments in the field of oncolytic or tumor-selective viruses have meant that the clinical applications of these agents are now being considered in more detail. Like most cancer therapies it is likely that they will be used primarily in combination with other therapeutics. Although several reports have shown that oncolytic viruses can synergize with chemotherapies within an infected cancer cell, it would be particularly important to determine whether factors released from infected cells could enhance the action of chemotherapies at a distance. Here, we demonstrate in vitro synergy between oncolytic vaccinia and taxanes. However, we also show, for the first time, that this synergy is at least partly due to the release of factors from the infected cells that are capable of sensitizing surrounding cells to chemotherapy. Several cellular factors were identified as being mediators of this bystander effect, including type I interferon released soon after infection and high-mobility group protein B1 (HMGB1) released after cell death. This represents the first description of these mechanisms for beneficial interactions between viral and traditional tumor therapies. These data may provide a direct basis for the design of clinical trials with agents currently in the clinic, as well as providing insight into the development of next generation viral vectors.