B16F10 Tumor Research Articles

A Multivalent Personalized Vaccine Orchestrating Two-Signal Activation Rebuilds the Bridge Between Innate and Adaptive Antitumor Immunity.

Personalized vaccines capable of circumventing tumor heterogeneity have exhibited compelling prospects. However, their therapeutic benefit is greatly hindered by the limited antigen repertoire and poor response of CD8+ T-cell immunity. Here, a double-signal coregulated cross-linking hydrogel-based vaccine (Bridge-Vax) is engineered to rebuild the bridge between innate and adaptive immunity for activating CD8+ T-cells against full repertoire of tumor antigens. Mechanistically, unlike prominent CD4+ T-cell responses in most cases, administration of Bridge-Vax encapsulated with granulocyte-macrophage colony-stimulating factor concentrates a wave of dendritic cells (DCs), which further promotes DCs activation with costimulatory signal by the self-adjuvanted nature of polysaccharide hydrogel. Simultaneously, synergy with the increased MHC-I epitopes by codelivered simvastatin for cross-presentation enhancement, Bridge-Vax endows DCs with necessary two signals for orchestrating CD8+ T-cell activation. Bridge-Vax elicits potent antigen-specific CD8+ T-cell responses in vivo, which not only shows efficacy in B16-OVA model but confers specific immunological memory to protect against tumor rechallenge. Moreover, personalized multivalent Bridge-Vax tailored by leveraging autologous tumor cell membranes as antigens inhibits postsurgical B16F10 tumor recurrence. Hence, this work provides a facile strategy to rebuild the bridge between innate and adaptive immunity for inducing potent CD8+ T-cell immunity and would be a powerful tool for personalized cancer immunotherapy.

Adoptive T cell transfer and host antigen-presenting cell recruitment with cryogel scaffolds promotes long-term protection against solid tumors

Although adoptive T cell therapy provides the T cell pool needed for immediate tumor debulking, the infused T cells generally have a narrow repertoire for antigen recognition and limited ability for long-term protection. Here, we present a hydrogel that locally delivers adoptively transferred T cells to the tumor site while recruiting and activating host antigen-presenting cells with GMCSF or FLT3L and CpG, respectively. T cells alone loaded into these localized cell depots provided significantly better control of subcutaneous B16-F10 tumors than T cells delivered through direct peritumoral injection or intravenous infusion. T cell delivery combined with biomaterial-driven accumulation and activation of host immune cells prolonged the activation of the delivered T cells, minimized host T cell exhaustion, and enabled long-term tumor control. These findings highlight how this integrated approach provide both immediate tumor debulking and long-term protection against solid tumors, including against tumor antigen escape.

Combination of AAV-delivered tumor suppressor PTEN with anti-PD-1 loaded depot gel for enhanced antitumor immunity

Recent clinical studies have shown that mutation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene in cancer cells may be associated with immunosuppressive tumor microenvironment (TME) and poor response to immune checkpoint blockade (ICB) therapy. Therefore, efficiently restoring PTEN gene expression in cancer cells is critical to improving the responding rate to ICB therapy. Here, we screened an adeno-associated virus (AAV) capsid for efficient PTEN gene delivery into B16F10 tumor cells. We demonstrated that intratumorally injected AAV6-PTEN successfully restored the tumor cell PTEN gene expression and effectively inhibited tumor progression by inducing tumor cell immunogenic cell death (ICD) and increasing immune cell infiltration. Moreover, we developed an anti-PD-1 loaded phospholipid-based phase separation gel (PPSG), which formed an in situ depot and sustainably release anti-PD-1 drugs within 42 days in vivo. In order to effectively inhibit the recurrence of melanoma, we further applied a triple therapy based on AAV6-PTEN, PPSG@anti-PD-1 and CpG, and showed that this triple therapy strategy enhanced the synergistic antitumor immune effect and also induced robust immune memory, which completely rejected tumor recurrence. We anticipate that this triple therapy could be used as a new tumor combination therapy with stronger immune activation capacity and tumor inhibition efficacy.

Aerobic Exercise Alters the Melanoma Microenvironment and Modulates ERK5 S496 Phosphorylation.

Exercise changes the tumor microenvironment by remodeling blood vessels and increasing infiltration by cytotoxic immune cells. The mechanisms driving these changes remain unclear. Herein, we demonstrate that exercise normalizes tumor vasculature and upregulates endothelial expression of VCAM1 in YUMMER 1.7 and B16F10 murine models of melanoma but differentially regulates tumor growth, hypoxia, and the immune response. We found that exercise suppressed tumor growth and increased CD8+ T-cell infiltration in YUMMER but not in B16F10 tumors. Single-cell RNA sequencing and flow cytometry revealed exercise modulated the number and phenotype of tumor-infiltrating CD8+ T cells and myeloid cells. Specifically, exercise caused a phenotypic shift in the tumor-associated macrophage population and increased the expression of MHC class II transcripts. We further demonstrated that ERK5 S496A knock-in mice, which are phosphorylation deficient at the S496 residue, "mimicked" the exercise effect when unexercised, yet when exercised, these mice displayed a reversal in the effect of exercise on tumor growth and macrophage polarization compared with wild-type mice. Taken together, our results reveal tumor-specific differences in the immune response to exercise and show that ERK5 signaling via the S496 residue plays a crucial role in exercise-induced tumor microenvironment changes. See related Spotlight by Betof Warner, p. 1158.

DNA based neoepitope vaccination induces tumor control in syngeneic mouse models

Recent findings have positioned tumor mutation-derived neoepitopes as attractive targets for cancer immunotherapy. Cancer vaccines that deliver neoepitopes via various vaccine formulations have demonstrated promising preliminary results in patients and animal models. In the presented work, we assessed the ability of plasmid DNA to confer neoepitope immunogenicity and anti-tumor effect in two murine syngeneic cancer models. We demonstrated that neoepitope DNA vaccination led to anti-tumor immunity in the CT26 and B16F10 tumor models, with the long-lasting presence of neoepitope-specific T-cell responses in blood, spleen, and tumors after immunization. We further observed that engagement of both the CD4+ and CD8+ T cell compartments was essential to hamper tumor growth. Additionally, combination therapy with immune checkpoint inhibition provided an additive effect, superior to either monotherapy. DNA vaccination offers a versatile platform that allows the encoding of multiple neoepitopes in a single formulation and is thus a feasible strategy for personalized immunotherapy via neoepitope vaccination.

The effectiveness of calcium electroporation combined with gene electrotransfer of a plasmid encoding IL-12 is tumor type-dependent.

In calcium electroporation (CaEP), electroporation enables the cellular uptake of supraphysiological concentrations of Ca2+, causing the induction of cell death. The effectiveness of CaEP has already been evaluated in clinical trials; however, confirmatory preclinical studies are still needed to further elucidate its effectiveness and underlying mechanisms. Here, we tested and compared its efficiency on two different tumor models to electrochemotherapy (ECT) and in combination with gene electrotransfer (GET) of a plasmid encoding interleukin-12 (IL-12). We hypothesized that IL-12 potentiates the antitumor effect of local ablative therapies as CaEP and ECT. The effect of CaEP was tested in vitro as well as in vivo in murine melanoma B16-F10 and murine mammary carcinoma 4T1 in comparison to ECT with bleomycin. Specifically, the treatment efficacy of CaEP with increasing calcium concentrations alone or in combination with IL-12 GET in different treatment protocols was investigated. We closely examined the tumor microenvironment by immunofluorescence staining of immune cells, as well as blood vessels and proliferating cells. In vitro, CaEP and ECT with bleomycin reduced cell viability in a dose-dependent manner. We observed no differences in sensitivity between the two cell lines. A dose-dependent response was also observed in vivo; however, the efficacy was better in 4T1 tumors than in B16-F10 tumors. In 4T1 tumors, CaEP with 250 mM Ca resulted in more than 30 days of growth delay, which was comparable to ECT with bleomycin. In contrast, adjuvant peritumoral application of IL-12 GET after CaEP prolonged the survival of B16-F10, but not 4T1-bearing mice. Moreover, CaEP with peritumoral IL-12 GET modified tumor immune cell populations and tumor vasculature. Mice bearing 4T1 tumors responded better to CaEP in vivo than mice bearing B16-F10 tumors, even though a similar response was observed in vitro. Namely, one of the most important factors might be involvement of the immune system. This was confirmed by the combination of CaEP or ECT with IL-12 GET, which further enhanced antitumor effectiveness. However, the potentiation of CaEP effectiveness was also highly dependent on tumor type; it was more pronounced in poorly immunogenic B16-F10 tumors compared to moderately immunogenic 4T1 tumors.

Carbohydrate Strengthens the Immunotherapeutic Effect of Small-Molecule PD-L1 Inhibitors.

PD-1/PD-L1 checkpoint blockade has demonstrated great success in cancer immunotherapy. Small-molecule PD-L1 inhibitors also attract significant research interests but remain challenging in the efficacy and safety. Carbohydrate moiety and carbohydrate-binding proteins (lectins) play important roles in immune modulation including antigen recognition and presenting. Herein, we reported a novel strategy to strengthen the immunotherapeutic effect of small-molecule PD-L1 inhibitors by introducing sugar motifs, which may utilize the carbohydrate-mediated immune enhancement for cancer treatment. The data revealed that glycoside compounds containing mannose or N-acetylglucosamine exhibited the best results in IFN-γ secretion. Moreover, compared to the nonglycosylated compounds, glycosides C3 and C15 demonstrated significant lower cytotoxicity and effective in vivo antitumor potency in the CT26 and melanoma B16-F10 tumor models with good tolerance. Notably, tumor-infiltrating lymphocyte (TIL) analysis validated increased CD3+, CD4+, CD8+, and granzyme B+ T cells after glycoside treatments. This work presents a new concept to improve the immunotherapy.

A General Biomineralization Strategy to Synthesize Autologous Cancer Vaccines with cGAS-STING Activating Capacity for Postsurgical Immunotherapy.

Autologous cancer vaccines constructed by nonproliferative whole tumor cells or tumor lysates together with appropriate adjuvants represent a promising strategy to suppress postsurgical tumor recurrence. Inspired by the potency of cytosolic double-stranded DNA (dsDNA) in initiating anticancer immunity by activating the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, we herein report the concise synthesis of a cGAS-STING agonist through dsDNA-templated biomineralization growth of calcium carbonate (CaCO3) microparticles. The yielded DNA@CaCO3 can activate the intracellular cGAS-STING pathway of dendritic cells (DCs) by promoting endosomal escape of dsDNA, triggering their maturation and activation as a potent immune stimulator. Upon intratumoral injection, DNA@CaCO3 can reverse the immunosuppressive tumor microenvironment by simultaneously provoking innate and adaptive antitumor immunity, thereby effectively suppressing the growth of murine CT26 and B16-F10 tumors in mice. Furthermore, via CaCO3-based biomineralization of complete tumor lysates, we constructed a personalized autologous cancer vaccine with intrinsic cGAS-STING activation capacity that could provoke tumor-specific immune responses to not only delay the growth of challenged tumors but also synergize with anti-PD-1 immunotherapy to suppress postsurgical tumor recurrence. This study highlights a CaCO3-based biomineralization method to prepare autologous cancer vaccines in a concise manner, which is promising for personalized immunotherapy and clinical translation.

TGF-β Type I Receptor Signaling in Melanoma Liver Metastases Increases Metastatic Outgrowth.

Despite advances in treatment for metastatic melanoma patients, patients with liver metastasis have an unfavorable prognosis. A better understanding of the development of liver metastasis is needed. The multifunctional cytokine Transforming Growth Factor β (TGF-β) plays various roles in melanoma tumors and metastasis, affecting both tumor cells and cells from the surrounding tumor microenvironment. To study the role of TGF-β in melanoma liver metastasis, we created a model to activate or repress the TGF-β receptor pathway in vitro and in vivo in an inducible manner. For this, we engineered B16F10 melanoma cells to have inducible ectopic expression of a constitutively active (ca) or kinase-inactive (ki) TGF-β receptor I, also termed activin receptor-like kinase (ALK5). In vitro, stimulation with TGF-β signaling and ectopic caALK5 expression reduced B16F10 cell proliferation and migration. Contrasting results were found in vivo; sustained caALK5 expression in B16F10 cells in vivo increased the metastatic outgrowth in liver. Blocking microenvironmental TGF-β did not affect metastatic liver outgrowth of both control and caALK5 expressing B16F10 cells. Upon characterizing the tumor microenvironment of control and caALk5 expressing B16F10 tumors, we observed reduced (cytotoxic) T cell presence and infiltration, as well as an increase in bone marrow-derived macrophages in caALK5 expressing B16F10 tumors. This suggests that caALK5 expression in B16F10 cells induces changes in the tumor microenvironment. A comparison of newly synthesized secreted proteins upon caALK5 expression by B16F10 cells revealed increased secretion of matrix remodeling proteins. Our results show that TGF-β receptor activation in B16F10 melanoma cells can increase metastatic outgrowth in liver in vivo, possibly through remodeling of the tumor microenvironment leading to altered infiltration of immune cells. These results provide insights in the role of TGF-β signaling in B16F10 liver metastasis and could have implications regarding the use of TGF-β inhibitors for the treatment of melanoma patients with liver metastasis.

Effects of clopidogrel bisulfate on B16-F10 cells and tumor development in a murine model of melanoma.

Metastatic melanoma is a very aggressive skin cancer. Platelets are constituents of the tumor microenvironment and, when activated, contribute to cancer progression, especially metastasis and inflammation. P2Y12 is an adenosine diphosphate receptor that triggers platelet activation. Inhibition of P2Y12 by clopidogrel bisulfate (CB) decreases platelet activation, which is also controlled by the extracellular concentration and the metabolism of purines by purinergic enzymes. We evaluated the effects of CB on the viability and proliferation of cultured B16-F10 cells. We also used a metastatic melanoma model with C57BL-6 mice to evaluate cancer development and purine metabolism modulation in platelets. B16-F10 cells were administered intraperitoneally to the mice. Two days later, the animals underwent a 12-day treatment with CB (30mg/kg by gavage). We have found that CB reduced cell viability and proliferation in B16-F10 culture in 72h at concentrations above 30µm. In vivo, CB decreased tumor nodule counts and lactate dehydrogenase levels and increased platelet purine metabolism. Our results showed that CB has significant effects on melanoma progression.

Cytotoxic activity of essential oil from Leaves of Myrcia splendens against A549 Lung Cancer cells

BackgroundPlants of the Myrcia genus have been widely used in folk medicine to treat various diseases, including cancer. Myrcia splendens species has a diverse chemical constitution, but the biological activities of its essential oil have not been well investigated. In this study to out the chemistry characterization of essential oil (EO) from the leaves of the species M. splendens from Brazil and evaluate cytotoxic effect in A549 lung cancer cells.MethodsM. splendens EO was obtained by hydrodistillation and analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). EO was isolated and evaluated for cellular viability in tumor cell lines by MTT assay. The evaluation of the formation of clones and the migratory capacity of the A549 cells treated with EO was done by the clonogenic assay and the wound healing assay. Morphological changes were observed in A549 cells by fluorescence using Phalloidin/FITC and DAPI.Results22 compounds were identified in the chemical analysis of EO, corresponding to 88% of the sample. Major compounds were the sesquiterpenic hydrocarbons bicyclogermacrene (15.4%), germacrene D (8.9%) and E-caryophyllene (10.1%). The biological analysis of the EO showed high cytotoxic activity with an IC50 below 20 µg/ml in the THP-1, A549 and B16-F10 tumor cells. The treatment with EO reduced colony formation and inhibited the migratory capacity of A549 cells. Furthermore, apoptotic morphological changes in the nucleus and cytoplasm of A549 cells was observed after of treatment with EO.ConclusionThe findings of this study suggest that the M. splendens EO has cytotoxic compounds for the A549 lung cancer cells. Treatment with the EO decreased the colony formation and reduced the ability of lung cancer cells to migrate. Future studies may be used to isolate compounds from the EO for the study of lung cancer.

In vivoimmune inhibitory role of Ly-6A protein

Abstract Ly-6A is a GPI-anchored cell surface protein with reported cell adhesion and cell signaling role in CD4 +T cells. Ly-6A shows low expression on naïve T cells; however, its expression is elevated upon T cell activation. Additionally, IFN-γ and IL-27 cytokines increase Ly-6A expression, a feature observed in immune checkpoint inhibitory (ICI) proteins, such as PD-1 and CTLA-4. Many reports using cell lines and primary cells show, as for ICI proteins, Ly-6A expression inhibiting clonal expansion of antigen receptor-stimulated CD4 +T cells. While the published in vitro and ex vivo studies provide strong evidence for Ly-6A as T cell inhibitory protein, in vivo role of Ly-6A is unknown. Using a tumor transplantation mouse model, we have found that Ly-6A-deficient mice support slower or no growth of B16-F10 melanoma and MC38 adenocarcinoma tumors compared to the wildtype C57BL/6 control mice. We report that Ly-6A-deficient mice injected with B16-F10 tumor cells have elevated number of activated T cells in the draining lymph nodes (DLN) on day-3 post-injection. Furthermore, significantly elevated proliferation of DLN cells from B16-F10 injected Ly-6A-deficient mice was observed on day-7, as assessed by ex vivo proliferation assays. Transcriptome analyses of the tumor tissues showed, among others, significantly elevated expression of IFN-γ inducible and chemokine genes. Additionally, CD73 expression, as assessed by confocal microscopy, was lower in B16-F10 tumor tissues from Ly-6A-deficient mice than the wildtype tumors. Our results reveal a novel immune inhibitory role of Ly-6A protein and potential ICI target for cancer immunotherapy, either as a standalone or combinatorial ICI blockade strategy. Supported by funds from Department of Biology, Villanova University (SRG) & Sigma Xi

Regulation of immune-recognition molecules following delivery of plasmid backbone

Abstract Local intratumor delivery with electroporation of low levels of plasmids encoding molecules, such as IL-12, IL-15, induces an antitumor effect without causing systemic toxicity. However, previous studies have predominately focused on the function of the delivered molecule encoded within the plasmid, and ignored the plasmid backbone. In this study, we found backbones pUMVC3 and pVax1 induced up-regulation of MHC class I (MHC-I), PD-L1, CD95 (Fas) and CD155 on tumor cell surface. These molecules participate in a considerable number of immunoregulatory functions through their interactions with activating and inhibitory immune cell receptors. MHC molecules are well-known for their role in antigen (cross-) presentation, thereby functioning as key players in the communication between immune cells and tumor cells. Increased PD-L1 expression on tumor cells is an important monitor of tumor growth and effectiveness of immune inhibitor therapy. During cancer progression CD95 is frequently downregulated, raising the possibility that loss of CD95 is part of a mechanism for tumor evasion. Due to its prominent endogenous and immune functions, CD155 has been gaining interest as a therapeutic target in the field of tumor immunology. Results from flow cytometry confirmed increased expression of MHC-I and PDL-1 on B16F10, 4T1 and KPC tumor cell lines. Preliminary animal data from tumor-bearing models, B16F10 melanoma, 4T1 breast cancer and KPC pancreatic cancer mouse models showed that tumor growth was attenuated after pUMVC3 intratumoral electroporation. Our data also documented pSTAT1-NF-κB signaling pathway might be associated with plasmid backbones’ function of up-regulating MHC-I, PD-L1, CD95 (Fas) and CD155 on tumor cells. R01 CA186730

Proteolysis-targeting chimera against NR4A1 for cancer immunotherapy

Abstract Modulating tumor-infiltrating immune cells is the key mission for the development of cancer immunotherapy. Based on recent literature and our bioinformatic analysis, nuclear receptor subfamily 4 group A member 1 (NR4A1) is an ideal target for cancer immunotherapy due to its important role in T cell exhaustion, regulatory T (Treg) cell function. Here we targets NR4A1 via proteolysis-targeting chimeric (PROTAC) technology, expecting to achieve immune activation and cancer clearance. We designed and synthesized over 80 NR4A1 PROTACs based on several reported NR4A1 ligands, such as celastrol. We found that several celastrol-based PROTACs achieved effective degradation of target protein NR4A1. We studied the therapeutic efficiency of the lead NR-V04, a celastrol- and von Hippel-Lindau (VHL)-based PROTAC. As expected, we confirmed that the NR-V04-induced NR4A1 degradation is through the uniquitin proteasome system with a tenery complex formation among NR4A1, VHL and NR-V04. The lead NR-V04 exhibits outstanding pharmacokinetics and effectively inhibits tumor growth at a low dose, 1.8mg/kg twice a week, in B16F10, Yummer 1.7, and MC38 mouse tumor models. We proved that NR-V04 significantly enhances anti-tumor immunity with seemingly distinct mechanisms of immune activation in a tumor-dependent manner. For example, NR-V04 induces the expansion of B cells and the reduction in myeloid-derived suppressor cells (MDSC) in B16F10; the reduction of Tregs, and the induction of CD8 effector population in Yummer 1.7 and MC38 tumors. we did not find obvious toxicity at these effective doses for up to 2 months. Therefore, the lead NR-V04 or other better ones under test have the potential for translation as a novel immunotherapeutic strategy. BC200100 - “Developing a Novel PROTAC-Based NR4A1 Degrader for Breast Cancer Therapy” (DEPARTMENT OF THE ARMY)

Innate Immune Memory for Improved Vaccine Response

Abstract Emerging diseases require generating new vaccines, which can often be time-consuming. An alternate method to boost host defense is by inducing nonspecific innate immune memory, called trained immunity, to develop novel prophylactics. Many molecules, most notably β-glucan, induce trained immunity, but their effects are often short-lived and uncontrolled. This lack of temporal control limits both the therapeutic ability of training and provides fundamental questions about its nature. To improve on the capabilities of β-glucan, we developed a method for temporal control of trained immunity and examined its use both as a prophylactic and as an additive for improve vaccine outcomes. We engineered controlled release nanoparticles encapsulating only 3.5% the standard dose of β-glucan to attain sustained release over a month. Nanoparticle trained mice exhibited prolonged training effects and improved resistance to a B16F10 tumor challenge compared to mice that received an equivalent amount of free β-glucan. We also showed that trained mice exhibited higher antibody responses upon vaccination. These results demonstrate that dosing and temporal control can substantially alter the trained response to unanticipated levels. As such, this approach using sustained release platforms might lead to a novel prophylactic strategy for improved disease resistance against a wide variety of diseases. HDTRA11810052

Design and Optimization of a Novel Proteomimetic Polymer Nanoplatform for the Co-delivery of Antigens with Adjuvants

Abstract We describe here a novel nanoplatform called the Protein-Like Polymer (PLP) with unique characteristics allowing for sustained delivery of tumor antigens in conjunction with STING agonists. Methods: A library of PLP formulations were synthesized via ROMP and characterized. Cell uptake/functional assays were conducted with payload-specific T Cells. Ability of PLPs to co-deliver adjuvants was tested by electrostatically coupling 2′3′ cGAMP, forming stable nanostructures. Results: Conjugating antigens to the polymer backbone using a cleavable disulfide linkage (reduces in APCs) resulted in increased endosomal localization and T cell proliferation/activation. Incorporating OEG side chains reduced enzymatic degradation while increasing immunogenicity and APC uptake. Increasing the density of side chains further improved vaccine efficacy and resistance to proteolysis. Antigen-PLP conjugates is effective only when paired with cells from their cognate system, demonstrating payload-specificity. Mice bearing established B16F10 tumors treated with gp100-PLPs resulted in increased survival time and reduced tumor burden with corresponding changes in immune cell profiles. Notably, STING-PLP complexes showed significantly smaller tumors at day 14 and allowed for subcutaneous administration of 2′3′ cGAMP. Studies looking at multiplexing pools of neoantigens onto one PLP and generation of immunological memory are ongoing. Conclusion: The modularity of the PLP platform allows for complex nano-architectures including systems capable of delivering challenging compounds, ie small molecule STING agonists, subcutaneously through electrostatic coupling, highlighting its potential to revolutionize cancer vaccinology. Supported by grants from the NIH (5F30CA257519-03)

YAP inhibition by CO promotes ferroptosis via ATF4-dependent REDD1 increase in melanoma

Abstract Ferroptotic cell death is a recently recognized form of non-apoptotic regulated cell death and exerts cytotoxicity in cancer cells. Carbon monoxide (CO) as an endogenous physiological mediator confers therapeutic potential in various cancer, while the therapeutic effects of CO on melanoma and its mechanism remain elusive. In this study, we suggest that CO induces ferroptosis via YAP inhibition and ATF4 activation in melanoma. YAP phosphorylation on serine 127 by CO-releasing molecule 2 (CORM2) caused cytoplasmic retention of YAP in B16F10 mouse melanoma cells, resulting in inhibition of YAP activity for transcription of target genes. Thus, CO-mediated YAP inhibition decreased SLC7A11 expression in melanoma cell lines, leading to ferroptosis. Moreover, CO increased REDD1 expression through PERK-ROS-ATF4 pathway. ATF4-REDD expression increased by CO became vulnerable to ferroptosis in melanoma. Thus, CO-induced YAP inhibition can induce ferroptosis viaATF4-mediated REDD1 increase and inhibition of SLC7A11. Furthermore, CO gas inhalation significantly inhibited B16F10 tumor size in xenograft mouse models resulting from ferroptosis induction via down-regulation of SLC7A11 and up-regulation of REDD1. These results suggest that CO acts as a potent inducer of ferroptosis in melanoma and that CO critically depends on YAP inhibition and ATF4 activation to promote ferroptosis.

Effects of Electrochemotherapy on Immunologically Important Modifications in Tumor Cells.

Electrochemotherapy (ECT) is a clinically acknowledged method that combines the use of anticancer drugs and electrical pulses. Electrochemotherapy with bleomycin (BLM) can induce immunogenic cell death (ICD) in certain settings. However, whether this is ubiquitous over different cancer types and for other clinically relevant chemotherapeutics used with electrochemotherapy is unknown. Here, we evaluated in vitro in the B16-F10, 4T1 and CT26 murine tumor cell lines, the electrochemotherapy triggered changes in the ICD-associated damage-associated molecular patterns (DAMPs): Calreticulin (CRT), ATP, High Mobility Group Box 1 (HMGB1), and four immunologically important cellular markers: MHCI, MHC II, PD-L1 and CD40. The changes in these markers were investigated in time up to 48 h after ECT. We showed that electrochemotherapy with all three tested chemotherapeutics induced ICD-associated DAMPs, but the induced DAMP signature was cell line and chemotherapeutic concentration specific. Similarly, electrochemotherapy with CDDP, OXA or BLM modified the expression of MHC I, MHC II, PD-L1 and CD40. The potential of electrochemotherapy to change their expression was also cell line and chemotherapeutic concentration specific. Our results thus put the electrochemotherapy with clinically relevant chemotherapeutics CDDP, OXA and BLM on the map of ICD inducing therapies.

Targeting the tumor microenvironment by liposomal Epacadostat in combination with liposomal gp100 vaccine

Indoleamine-2,3-dioxygenase (IDO1) pathway has vital role in cancer immune escape and its upregulation leads to immunosuppressive environment which is associated with poor prognosis and progression in various cancers like melanoma. Previously, we showed the antitumoral efficacy of nanoliposomal form of Epacadostat (Lip-EPA), as an IDO1 inhibitor. Herein, we used Lip-EPA as a combination approach with liposomal gp100 (Lip-gp100) anti-cancer vaccine in melanoma model. Here, we showed that B16F10 tumor express IDO1 so using Lip-EPA will enhance the efficacy of vaccine therapy. The biodistribution of ICG-labelled liposomal form of EPA showed the remarkable accumulation of drug at tumor site. In an in vivo study, Lip-EPA enhanced the antitumor efficacy of Lip-gp100 in which the IDO mRNA expression was decreased (~ fourfold) in tumor samples. Also, we identified a significant increase in the number of infiltrated T lymphocytes (p < 0.0001) with enhanced in interferon gamma (IFN-γ) production (p < 0.0001). Additionally, Lip-EPA + Lip-gp100 significantly modulated intratumoral regulatory T cells which altogether resulted in the highest delay in tumor growth (TGD = 56.54%) and increased life span (ILS > 47.36%) in treated mice. Our study demonstrated that novel combination of Lip-EPA and Lip-gp100 was effective treatment with capability of being used in further clinical studies.

Abstract 6408: Anti-tumor immunity evolved by novel titanium peroxide nanoparticles (TiOxNPs) as a radiosensitizer and PD-1 blockade

Abstract Abstract: Release and presentation of tumor antigens are the preconditions of cellular immune response. Radiotherapy have been reported to release endogenous tumor antigens and enhanced the adjuvanticity from dying cancer cells via endoplasmic reticulum (ER) stress, known as immunogenic cell death. We developed a novel radiotherapy using radiosensitizer (TiOxNPs) that could drive effective anti-tumor responses. The TiOxNPs were robustly radiation-induced reactive oxygen species (ROS) generation. This burst therapy could enhance to release and presentation of endogenous tumor antigens, subsequently cross-presentation of antigens by Dendritic cells (DCs) for T-cell activation in secondary lymphoid organs (SLO). Methods: B16-F10 murine melanoma cells (5 × 105) were subcutaneously injected into the right flank of C57BL/6J respectively. Mice were divided into five treatment groups: 1) untreated; 2) XRT; 3) XRT+ TiOxNPs; 4) XRT+anti-PD-1Ab; and 5) XRT+ TiOxNPs +anti-PD-1Ab. B16-F10 tumors were intratumorally injected with TiOxNPs (8mg/kg) on days 10 and received 5Gy or 10Gy X-ray radiation approximately 1 hour after the injection. Anti-PD-1 antibody (200µg) intraperitoneally administered to the mice on the first day of X-ray irradiation, days 13 and 16. The tumor volume were assessed every 2-3 days after treatment. Mice were sacrificed after days 17 and isolated spleen and tumor. Results: TiOxNPs acted as a potent radiosensitizer, which enhanced cytotoxic ROS-mediated cell death. This combination effect could not only a local treatment that directly kills cancer cells but also could enhance the adjuvanticity from dying cancer cells by DAMPs, and significantly upregulate expression of MHC class I on tumor cells. Treatment of B16-F10 melanoma tumor model with burst therapy resulted in tumor shrinkage and extended overall survival. Burst therapy facilitated the recruitment and maturation of DCs into SLO, and improved antigen presentation to naive T cells. Infiltration into the tumor site of CTL were correlated with increasing of migration DCs into SLO. Conclusion: A novel radiotherapy combining with TiOxNPs played an important role in release of endogenous tumor antigens and DAMPs. The upregulation of MHC class I expression on tumor cells after X-ray irradiation facilitated the recognition of the tumor cells by T cells, leading to significantly enhanced antitumor immune response compared to exisiting immunoradiotherapy. This novel nanoparticles exposed to X-ray irradiation could trigger of antigen-specific immune responses and transform a tumor into a site for activation of antitumor immune response. Citation Format: Yoshiko Fujita, Hikaru Kubota, Chiaki Ogino, Ryohei Sasaki. Anti-tumor immunity evolved by novel titanium peroxide nanoparticles (TiOxNPs) as a radiosensitizer and PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6408.