Mechanistic Study into Free Radical-Activated Glycan Dissociations through Isotope-Labeled Cellobioses.

Inspired by the electron-activated dissociation technique, the most potent tool for glycan characterization, we recently developed free radical reagents for glycan structural elucidation. However, the underlying mechanisms of free radical-induced glycan dissociation remain unclear and, therefore, hinder the rational optimization of the free radical reagents and the interpretation of tandem mass spectra, especially the accurate assignment of the relatively low-abundant but information-rich ions. In this work, we selectively incorporate the 13C and/or 18O isotopes into cellobiose to study the mechanisms for free radical-induced dissociation of glycans. The eight isotope-labeled cellobioses include 1-13C, 3-13C, 1'-13C, 2'-13C, 3'-13C, 4'-13C, 5'-13C, and 1'-13C-4-18O-cellobioses. Upon one-step collisional activation, cross-ring (X ions), glycosidic bond (Y-, Z-, and B-related ions), and combinational (Y1 + 0,4X0 ion) cleavages are generated. These fragment ions can be unambiguously assigned and confirmed by the mass difference of isotope labeling. Importantly, the relatively low-abundant but information-rich ions, such as 1,5X0 + H, 1,4X0 + H, 2,4X0 + H-OH, Y1 + 0,4X0, 2,5X1-H, 3,5X0-H, 0,3X0-H, 1,4X0-H, and B2-3H, are confidently assigned. The mechanisms for the formations of these ions are investigated and supported by quantum chemical calculations. These ions are generally initiated by hydrogen abstraction followed by sequential ß-elimination and/or radical migration. Here, the mechanistic study for free radical-induced glycan dissociation allows us to interpret all of the free radical-induced fragment ions accurately and, therefore, enables the differentiation of stereochemical isomers. Moreover, it provides fundamental knowledge for the subsequent development of bioinformatics tools to interpret the complex free radical-induced glycan spectra.

Systemic delivery of SapC-DOPS has antiangiogenic and antitumor effects against glioblastoma.

Saposin C-dioleoylphosphatidylserine (SapC-DOPS) nanovesicles are a nanotherapeutic which effectively target and destroy cancer cells. Here, we explore the systemic use of SapC-DOPS in several models of brain cancer, including glioblastoma multiforme (GBM), and the molecular mechanism behind its tumor-selective targeting specificity. Using two validated spontaneous brain tumor models, we demonstrate the ability of SapC-DOPS to selectively and effectively cross the blood-brain tumor barrier (BBTB) to target brain tumors in vivo and reveal the targeting to be contingent on the exposure of the anionic phospholipid phosphatidylserine (PtdSer). Increased cell surface expression of PtdSer levels was found to correlate with SapC-DOPS-induced killing efficacy, and tumor targeting in vivo was inhibited by blocking PtdSer exposed on cells. Apart from cancer cell killing, SapC-DOPS also exerted a strong antiangiogenic activity in vitro and in vivo. Interestingly, unlike traditional chemotherapy, hypoxic cells were sensitized to SapC-DOPS-mediated killing. This study emphasizes the importance of PtdSer exposure for SapC-DOPS targeting and supports the further development of SapC-DOPS as a novel antitumor and antiangiogenic agent for brain tumors.

Inquiry-Team-Based Lab Course Design Enhances Underrepresented Undergraduate Predictors of Persistence in the Sciences.

Introduction: Persistence in Science, Technology, Engineering, and Mathematics (STEM) may be promoted in underrepresented student populations by implementing an authentic inquiry-team-based learning (ITBL) STEM laboratory course design. Methods: Between Spring 2021 and Spring 2022, the research team compared junior and senior undergraduates enrolled in an ITBL-based pharmaceutical science lab course to a comparative student population enrolled in a traditionally designed biology lab course. At the end of either STEM lab course, students completed the experimentally validated Persistence in the Sciences (PITS) survey and an open-ended question asking them to recount a moment that validated or questioned their science identity determined the effect of the ITBL STEM lab course design on factors that may impact underrepresented students' indicators of science identity formation and persistence in STEM. Results: Students taking an ITBL-based pharmaceutical sciences lab course demonstrated higher scores on the persistence in the sciences instrument compared to students in the traditionally designed biology lab. Interestingly, different underrepresented student communities scored differently among the six factors. Multiple mechanisms of validating science identity were cited by students such as through gaining confidence in individualistic laboratory performance, collaborating through learning barriers, and fostering confidence and societal impact in a future career in pharmacy. Conclusion: The pharmaceutical sciences ITBL lab offered a collaborative, growth-promoting environment with experiments that are authentic to perspective pharmacists, which resulted in students reporting higher persistence in the sciences scores indicative of feeling like a pharmacist such as project ownership content/emotion, science identity, and networking across various student demographics.

Pediatric Cancer Immunotherapy: Opportunities and Challenges.

Cancer immunotherapies, widely heralded as transformational for many adult cancer patients, are becoming viable options for selected subsets of pediatric cancer patients. Many therapies are currently being investigated, from immunomodulatory agents to adoptive cell therapy, bispecific T-cell engagers, oncolytic virotherapy, and checkpoint inhibition. One of the most exciting immunotherapies recently FDA approved is the use of CD19 chimeric antigen receptor T cells for pre-B-cell acute lymphoblastic leukemia. With this approval and others, immunotherapy for pediatric cancers is gaining traction. One of the caveats to many of these immunotherapies is the challenge of predictive biomarkers; determining which patients will respond to a given therapy is not yet possible. Much research is being focused on which biomarkers will be predictive and prognostic for these patients. Despite many benefits of immunotherapy, including less long-term side effects, some treatments are fraught with immediate side effects that range from mild to severe, although most are manageable. With few downsides and the potential for disease cures, immunotherapy in the pediatric population has the potential to move to the front-line of therapeutic options.

Myelolytic Treatments Enhance Oncolytic Herpes Virotherapy in Models of Ewing Sarcoma by Modulating the Immune Microenvironment.

Ewing sarcoma is a highly aggressive cancer that promotes the infiltration and activation of pro-tumor M2-like macrophages. Oncolytic virotherapy that selectively infects and destroys cancer cells is a promising option for treating Ewing sarcoma. The effect of tumor macrophages on oncolytic virus therapy, however, is variable among solid tumors and is unknown in Ewing sarcoma. We tested the effects of macrophage reduction using liposomal clodronate (Clodrosome) and trabectedin on the antitumor efficacy of intratumoral oncolytic herpes simplex virus, rRp450, in two Ewing sarcoma xenograft models. Both agents enhanced antitumor efficacy without increasing virus replication. The most profound effects were in A673 with only a transient effect on response rates in TC71. Interestingly, A673 was more dependent than TC71 on macrophages for its tumorigenesis. We found Clodrosome and virus together induced expression of antitumorigenic genes and reduced expression of protumorigenic genes in both the tumor-associated macrophages and the overall tumor stroma. Trabectedin reduced intratumoral natural killer (NK) cells, myeloid-derived suppressor cells, and M2-like macrophages, and prevented their increase following virotherapy. Our data suggest that a combination of trabectedin and oncolytic herpes virotherapy warrants testing in the clinical setting.

Tumor-Associated Macrophages in Oncolytic Virotherapy: Friend or Foe?

Cancer therapy remains a challenge due to toxicity limitations of chemotherapy and radiation therapy. Oncolytic viruses that selectively replicate and destroy cancer cells are of increasing interest. In addition to direct cell lysis, these vectors stimulate an anti-tumor immune response. A key regulator of tumor immunity is the tumor-associated macrophage population. Macrophages can either support oncolytic virus therapy through pro-inflammatory stimulation of the anti-tumor response at the cost of hindering direct oncolysis or through immunosuppressive protection of virus replication at the cost of hindering the anti-tumor immune response. Despite similarities in macrophage interaction between adult and pediatric tumors and the abundance of research supporting macrophage modulation in adult tumors, there are few studies investigating macrophage modulation in pediatric cancers or modulation of immunotherapy. We review the current state of knowledge regarding macrophages in cancers and their influence on oncolytic virotherapy.

Pediatric cancer gone viral. Part II: potential clinical application of oncolytic herpes simplex virus-1 in children.

Oncolytic engineered herpes simplex viruses (HSVs) possess many biologic and functional attributes that support their use in clinical trials in children with solid tumors. Tumor cells, in an effort to escape regulatory mechanisms that would impair their growth and progression, have removed many mechanisms that would have protected them from virus infection and eventual virus-mediated destruction. Viruses engineered to exploit this weakness, like mutant HSV, can be safely employed as tumor cell killers, since normal cells retain these antiviral strategies. Many preclinical studies and early phase trials in adults demonstrated that oncolytic HSV can be safely used and are highly effective in killing tumor cells that comprise pediatric malignancies, without generating the toxic side effects of nondiscriminatory chemotherapy or radiation therapy. A variety of engineered viruses have been developed and tested in numerous preclinical models of pediatric cancers and initial trials in patients are underway. In Part II of this review series, we examine the preclinical evidence to support the further advancement of oncolytic HSV in the pediatric population. We discuss clinical advances made to date in this emerging era of oncolytic virotherapy.

Pediatric cancer gone viral. Part I: strategies for utilizing oncolytic herpes simplex virus-1 in children.

Progress for improving outcomes in pediatric patients with solid tumors remains slow. In addition, currently available therapies are fraught with numerous side effects, often causing significant life-long morbidity for long-term survivors. The use of viruses to kill tumor cells based on their increased vulnerability to infection is gaining traction, with several viruses moving through early and advanced phase clinical testing. The prospect of increased efficacy and decreased toxicity with these agents is thus attractive for pediatric cancer. In part I of this two-part review, we focus on strategies for utilizing oncolytic engineered herpes simplex virus (HSV) to target pediatric malignancies. We discuss mechanisms of action, routes of delivery, and the role of preexisting immunity on antitumor efficacy. Challenges to maximizing oncolytic HSV in children are examined, and we highlight how these may be overcome through various arming strategies. We review the preclinical and clinical evidence demonstrating safety of a variety of oncolytic HSVs. In Part II, we focus on the antitumor efficacy of oncolytic HSV in pediatric tumor types, pediatric clinical advances made to date, and future prospects for utilizing HSV in pediatric patients with solid tumors.

Oncolytic HSV virotherapy in murine sarcomas differentially triggers an antitumor T-cell response in the absence of virus permissivity.

Multiple studies have indicated that in addition to direct oncolysis, virotherapy promotes an antitumor cytotoxic T cell response important for efficacy. To study this phenomenon further, we tested three syngeneic murine sarcoma models that displayed varied degrees of permissiveness to oncolytic herpes simplex virus replication and cytotoxicity in vitro, with the most permissive being comparable to some human sarcoma tumor lines. The in vivo antitumor effect ranged from no or modest response to complete tumor regression and protection from tumor rechallenge. The in vitro permissiveness to viral oncolysis was not predictive of the in vivo antitumor effect, as all three tumors showed intact interferon signaling and minimal permissiveness to virus in vivo. Tumor shrinkage was T-cell mediated with a tumor-specific antigen response required for maximal antitumor activity. Further analysis of the innate and adaptive immune microenvironment revealed potential correlates of susceptibility and resistance, including favorable and unfavorable cytokine profiles, differential composition of intratumoral myeloid cells, and baseline differences in tumor cell immunogenicity and tumor-infiltrating T-cell subsets. It is likely that a more complete understanding of the interplay between the immunologic immune microenvironment and virus infection will be necessary to fully leverage the antitumor effects of this therapeutic platform.

SapC-DOPS-induced lysosomal cell death synergizes with TMZ in glioblastoma.

SapC-DOPS is a novel nanotherapeutic that has been shown to target and induce cell death in a variety of cancers, including glioblastoma (GBM). GBM is a primary brain tumor known to frequently demonstrate resistance to apoptosis-inducing therapeutics. Here we explore the mode of action for SapC-DOPS in GBM, a treatment being developed by Bexion Pharmaceuticals for clinical testing in patients. SapC-DOPS treatment was observed to induce lysosomal dysfunction of GBM cells characterized by decreased glycosylation of LAMP1 and altered proteolytic processing of cathepsin D independent of apoptosis and autophagic cell death. We observed that SapC-DOPS induced lysosomal membrane permeability (LMP) as shown by LysoTracker Red and Acridine Orange staining along with an increase of sphingosine, a known inducer of LMP. Additionally, SapC-DOPS displayed strong synergistic interactions with the apoptosis-inducing agent TMZ. Collectively our data suggest that SapC-DOPS induces lysosomal cell death in GBM cells, providing a new approach for treating tumors resistant to traditional apoptosis-inducing agents.