MB2033, an anti-PD-L1 × IL-2 variant fusion protein, demonstrates robust anti-tumor efficacy with minimal peripheral toxicity.

Interleukin-2 (IL-2), a cytokine with pleiotropic immune effects, was the first approved cancer immunotherapy agent. However, IL-2 is associated with systemic toxicity due to binding with its ligand IL-2Rα, such as vascular leakage syndrome, limiting its clinical applications. Despite efforts to extend the half-life of IL-2 and abolish IL-2Rα interactions, the risk of toxicity remains unresolved. In this study, we developed the bispecific fusion protein MB2033, comprising a novel IL-2 variant (IL-2v) connected to anti-programmed death ligand 1 (PD-L1) via a silenced Fc domain. The IL-2v of MB2033 exhibits attenuated affinity for IL-2Rßγ without binding to IL-2Rα. The binding affinity of MB2033 for PD-L1 is greater than that for IL-2Rßγ, indicating its preferential targeting of PD-L1+ tumor cells to induce tumor-specific immune activation. Accordingly, MB2033 exhibited significantly reduced regulatory T cell activation, while inducing comparable CD8+ T cell activation to recombinant human IL-2 (rhIL-2). MB2033 induced lower immune cell expansion and reduced cytokine levels compared with rhIL-2 in human peripheral blood mononuclear cells, indicating a decreased risk of peripheral toxicity. MB2033 exhibited superior anti-tumor efficacy, including tumor growth inhibition and complete responses, compared with avelumab monotherapy in an MC38 syngeneic mouse model. In normal mice, MB2033 was safer than non-α IL-2v and tolerable up to 30 mg/kg. These preclinical results provide evidence of the dual advantages of MB2033 with an enhanced safety and potent clinical efficacy for cancer treatment.

Bacillus Calmette-Guérin immunotherapy induces an efficient antitumor response to control murine melanoma depending on MyD88 signaling.

Bacillus Calmette-Guérin (BCG) is the first line treatment for bladder cancer and it is also proposed for melanoma immunotherapy. BCG modulates the tumor microenvironment (TME) inducing an antitumor effective response, but the immune mechanisms involved still poorly understood. The immune profile of B16-F10 murine melanoma cells was assessed by infecting these cells with BCG or stimulating them with agonists for different innate immune pathways such as TLRs, inflammasome, cGAS-STING and type I IFN. B16-F10 did not respond to any of those stimuli, except for type I IFN agonists, contrasting with bone marrow-derived macrophages (BMDMs) that showed high production of proinflammatory cytokines. Additionally, we confirmed that BCG is able to infect B16-F10, which in turn can activate macrophages and spleen cells from mice in co-culture experiments. Furthermore, we established a subcutaneous B16-F10 melanoma model for intratumoral BCG treatment and compared wild type mice to TLR2-/-, TLR3-/-, TLR4-/-, TLR7-/-, TLR3/7/9-/-, caspase 1-/-, caspase 11-/-, IL-1R-/-, cGAS-/-, STING-/-, IFNAR-/-, MyD88-/-deficient animals. These results in vivo demonstrate that MyD88 signaling is important for BCG immunotherapy to control melanoma in mice. Also, BCG fails to induce cytokine production in the co-culture experiments using B16-F10 and BMDMs or spleen cells derived from MyD88-/- compared to wild-type (WT) animals. Immunotherapy with BCG was not able to induce the recruitment of inflammatory cells in the TME from MyD88-/- mice, impairing tumor control and IFN-γ production by T cells. In conclusion, MyD88 impacts on both innate and adaptive responses to BCG leading to an efficient antitumor response against melanoma.

IFNγ at the early stage induced after cryo-thermal therapy maintains CD4+ Th1-prone differentiation, leading to long-term antitumor immunity.

Introduction: Recently, more and more research illustrated the importance of inducing CD4+ T helper type (Th)-1 dominant immunity for the success of tumor immunotherapy. Our prior studies revealed the crucial role of CD4+ Th1 cells in orchestrating systemic and durable antitumor immunity, which contributes to the satisfactory outcomes of the novel cryo-thermal therapy in the B16F10 tumor model. However, the mechanism for maintaining the cryo-thermal therapy-mediated durable CD4+ Th1-dominant response remains uncovered. Additionally, cryo-thermal-induced early-stage CD4+ Th1-dominant T cell response showed a correlation with the favorable prognosis in patients with colorectal cancer liver metastasis (CRCLM). We hypothesized that CD4+ Th1-dominant differentiation induced during the early stage post cryo-thermal therapy would affect the balance of CD4+ subsets at the late phase. Methods: To understand the role of interferon (IFN)-γ, the major effector of Th1 subsets, in maintaining long-term CD4+ Th1-prone polarization, B16F10 melanoma model was established in this study and a monoclonal antibody was used at the early stage post cryo-thermal therapy for interferon (IFN)-γ signaling blockade, and the influence on the phenotypic and functional change of immune cells was evaluated. Results: IFNγ at the early stage after cryo-thermal therapy maintained long-lasting CD4+ Th1-prone immunity by directly controlling Th17, Tfh, and Tregs polarization, leading to the hyperactivation of Myeloid-derived suppressor cells (MDSCs) represented by abundant interleukin (IL)-1ß generation, and thereby further amplifying Th1 response. Discussion: Our finding emphasized the key role of early-phase IFNγ abundance post cryo-thermal therapy, which could be a biomarker for better prognosis after cryo-thermal therapy.

Combination of STING agonist with anti-vascular RGD-(KLAKLAK)2 peptide as a novel anti-tumor therapy.

Immunotherapy is one of the most promising anti-cancer treatment. It involves activating the host's own immune system to eliminate cancer cells. Activation of cGAS-STING pathway is promising therapeutic approach for cancer immunotherapy. However, in human clinical trials, targeting cGAS-STING pathway results in insufficient or unsustainable anti-tumor response. To enhance its effectiveness, combination with other anti-cancer therapies seems essential to achieve synergistic systemic anti-tumor response.The aim of this study was to evaluate whether the combination of STING agonist-cGAMP with anti-vascular RGD-(KLAKLAK)2 peptide results in a better anti-tumor response in poorly immunogenic tumors with various STING protein and αvß3 integrin status.Combination therapy inhibited growth of murine breast carcinoma more effectively than melanoma. In melanoma, the administration of STING agonist alone was sufficient to obtain a satisfactory therapeutic effect. In both tumor models we have noted stimulation of innate immune response following cGAMP administration alone or in combination. The largest population of immune cells infiltrating the TME after therapy were activated NK cells. Increased infiltration of cytotoxic CD8+ T lymphocytes within the TME was only observed in melanoma tumors. However, they also expressed the "exhaustion" PD-1 receptor. In contrast, in breast carcinoma tumors each therapy caused the drop in the number of infiltrating CD8+ T cells.The obtained results indicate an additional therapeutic benefit from combining STING agonist with an anti-vascular agent. However, this effect depends on the type of tumor, the status of its microenvironment and the expression of specific proteins such as STING and αvß3 family integrin.

Construction of lymph nodes-targeting tumor vaccines by using the principle of DNA base complementary pairing to enhance anti-tumor cellular immune response.

Tumor vaccines, a crucial immunotherapy, have gained growing interest because of their unique capability to initiate precise anti-tumor immune responses and establish enduring immune memory. Injected tumor vaccines passively diffuse to the adjacent draining lymph nodes, where the residing antigen-presenting cells capture and present tumor antigens to T cells. This process represents the initial phase of the immune response to the tumor vaccines and constitutes a pivotal determinant of their effectiveness. Nevertheless, the granularity paradox, arising from the different requirements between the passive targeting delivery of tumor vaccines to lymph nodes and the uptake by antigen-presenting cells, diminishes the efficacy of lymph node-targeting tumor vaccines. This study addressed this challenge by employing a vaccine formulation with a tunable, controlled particle size. Manganese dioxide (MnO2) nanoparticles were synthesized, loaded with ovalbumin (OVA), and modified with A50 or T20 DNA single strands to obtain MnO2/OVA/A50 and MnO2/OVA/T20, respectively. Administering the vaccines sequentially, upon reaching the lymph nodes, the two vaccines converge and simultaneously aggregate into MnO2/OVA/A50-T20 particles through base pairing. This process enhances both vaccine uptake and antigen delivery. In vitro and in vivo studies demonstrated that, the combined vaccine, comprising MnO2/OVA/A50 and MnO2/OVA/T20, exhibited robust immunization effects and remarkable anti-tumor efficacy in the melanoma animal models. The strategy of controlling tumor vaccine size and consequently improving tumor antigen presentation efficiency and vaccine efficacy via the DNA base-pairing principle, provides novel concepts for the development of efficient tumor vaccines.

A lactate-SREBP2 signaling axis drives tolerogenic dendritic cell maturation and promotes cancer progression.

Conventional dendritic cells (DCs) are essential mediators of antitumor immunity. As a result, cancers have developed poorly understood mechanisms to render DCs dysfunctional within the tumor microenvironment (TME). After identification of CD63 as a specific surface marker, we demonstrate that mature regulatory DCs (mregDCs) migrate to tumor-draining lymph node tissues and suppress DC antigen cross-presentation in trans while promoting T helper 2 and regulatory T cell differentiation. Transcriptional and metabolic studies showed that mregDC functionality is dependent on the mevalonate biosynthetic pathway and its master transcription factor, SREBP2. We found that melanoma-derived lactate activates SREBP2 in tumor DCs and drives conventional DC transformation into mregDCs via homeostatic or tolerogenic maturation. DC-specific genetic silencing and pharmacologic inhibition of SREBP2 promoted antitumor CD8+ T cell activation and suppressed melanoma progression. CD63+ mregDCs were found to reside within the lymph nodes of several preclinical tumor models and in the sentinel lymph nodes of patients with melanoma. Collectively, this work suggests that a tumor lactate-stimulated SREBP2-dependent program promotes CD63+ mregDC development and function while serving as a promising therapeutic target for overcoming immune tolerance in the TME.

Combination therapy with oncolytic virus and T cells or mRNA vaccine amplifies antitumor effects.

Antitumor therapies based on adoptively transferred T cells or oncolytic viruses have made significant progress in recent years, but the limited efficiency of their infiltration into solid tumors makes it difficult to achieve desired antitumor effects when used alone. In this study, an oncolytic virus (rVSV-LCMVG) that is not prone to induce virus-neutralizing antibodies was designed and combined with adoptively transferred T cells. By transforming the immunosuppressive tumor microenvironment into an immunosensitive one, in B16 tumor-bearing mice, combination therapy showed superior antitumor effects than monotherapy. This occurred whether the OV was administered intratumorally or intravenously. Combination therapy significantly increased cytokine and chemokine levels within tumors and recruited CD8+ T cells to the TME to trigger antitumor immune responses. Pretreatment with adoptively transferred T cells and subsequent oncolytic virotherapy sensitizes refractory tumors by boosting T-cell recruitment, down-regulating the expression of PD-1, and restoring effector T-cell function. To offer a combination therapy with greater translational value, mRNA vaccines were introduced to induce tumor-specific T cells instead of adoptively transferred T cells. The combination of OVs and mRNA vaccine also displays a significant reduction in tumor burden and prolonged survival. This study proposed a rational combination therapy of OVs with adoptive T-cell transfer or mRNA vaccines encoding tumor-associated antigens, in terms of synergistic efficacy and mechanism.

In vitro effects and mathematical modelling of CTCE-9908 (a chemokine receptor 4 antagonist) on melanoma cell survival.

CTCE-9908, a CXC chemokine receptor 4 (CXCR4) antagonist, prevents CXCR4 phosphorylation and inhibits the interaction with chemokine ligand 12 (CXCL12) and downstream signalling pathways associated with metastasis. This study evaluated the in vitro effects of CTCE-9908 on B16 F10 melanoma cells with the use of mathematical modelling. Crystal violet staining was used to construct a mathematical model of CTCE-9908 B16 F10 (melanoma) and RAW 264.7 (non-cancerous macrophage) cell lines on cell viability to predict the half-maximal inhibitory concentration (IC50). Morphological changes were assessed using transmission electron microscopy. Flow cytometry was used to assess changes in cell cycle distribution, apoptosis via caspase-3, cell survival via extracellular signal-regulated kinase1/2 activation, CXCR4 activation and CXCL12 expression. Mathematical modelling predicted IC50 values from 0 to 100 h. At IC50, similar cytotoxicity between the two cell lines and ultrastructural morphological changes indicative of cell death were observed. At a concentration 10 times lower than IC50, CTCE-9908 induced inhibition of cell survival (p = 0.0133) in B16 F10 cells but did not affect caspase-3 or cell cycle distribution in either cell line. This study predicts CTCE-9908 IC50 values at various time points using mathematical modelling, revealing cytotoxicity in melanoma and non-cancerous cells. CTCE-9908 significantly inhibited melanoma cell survival at a concentration 10 times lower than the IC50 in B16 F10 cells but not RAW 264.7 cells. However, CTCE-9908 did not affect CXCR4 phosphorylation, apoptosis,\ or cell cycle distribution in either cell line.

Combining large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers for promoting protective melanosome autophagy via the PI3K/Akt/mTOR signalling pathway for the treatment of melasma.

Melasma is a common condition of hyperpigmented facial skin. Picosecond lasers are reported to be effective for the treatment of melasma. We aimed to identify the most effective therapeutic mode and elucidate the potential molecular mechanisms of picosecond lasers for the treatment of melasma. Female Kunming mice with melasma-like conditions were treated using four different picosecond laser modes. Concurrently, in vitro experiments were conducted to assess changes in melanin and autophagy in mouse melanoma B16-F10 cells treated with these laser modes. Changes in melanin in mouse skin were detected via Fontana-Masson staining, and melanin particles were evaluated in B16-F10 cells. Real-time polymerase chain reaction and western blotting were used to analyse the expression levels of melanosome and autophagy-related messenger ribonucleic acid (mRNA) and proteins. A combination of large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers resulted insignificant decreases in melanin as well as in mRNA and protein expression of melanin-synthesizing enzymes (TYR, TRP-1 and MITF). This combination also led to increased expression of the autophagy-related proteins, Beclin1 and ATG5, with a marked decrease in p62 expression. Intervention with the PI3K activator, 740 Y-P, increased TYR, TRP-1, MITF, p-PI3K, p-AKT, p-mTOR and p62 expression but decreased the expression of LC3, ATG5 and Beclin1. A combination of large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers proved more effective and safer. It inhibits melanin production, downregulates the PI3K/AKT/mTOR pathway, enhances melanocyte autophagy and accelerates melanin metabolism, thereby reducing melanin content.

The Expression of Markers of Acute Kidney Injury Kim1 and NGAL after Administration of High Doses of Lithium Carbonate in Mice with Engrafted Skin Melanoma B16.

The expression of marker proteins of acute kidney injury after administration of high doses of lithium carbonate was assessed to evaluate the possibility of lithium use in neutron capture therapy. In mice with implanted skin melanoma B16, the expression of Kim1 (kidney injury molecule 1) and NGAL (neutrophil gelatinase-associated lipocalin) proteins in the kidneys was evaluated immunohistochemically 15, 30, 90, 180 min, and 7 days after peroral administration of lithium carbonate at single doses of 300 and 400 mg/kg. An increase in the expression of the studied proteins was found in 30 and 90 min after administration of 400 mg/kg lithium carbonate, however, 7 days after the drug administration, the expression returned to the level observed in the control group. It can be suggested that single administration of lithium carbonate in the studied doses effective for lithium neutron capture therapy will not significantly affect the renal function.

Probiotic-derived amphiphilic exopolysaccharide self-assembling adjuvant delivery platform for enhancing immune responses.

Enhancing immune response activation through the synergy of effective antigen delivery and immune enhancement using natural, biodegradable materials with immune-adjuvant capabilities is challenging. Here, we present NAPSL.p that can activate the Toll-like receptor 4 (TLR4) pathway, an amphiphilic exopolysaccharide, as a potential self-assembly adjuvant delivery platform. Its molecular structure and unique properties exhibited remarkable self-assembly, forming a homogeneous nanovaccine with ovalbumin (OVA) as the model antigen. When used as an adjuvant, NAPSL.p significantly increased OVA uptake by dendritic cells. In vivo imaging revealed prolonged pharmacokinetics of NAPSL. p-delivered OVA compared to OVA alone. Notably, NAPSL.p induced elevated levels of specific serum IgG and isotype titers, enhancing rejection of B16-OVA melanoma xenografts in vaccinated mice. Additionally, NAPSL.p formulation improved therapeutic effects, inhibiting tumor growth, and increasing animal survival rates. The nanovaccine elicited CD4+ and CD8+ T cell-based immune responses, demonstrating the potential for melanoma prevention. Furthermore, NAPSL.p-based vaccination showed stronger protective effects against influenza compared to Al (OH)3 adjuvant. Our findings suggest NAPSL.p as a promising, natural self-adjuvanting delivery platform to enhance vaccine design across applications.

Transcriptome analysis of immune-inflammatory regulation in Tremella fuciformis-derived polysaccharide reeducated B16 cells: A subcutaneous model.

BACKGROUND: Circulating cancer cells have characteristics of tumor self-targeting. Modified circulating tumor cells may serve as tumor-targeted cellular drugs. Tremella fuciformis-derived polysaccharide (TFP) is related to immune regulation and tumor inhibition, so could B16 cells reeducated by TFP be an effective anti-tumor drug? OBJECTIVES: To evaluate the intrinsic therapeutic potential of B16 cells exposed to TFP and clarify the therapeutic molecules or pathways altered by this process. MATERIAL AND METHODS: RNA-seq technology was used to study the effect of TFP-reeducated B16 cells on the immune and inflammatory system by placing the allograft subcutaneously in C57BL/6 mice. RESULTS: Tremella fuciformis-derived polysaccharide-reeducated B16 cells recruited leukocytes, neutrophils, dendritic cells (DCs), and mast cells into the subcutaneous region and promoted the infiltration of several cytokines such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), interleukin 1ß (IL-1ß), and interleukin 1 (IL-1). Tumor necrosis factor alpha also activated Th17 lymphocytes to secrete interleukin 17 (IL-17) and interferon gamma (IFN-γ). The co-expression of IFN-γ and IL-17 was favorable for tumor immunity to shrink tumors. In short, TFP-reeducated B16 cells activated the innate and adaptive immune responses, especially Th17 cell differentiation and IFN-γ production, as well as the TNF-α signaling pathway, which re-regulated the inflammatory and immune systems. CONCLUSION: B16 cells subcutaneously exposed to TFP in mice induced an immune and inflammatory response to inhibit tumors. The study of the function of TFP-reeducated B16 cells to improve cancer immunotherapy may be of particular research interest. This approach could be an alternative and more efficient strategy to deliver cytokines and open up new possibilities for long-lasting, multi-level tumor control.

Anti-Melanogenic Effects of Takifugu flavidus Muscle Hydrolysate in B16F10 Melanoma Cells and Zebrafish.

Abnormal melanogenesis can lead to hyperpigmentation. Tyrosinase (TYR), a key rate-limiting enzyme in melanin production, is an important therapeutic target for these disorders. We investigated the TYR inhibitory activity of hydrolysates extracted from the muscle tissue of Takifugu flavidus (TFMH). We used computer-aided virtual screening to identify a novel peptide that potently inhibited melanin synthesis, simulated its binding mode to TYR, and evaluated functional efficacy in vitro and in vivo. TFMH inhibited the diphenolase activities of mTYR, reducing TYR substrate binding activity and effectively inhibiting melanin synthesis. TFMH indirectly reduced cAMP response element-binding protein phosphorylation in vitro by downregulating melanocortin 1 receptor expression, thereby inhibiting expression of the microphthalmia-associated transcription factor, further decreasing TYR, tyrosinase related protein 1, and dopachrome tautomerase expression and ultimately impeding melanin synthesis. In zebrafish, TFMH significantly reduced black spot formation. TFMH (200 µg/mL) decreased zebrafish TYR activity by 43% and melanin content by 52%. Molecular dynamics simulations over 100 ns revealed that the FGFRSP (T-6) peptide stably binds mushroom TYR via hydrogen bonds and ionic interactions. T-6 (400 µmol/L) reduced melanin content in B16F10 melanoma cells by 71% and TYR activity by 79%. In zebrafish, T-6 (200 µmol/L) inhibited melanin production by 64%. TFMH and T-6 exhibit good potential for the development of natural skin-whitening cosmetic products.

Immunotherapeutic Potential of Mollusk Hemocyanins in Murine Model of Melanoma.

The development of antitumor drugs and therapy requires new approaches and molecules, and products of natural origin provide intriguing alternatives for antitumor research. Gastropodan hemocyanins-multimeric copper-containing glycoproteins have been used in therapeutic vaccines and antitumor agents in many cancer models. MATERIALS AND METHODS: We established a murine model of melanoma by challenging C57BL/6 mice with a B16F10 cell line for solid tumor formation in experimental animals. The anticancer properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) were evaluated in this melanoma model using various schemes of therapy. Flow cytometry, ELISA, proliferation, and cytotoxicity assays, as well as histology investigations, were also performed. RESULTS: Beneficial effects on tumor growth, tumor incidence, and survival of tumor-bearing C57BL/6 mice after administration of the RtH or HaH were observed. The generation of high titers of melanoma-specific IgM antibodies, pro-inflammatory cytokines, and tumor-specific CTLs, and high levels of tumor-infiltrated M1 macrophages enhanced the immune reaction and tumor suppression. DISCUSSION: Both RtH and HaH exhibited promising properties for applications as antitumor therapeutic agents and future experiments with humans.

Pentoxifylline and Norcantharidin Modify p62 Expression in 2D and 3D Cultures of B16F1 Cells.

Three-dimensional cell cultures have improved the evaluation of drugs for cancer therapy, due to their high similarity to solid tumors. In melanoma, autophagy appears to show a dual role depending on the progression of the disease. p62 protein has been proposed for the evaluation of autophagic flux since its expression is an indicator of the state of autophagy. Pentoxifylline (PTX) and Norcantharidin (NCTD) are drugs that have been shown to possess anticancer effects. In this work, we used B16F1 mouse melanoma cells in two-dimensional (2D) monolayer cultures and three-dimensional (3D) spheroids to test the effect of PTX and NCTD over the p62 expression. We analyzed the effect on p62 expression through Western blot and immunofluorescence assays. Our results indicate that PTX decreases p62 expression in both cell culture models, while Norcantharidin increases its expression in 3D cultures at 24 h. Therefore, these drugs could have a potential therapeutic use for the regulation of autophagy in melanoma, depending on the state of evolution of the disease.

Tumor- and Fibroblast-Derived Cell-Free DNAs Differently Affect the Progression of B16 Melanoma In Vitro and In Vivo.

It is widely postulated that the majority of pathologically elevated extracellular or cell-free DNA (cfDNA) in cancer originates from tumor cells; however, evidence has emerged regarding the significant contributions of other cells from the tumor microenvironment. Here, the effect of cfDNA originating from murine B16 melanoma cells and L929 fibroblasts on B16 cells was investigated. It was found that cfDNAL929 increased the viability and migration properties of B16 cells in vitro and their invasiveness in vivo. In contrast, cfDNAB16 exhibited a negative effect on B16 cells, reducing their viability and migration in vitro, which in vivo led to decreased tumor size and metastasis number. It was shown that cell treatment with both cfDNAs resulted in an increase in the expression of genes encoding DNases and the oncogenes Braf, Kras, and Myc. cfDNAL929-treated cells were shown to experience oxidative stress. Gene expression changes in the case of cfDNAB16 treatment are well correlated with the observed decrease in proliferation and migration of B16 cells. The obtained data may indicate the possible involvement of fibroblast DNA in the tumor microenvironment in tumor progression and, potentially, in the formation of new tumor foci due to the transformation of normal cells.

The cholera toxin B subunit induces trained immunity in dendritic cells and promotes CD8 T cell antitumor immunity.

Introduction: Innate immune training is a metabolic, functional, and epigenetic long-term reprogramming of innate cells triggered by different stimuli. This imprinting also reaches hematopoietic precursors in the bone marrow to sustain a memory-like phenotype. Dendritic cells (DCs) can exhibit memory-like responses, enhanced upon subsequent exposure to a pathogen; however, whether this imprinting is lineage and stimulus-restricted is still being determined. Nevertheless, the functional consequences of DCs training on the adaptive and protective immune response against non-infectious diseases remain unresolved. Methods: We evaluated the effect of the nontoxic cholera B subunit (CTB), LPS and LTA in the induction of trained immunity in murine DCs revealed by TNFa and LDH expression, through confocal microscopy. Additionally, we obtained bone marrow DCs (BMDCs) from mice treated with CTB, LPS, and LTA and evaluated training features in DCs and their antigen-presenting cell capability using multiparametric cytometry. Finally, we design an experimental melanoma mouse model to demonstrate protection induced by CTB-trained DCs in vivo. Results: CTB-trained DCs exhibit increased expression of TNFa, and metabolic reprogramming indicated by LDH expression. Moreover, CTB training has an imprint on DC precursors, increasing the number and antigen-presenting function in BMDCs. We found that training by CTB stimulates the recruitment of DC precursors and DCs infiltration at the skin and lymph nodes. Interestingly, training-induced by CTB promotes a highly co-stimulatory phenotype in tumor-infiltrating DCs (CD86+) and a heightened functionality of exhausted CD8 T cells (Ki67+, GZMB+), which were associated with a protective response against melanoma challenge in vivo. Conclusion: Our work indicates that CTB can induce innate immune training on DCs, which turns into an efficient adaptive immune response in the melanoma model and might be a potential immunotherapeutic approach for tumor growth control.

Epigallocatechin gallate protects against fat and muscle atrophy in B16BL6 melanoma-bearing mice on a high-fat diet.

AIMS: Epidemiological evidence indicates that there is a substantial association between body mass index (BMI) and at least ten forms of cancer, including melanoma, and BMI imbalance contributes to the poor survival rate of cancer patients before and after therapy. Nevertheless, few pharmacological studies on models of obesity and cancer have been reported. In this study, we administered epigallocatechin gallate (EGCG) to B16BL6 tumor-bearing mice that received a high-fat diet (HFD) to examine its impact. METHODS: B16BL6 tumor-bearing mice were fed a HFD. Body weight and food intake were documented every week. We conducted a Western blot analysis to examine the protein levels in the tumor, gastrocnemius (GAS), and tibialis anterior (TA) muscles, as well as the inguinal and epididymal white adipose tissues (iWAT and eWAT). KEY FINDINGS: EGCG has been shown to have anti-cancer effects equivalent to those of cisplatin, a chemotherapy drug. Furthermore, EGCG protected against the loss of epidydimal white adipose tissue by regulating protein levels of lipolysis factors of adipose triglyceride lipase and hormone-sensitive lipase as well as WAT browning factors of uncoupling protein 1, as opposed to cisplatin. EGCG was shown to reduce the protein levels of muscular atrophy factors of muscle RING-finger protein-1, whereas cisplatin did not contribute to rescuing the atrophy of TA and GAS muscles. CONCLUSION: Taken together, our findings indicate that EGCG has a preventive effect against cachexia symptoms and has anti-cancer effects similar to those of cisplatin in tumor-bearing mice fed a high-fat diet.

Experimental evidence that carbon-ion radiotherapy utilizes cytotoxic T lymphocyte-mediated anti-tumor immunity for shrinking tumors compared to X-ray therapy.

The therapeutic efficacy of radiotherapy (RT) is primarily driven by two factors: biophysical DNA damage in cancer cells and radiation-induced anti-tumor immunity. However, Anti-tumor immune responses between X-ray RT (XRT) and carbon-ion RT (CIRT) remain unclear. In this study, we, employed mouse models to assess the immunological contribution, especially cytotoxic T-lymphocyte (CTL)-mediated immunity, to the therapeutic effectiveness of XRT and CIRT in shrinking tumors. We irradiated mouse intradermal tumors of B16F10-ovalbumin (OVA) mouse melanoma cells and 3LL-OVA mouse lung cancer cells with carbon-ion beams or X-rays in the presence or absence of CTLs. CTL removal was performed by administration of anti-CD8 monoclonal antibody (mAb) in mice. Based on tumor growth delay, we determined the tumor growth and regression curves. The enhancement ratio (ER) of the slope of regression lines in the presence of CTLs, relative to the absence of CTLs, indicates the dependency of RT on CTLs for shrinking mouse tumors, and the biological effectiveness (RBE) of CIRT relative to XRT were calculated. Tumor growth curves revealed that the elimination of CD8+ CTLs by administrating anti-CD8 mAb accelerated tumor growth compared to the presence of CTLs in both RTs. The ERs were larger in CIRT compared to XRT in the B16F10-OVA tumor models, but not in the 3LL-OVA models, suggesting a greater contribution of CTL-mediated anti-tumor immunity to tumor reduction in CIRT compared to XRT in the B16F10-OVA tumor model. In addition, the RBE values for both models were larger in the presence of CTLs compared to models without CTLs, suggesting that CIRT may utilize CTL-mediated anti-tumor immunity more than X-ray. The findings from this study suggest that although immunological contribution to therapeutic efficacy may vary depending on the type of tumor cell, CIRT utilizes CTL-mediated immunity to a greater extent compared to XRT.

Enhanced Therapeutic Efficacy Against Melanoma through Exosomal Delivery of Hesperidin.

Melanoma, characterized as the most aggressive and metastatic form of skin cancer, currently has limited treatment options, predominantly chemotherapy and radiation therapy. However, the drawbacks associated with parenterally administered chemotherapy underscore the urgent need for alternative compounds to combat melanoma effectively. Hesperidin (HES), a flavonoid present in various citrus fruits, exhibits promising anticancer activity. Nevertheless, the clinical utility of HES is hindered by challenges such as poor water solubility, a short half-life, and low oral bioavailability. In response to these limitations, we introduced a novel approach by formulating HES-loaded exosomes (Exo-HES). Isolation of exosomes was achieved through the ultracentrifugation method, and HES was efficiently loaded using the sonication method. The resulting formulations displayed a desirable particle size (∼106 nm) and exhibited a spherical morphology, as confirmed by scanning electron and atomic force microscopy. In vitro studies conducted on B16F10 cell lines demonstrated higher cytotoxicity of Exo-HES compared to free HES, supported by enhanced cellular uptake validated through coumarin-6-loaded exosomes. This superior cytotoxicity was further evidenced by DNA fragmentation, increased generation of free radicals (ROS), loss of mitochondrial membrane potential, and effective inhibition of colony formation. The antimetastatic properties of Exo-HES were confirmed through wound healing and transwell migration assays. Oral pharmacokinetics studies revealed a remarkable increase of approximately 2.5 times in oral bioavailability and half-life of HES when loaded into exosomes. Subsequent in vivo experiments utilizing a B16F10-induced melanoma model in Swiss mice established that Exo-HES exhibited superior anticancer activity compared to HES after oral administration. Importantly, no biochemical, hematological, or histological toxicities were observed in tumor-bearing mice treated with Exo-HES. These findings suggest that exosomes loaded with HES represent a promising nanocarrier strategy to enhance the therapeutic effectiveness of hesperidin in melanoma treatment.