To understand how extracellular potassium ions influence both anti-tumor immunity and virotherapy in cancer progression, we construct two mathematical models informed by our experimental results. For the tumor immune system, our analysis shows a high concentration of extracellular potassium ions diminishes the killing rate of tumor cells by immune cells. Our model confirms that the stimulation coefficient of immune cells by tumor cells remains a crucial parameter in the presence of extracellular potassium ions, which largely controls the overall tumor growth. For virotherapy, we obtain a formula for the basic viral reproduction number which combines several parameters including potassium ions. When this number is greater than one, virotherapy achieves some partial success, where the tumor load is an increasing function of the potassium ion concentration. Therefore, the tumor load is reduced if the potassium ion concentration is lowered. The delay parameter of the viral lytic cycle also affects the basic reproduction number, and we find a critical delay time which determines when the reproduction number is greater than one. We performed some numerical analysis. A two-parameter bifurcation analysis reveals a positive correlation between viral burst size and the potassium-ion absorption rate, suggesting that higher absorption rates can enhance the success of virotherapy.

Immuno-oncology has revolutionized cancer treatment by mobilizing the immune system to eliminate tumors. Although immune checkpoint inhibitors and T cell therapies have mediated durable responses in hematologic malignancies and select solid tumors, most patients still relapse or fail to respond. To overcome these limitations, novel, next-generation immune constructs, including bispecific-cell therapy combinations, armored cells, tethered cytokines, immune-stimulatory antibody conjugates, and in vivo gene editing, are being developed to enhance specificity, persistence, and immune activation. This Society for Immunotherapy of Cancer roadmap highlights emerging technologies that integrate, redirect, or potentiate immune response. We examine advances in construct design, strategies for clinical translation, and opportunities for combinatorial approaches. By addressing translational barriers and real-world challenges, we outline how innovative engineering can unlock a new era of safe, durable, and accessible immunotherapies. Realizing this potential will require coordinated efforts from researchers, clinicians, industry leaders, and policymakers to deliver curative outcomes to patients worldwide.

The oral epithelium, a dynamic interface constantly facing environmental challenges, relies on intricate molecular pathways to maintain its homeostasis. This comprehensive review delves into the nuanced interplay between T-lymphocytic cells (T cells) and RNA-binding proteins (RBPs) within the oral epithelium, elucidating their roles in orchestrating immune responses and influencing tissue plasticity. By synthesizing current knowledge, we aim to unravel the molecular intricacies that govern this interplay, with a focus on potential therapeutic implications for oral health and diseases. Understanding the regulatory networks shaped by T cells and RBPs in the oral epithelial microenvironment holds promise for innovative strategies in managing conditions associated with epithelial dysfunction.

Oncolytic virotherapy uses replication-competent viruses to treat various solid tumors. While much of the clinical efficacy of oncolytic virotherapy is mediated by anti-tumor T cell responses, most of these therapies still rely on the in vivo replication of the viral agents within infected tumor cells. Understanding the fundamental mechanisms that govern this replication therefore remains essential to the clinical application of these therapies. As viruses, oncolytic agents rely entirely on host metabolites and resources for their propagation. To address this gap in knowledge, we asked which cells impacted the intratumoral replication of oncolytic myxoma virus during treatment of B16F10 melanomas. Our results demonstrate that myxoma replication is potently restricted by the presence of intratumoral arginase-1+ myeloid-derived suppressor cells, which prevent the spread of oncolytic infection by catabolizing intratumoral arginine supplies. Additionally, either pharmacological depletion of these cells or genetic ablation of their arginase-1 expression markedly improves intratumoral myxoma infection and enhances the therapeutic efficacy of viruses. Collectively, these results suggest that the clinical application of oncolytic viruses is likely to be impacted by the unique metabolic state of the tumor microenvironment and that myeloid-derived suppressor cell-mediated depression of arginine within tumors may play a critical role in suppressing these treatments.

Cytokine therapy represents an attractive option to improve the outcomes of cancer patients. However, the systemic delivery of these agents often leads to severe immune-related toxicities, which can prevent their efficient clinical use. One approach to address this issue is the use of recombinant oncolytic viruses to deliver various cytokines directly to the tumor. This improves the biodistribution of the secreted cytokine-transgenes, both augmenting antitumor immune responses and decreasing systemic toxicities. We have shown recently that a doubly recombinant oncolytic myxoma virus that secretes a soluble version of PD1 as well as an interleukin-12 (IL-12) fusion protein (vPD1/IL-12) can cause potent regression of disseminated cancers. Here we show that, despite the predominant localization of both transgenes within the infected tumor, treatment with vPD1/IL-12 still results in systemic, IL-12-mediated toxicities. Interestingly, these toxicities are independent of interferon-γ and instead appear to be mediated by the interaction of tumor necrosis factor α with tumor necrosis factor receptor 2 on hematopoietic cells. Critically, this unique mechanism allows for vPD1/IL-12-mediated toxicities to be alleviated through the use of US Food and Drug Administration (FDA)-approved tumor necrosis factor (TNF) blockers such as etanercept.

Intraperitoneal (IP) administration of immunogenic mesoporous silica nanoparticles (iMSN) in a mouse model of metastatic ovarian cancer promotes the development of tumor-specific CD8+ T cells and protective immunity. IP delivery of iMSN functionalized with the Toll-like receptor (TLR) agonists polyethyleneimine (PEI), CpG oligonucleotide, and monophosphoryl lipid A (MPLA) stimulated rapid uptake by all peritoneal myeloid subsets. Myeloid cells quickly transported iMSN to milky spots and fat-associated lymphoid clusters (FALCs) present in tumor-burdened adipose tissues, leading to a reduction in suppressive T cells and an increase in activated memory T cells. Two doses of iMSN cleared or reduced ovarian and colorectal cancer and protected against future tumor engraftment. In contrast, subcutaneous (SC) and intravenous (IV) delivery of iMSN were without therapeutic effect in mice with peritoneal metastases, supporting the need for activation of regional immune cells. Remarkably, intraperitoneal delivery of iMSN cleared subcutaneously implanted ovarian cancer, supporting homing of antigen specific T cells to extraperitoneal tumor sites.

Widespread peritoneal dissemination is common in patients with gynecologic or gastrointestinal cancers. Accumulating evidence of a central role for regional immunity in cancer control indicates that intraperitoneal immunotherapy may have treatment advantages. This study delineates immune mechanisms engaged by intraperitoneal delivery of a cell-based vaccine comprised of silicified ovarian cancer cells associated with enhanced survival. Vaccine trafficking from the site of injection to milky spots and other fat-associated lymphoid clusters was studied in syngeneic cancer models using bioluminescent and fluorescent imaging, microscopy, and flow cytometry. Spectral flow cytometry was used to phenotype peritoneal immune cell populations, while bioluminescent imaging of cancer was used to study myeloid and T cell dependency, systemic immunity, and vaccine efficacy in models of disseminated high-grade serous ovarian and DNA mismatch-repair proficient microsatellite-stable colorectal cancer. Following intraperitoneal vaccination of mice with ovarian cancer, vaccine cells were rapidly internalized by myeloid cells, with subsequent trafficking to fat-associated lymphoid clusters. Tumor clearance was confirmed to be T cell-mediated, leading to the establishment of local and systemic immunity. Combination immune checkpoint inhibitor and vaccine therapy in mice with advanced disease, characterized by an established suppressive tumor microenvironment, increased the number of mice with non-detectable tumors, however, change in tumor burden compared to vaccine monotherapy was not significant. Vaccination also resulted in tumor clearance in mouse models of metastatic colorectal cancer. This study demonstrates that intraperitoneal vaccine delivery has the potential to enhance vaccine efficacy by activating resident immune cells with the subsequent establishment of protective systemic anti-tumor immunity.

Background For the majority of women with ovarian cancer, the disease remains undetectable until later stages, where it is largely uncurable. Fortunately, some patients have experienced remarkable responses to immune therapy. We have fabricated mesoporous silica nanoparticles (MSN) that present the Toll-like receptor (TLR) agonists polyethyleneimine (PEI), CpG oligonucleotide, and monophosphoryl lipid A (MPL-A), creating microbial mimetic MSN. The efficacy and fate of these MSN following intraperitoneal (IP) administration into the tumor microenvironment was studied in a mouse model of serous epithelial ovarian cancer. Methods Fluorescent MSN or MSN-PEI-CpG-MPL-A (100 or 600 nm) were IP injected into mice 19 days post IP BR5-Akt-Luc cancer challenge. Cell association in ascites fluid and tissues were studied from 10 minutes to 24 hours post injection using fluorescent animal and tissue imaging (IVIS Spectrum), high throughput flow cytometry (Cytek), Velocyt acoustic cytometry (Bennubio), and confocal microscopy (Leica SP8). In addition, the therapeutic efficacy of MSN-PEI-CpG-MPL-A was assessed following 2 weekly injections beginning 4 days post tumor challenge by measuring weight, tumor bioluminescence (IVIS Spectrum), and survival. Results MSN were rapidly (within 10 minutes) associated with ascites spheroids, followed by steady declines within the first hour (figure 1). In mice, two major subsets of myeloid-derived suppressor cells (MDSCs) exist, monocytic (M-MDSC) and polymorphonuclear (PMN-MDSC) with surface expression of CD11b+Ly6C++Ly6G- and CD11b+Ly6CloLy6G+, respectively.1 2 Within one-hour post IP injection in mice, MSN colocalized with CD11b+Ly6C+Ly6G- and CD11b+Ly6CloLy6G+ cells. Within 24 hours of injection, approximately 85% of MSN were located in peritoneal tumor-burdened tissues. Tumor-specific accumulation was independent of particle size (100 vs 600 nm) and the presence of surface TLR agonists. Studies also examined the therapeutic impact of adjuvanted MSN in mice with ovarian cancer. IP delivery of MSN-PEI-CpG-MPL-A or MSN-PEI-CpG (2 weekly doses) reduced tumor burden, resulting in tumor-free survival. Conclusions The tumor immune microenvironment is a key contributing factor to tumor progression. Immature myeloid cells become MDSC, supporting tumor progression by suppressing T cells. Specific localization of MSN-PEI-CpG-MPL-A in MDSCs and functional plasticity within this population provides opportunities to covert tumor-promoting myeloid cells into tumor-fighting immune cells. Acknowledgements We are grateful for assistance from the University of New Mexico Comprehensive Cancer Center Animal Models (Irina Lagutina and Lillian Fitzpatrick), Fluorescence Microscopy (Michael Paffett), Flow Cytometry (Wade Johnson) and Histology Shared Resources, supported by NIH grant NCI 2P30 CA118100. References K Movahedi, M Guilliams, J Van den Bossche, R Van den Bergh, C Gysemans, A Beschin, P De Baetselier, JA Van Ginderachter, Identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct T cell-suppressive activity, Blood 2008;111(8):4233–44. E Kallberg, M Stenstrom, D Liberg, F Ivars, T Leanderson. CD11b+Ly6C++Ly6G- cells show distinct function in mice with chronic inflammation or tumor burden, BMC Immunol 2012;13:69. Ethics Approval The study was approved by the University of New Mexico IACUC, approval number 20–201067-HSC.

Expression of PDL1 on B16/F10 tumor cells in vivo following MYXV treatment.

Background Arginine (Arg) is a semiessential amino acid whose bioavailability is required for the in vitro replication of several oncolytic viruses. In vivo, Arg bioavailability is regulated by a combination of dietary intake, protein catabolism, and limited biosynthesis through portions of the urea cycle. Interestingly, despite the importance of bioavailable Arg to support cellular proliferation, many forms of cancer are functionally auxotrophic for this amino acid due to the epigenetic silencing of argininosuccinate synthetase 1 (ASS1), an enzyme responsible for the conversion of citrulline and aspartate into the Arg precursor argininosuccinate. The impact of this silencing on oncolytic virotherapy (OV), however, has never been examined. Methods To address this gap in knowledge, we generated tumor cells lacking ASS1 and examined how loss of this enzyme impacted the in vivo replication and therapeutic efficacy of oncolytic myxoma virus (MYXV). We also generated a series of recombinant MYXV constructs expressing exogenous ASS1 to evaluate the therapeutic benefit of virally reconstituting Arg biosynthesis in ASS1 −/− tumors. Results Our results show that the in vitro replication of oncolytic MYXV is dependent on the presence of bioavailable Arg. This dependence can be overcome by the addition of the metabolic precursor citrulline, however, this rescue requires expression of ASS1. Because of this, tumors formed from functionally ASS1 −/− cells display significantly reduced MYXV replication as well as poorer therapeutic responses. Critically, both defects could be partially rescued by expressing exogenous ASS1 from recombinant oncolytic MYXVs. Conclusions These results demonstrate that intratumoral defects to Arg metabolism can serve as a novel barrier to virally induced immunotherapy and that the exogenous expression of ASS1 can improve the efficacy of OV in Arg-auxotrophic tumors.

Oncolytic viruses are being heavily investigated as novel methods to treat cancers; however, predicting their therapeutic efficacy remains challenging. The most commonly used predic-tive tests involve determining the in vitro susceptibility of a tu-mor's malignant cells to infection with an oncolytic agent. Whether these tests are truly predictive of in vivo efficacy, how-ever, remains unclear. Here we demonstrate that a recombi-nant, oncolytic myxoma virus shows efficacy in two murine models of triple negative breast cancer despite extremely low permissivity of these models to viral infection. These data demonstrate that in vitro infectivity studies are not an accurate surrogate for therapeutic efficacy and suggest that other tests need to be developed.

T-cell immunoglobulin and mucin domain 3 (TIM3) is emerging as a potential target for antibody-based checkpoint blockade. However, the efficacy of TIM3 blockade in combination with other treatment modalities, has not been extensively studied. In the current work we combined TIM3 blockade with myxoma virus-based oncolytic virotherapy (OV). Our results demonstrate that myxoma virus's ability to initiate an immense antitumor immune response complements the ability of TIM3 blockade to shift the tumor microenvironment to a more proinflammatory state. As a result, the combination of TIM3 blockade and OV is able to completely eradicate established disease, while neither monotherapy is effective. These data represent the first demonstration that OV can enhance the efficacy of TIM3 blockade and suggest that this treatment may need to be incorporated into more aggressive, combinatorial regimens in order to fulfill its potential as an immunotherapeutic.

Oncogenes destabilize STING in epithelial cell-derived cancer cells, such as head and neck squamous cell carcinomas (HNSCCs), to promote immune escape. Despite the abundance of tumor-infiltrating myeloid cells, HNSCC presents notable resistance to STING stimulation. Here, we show how saturated fatty acids in the microenvironment dampen tumor response to STING stimulation. Using single-cell analysis, we found that obesity creates an IFN-I-deprived tumor microenvironment with a massive expansion of suppressive myeloid cell clusters and contraction of effector T cells. Saturated fatty acids, but not unsaturated fatty acids, potently inhibit the STING-IFN-I pathway in HNSCC cells. Myeloid cells from obese mice show dampened re-sponses to STING stimulation and are more suppressive of T cell activation. In agreement, obese hosts ex-hibited increased tumor burden and lower responsiveness to STING agonist. As a mechanism, saturated fatty acids induce the expression of NLRC3, depletion of which results in a T cell inflamed tumor microenvironment and IFN-I-dependent tumor control.

Legends for supplemental figures.

Efficacy and Specificity of antibody depletion in vivo.

Immunological response of mice eradicated of B16/F10 tumors by vPD1

Gross histological examination of B16/F10 tumors treated with MYXV

Intracellular K + homeostasis has been shown to play a major role in the replication of numerous viral families. However, the potential impact of altered extracellular K + concentrations is less well understood.