B16 Melanoma Research Articles

Abstract B048: Neutrophil extracellular traps-targeting DNase I enhances the therapeutic efficacy of CAR-T cell adoptive immunotherapy in a syngeneic murine metastasis model

Abstract In recent years, chimeric antigen receptor (CAR) T cell therapy has emerged as a promising approach for treating various malignancies, most notably in hematologic cancers. However, its efficacy in solid tumors is often hindered by an immunosuppressive tumor microenvironment (TME), which, in particular, is frequently characterized by the presence of tumor-associated cell-free DNA (cfDNA), especially in the form of neutrophil extracellular traps (NETs). In adoptive immunotherapies NETs, composed primarily of neutrophil-derived DNA, histones and neutrophil granule enzymes (e.g., MPO, NE), act as physical barriers and induce the secretion of immunosuppressive factors that impair CAR T cell function. This study investigates the potential of co-administration of deoxyribonuclease I (DNase I) with CAR T cells in a syngeneic B16 melanoma murine model of lung metastasis. Bioluminescent imaging of melanoma metastatic processes has shown that a single injection of DNase I (10 mg/kg) suppresses B16-EGFR lung metastasis at early stages in comparison to the vehicle control group but does not improve survival. However, co-administration of DNase I with murine EGFR-CAR T cells significantly suppresses tumor burden, decreases the number of metastatic foci, and substantially prolongs survival compared to the CAR T cell monotherapy group. Degrading of NETs [RB1] by DNase I increases the amount of tumor-infiltrating T and CAR T cells and reduces the immunosuppressive effects of the TME. This is reflected in the CD8 population of tumor-infiltrating CAR T cells from the DNase I treated group, which have lower expression of PD-1 and TIM-3 exhaustion markers. This research highlights the critical role of the NETs in modulating CAR T cell efficacy and provides a compelling rationale for incorporating DNase I as an adjunctive treatment to improve therapeutic responses in patients undergoing CAR T cell therapy. However, further clinical studies are warranted to validate these findings and explore the translational potential of this combinatorial approach in enhancing cancer treatment. [RB1]Again - this takes the emphasis and the reader away from NETs and into the cfDNA universe. Citation Format: Alexey Stepanov, Wenjian Wang, Yingqin Hou, Ivan Chernikov Ivan Chernikov, Reid Bissonnette, George Tetz, Grigory Borisenko, Dmitry Genkin. Neutrophil extracellular traps-targeting DNase I enhances the therapeutic efficacy of CAR-T cell adoptive immunotherapy in a syngeneic murine metastasis model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B048.

TMIC-32. INFIGRATINIB INHIBITION OF THE THBS1 PATHWAY IN MELANOMA BRAIN METASTASIS

Abstract INTRODUCTION Despite promising preclinical studies, BRAF/MEK inhibition with vemurafenib in melanoma brain metastases (MBM) has suboptimal durable response in humans. Thrombospondin 1 (THBS1), a matricellular protein, is upregulated in vemurafenib-resistant metastatic melanoma, and its inhibition reverses BRAF inhibitor chemoresistance. However, the characteristics of THBS1-mediated signaling pathways as well as methods of inhibition require further exploration in the context of MBM. OBJECTIVE To characterize THBS1-mediated signaling pathways in MBM and evaluate the therapeutic potential of targeting THBS1 pathways. METHODS We analyzed human parenchymal MBM samples using publicly available single-cell RNA sequencing (scRNA-seq) data. Protein-protein interaction networks were assessed using the STRING database. Syngeneic B16 melanoma cells were intracranially implanted in C57BL/6J mice and treated with intraperitoneal injection of FGF7/FGFR2 small-molecule inhibitor Infigratinib or PBS (sham). Mice were immunophenotyped using flow cytometry and monitored for survival. RESULTS THBS1 is highly upregulated in immunosuppressive MBM-associated cell populations such as S100A8+ Metastasis-Associated Macrophages and Mesenchymal Stromal Cell-like cells. Gene set enrichment analysis of THBS1-positive cells paradoxically revealed upregulation of inflammation-associated gene sets. Differential expression analysis identified Fibroblast Growth Factor 7 (FGF7) as strongly co-expressed in THBS1-positive MBM cells, and STRING-DB confirmed close protein-protein interactions between FGF7 and THBS1. In vivo inhibition using Infigratinib for FGF7’s receptor, FGFR2, decreased the proportion of monocytic myeloid-derived suppressor cells (M-MDSCs) (P=0.030), increased T-cell activation marker IFN-y (P=0.027) and proliferation marker CD69 (P=0.047), and resulted in improved overall survival (P=0.0001). CONCLUSION MBM scRNA-seq data suggests that THBS1 is associated with inflammation-induced immunosuppression. Inhibiting the FGF7/FGFR2-THBS1 axis with Infigratinib modulates the tumor microenvironment by reducing immunosuppressive M-MDSCs and increasing T cell activation and proliferation. This data supports THBS1 inhibition as a viable therapeutic option that in the future can also be combined with other current immunotherapeutic strategies.

Differential T cell accumulation within intracranial and subcutaneous melanomas is associated with differences in intratumoral myeloid cells

Patients with metastatic brain melanomas (MBM) experience shorter-lasting survival than patients with extracranial metastases, and this is associated with a higher fraction of dysfunctional CD8 T cells. The goal of this study was to understand the underlying cause of T cell dysfunction in MBM. To accomplish this, we compared murine B16 melanomas implanted intracranially (IC) or subcutaneously (SC). CD8 T cell activation was not altered, but representation in IC tumors was lower. Transferred activated or naïve CD8 T cells accumulated in similar numbers in both tumors, suggesting that the vasculature does not differentially impair T cell presence. Surprisingly, we found no evidence for T cell activation in draining lymph nodes of SC or IC tumor-bearing mice, consistent with the fact that dendritic cells (DC) that had acquired tumor antigen showed an immature phenotype. Instead, T cell activation occurred within both tumors, where the majority of tumor antigen+ myeloid cells were found. While, the numbers of intratumoral DC were comparable, those in IC tumors acquired less tumor antigen, and were alternatively matured based on upregulation of MHCII without upregulation of CD86. Additionally, in IC tumors, the largest population of tumor antigen+ myeloid cells were microglia. However, their presence did not influence either antigen acquisition or the phenotype of other myeloid cell populations. Overall, our data suggest that diminished representation of CD8 T cells in IC tumors is a consequence of alternatively matured DC and/or microglia that induce distinctly activated T cells, which ultimately fail to continue to accumulate inside the tumor.

P-type pilus PapG protein elicits toll-like receptor 2-mediated immune activation during cancer immunotherapy

The immune activation ability of FimH, an adhesion protein in pili of Escherichia coli (E. coli), has been recently reported. However, studies on the immune activity of PapG, another major pili terminal protein, have not been well explored. In this study, the immune stimulatory effect of purified recombinant PapG was evaluated. PapG treatment promoted dramatic changes in dendritic morphology of the bone marrow-derived dendritic cells (BMDCs) and induced upregulation of co-stimulatory molecule levels, major histocompatibility complex (MHC) I and II expression, and pro-inflammatory cytokine production in BMDCs. To identify the stimulatory receptor of PapG, an in silico study was performed. PapG exhibited strong binding affinity with murine toll-like receptor 2 (TLR2). In addition, PapG-induced activation of splenic DC and its subsets was unsuccessful in TLR2-knock out mice. Combination of PapG and ovalbumin (OVA) elicited OVA-specific T cell proliferation and cytokine production and cytotoxicity that consequently promoted anti-cancer immune responses against OVA-expressing B16 melanoma. Furthermore, PapG treatment induced activation of peripheral blood DCs and its subsets in humans in a TLR2 dependent manner. PapG-stimulated human conventional DC2 promoted syngeneic T cell proliferation and activation. The findings of this study demonstrated that PapG could be a useful immune stimulator for immunotherapy against cancer.

Caerin 1.1 and 1.9 peptides halt B16 melanoma metastatic tumours via expanding cDC1 and reprogramming tumour macrophages

BackgroundCancer immunotherapy, particularly immune checkpoint inhibitors (ICBs) such as anti-PD-1 antibodies, has revolutionised cancer treatment, although response rates vary among patients. Previous studies have demonstrated that caerin 1.1 and 1.9, host-defence peptides from the Australian tree frog, enhance the effectiveness of anti-PD-1 and therapeutic vaccines in a murine TC-1 model by activating tumour-associated macrophages intratumorally.MethodsWe employed a murine B16 melanoma model to investigate the therapeutic potential of caerin 1.1 and 1.9 in combination with anti-CD47 and a therapeutic vaccine (triple therapy, TT). Tumour growth of caerin-injected primary tumours and distant metastatic tumours was assessed, and survival analysis conducted. Single-cell RNA sequencing (scRNAseq) of CD45+ cells isolated from distant tumours was performed to elucidate changes in the tumour microenvironment induced by TT.ResultsThe TT treatment significantly reduced tumour volumes on the treated side compared to untreated and control groups, with notable effects observed by Day 21. Survival analysis indicated extended survival in mice receiving TT, both on the treated and distant sides. scRNAseq revealed a notable expansion of conventional type 1 dendritic cells (cDC1s) and CD4+CD8+ T cells in the TT group. Tumour-associated macrophages in the TT group shifted toward a more immune-responsive M1 phenotype, with enhanced communication observed between cDC1s and CD8+ and CD4+CD25+ T cells. Additionally, TT downregulated M2-like macrophage marker genes, particularly in MHCIIhi and tissue-resident macrophages, suppressing Cd68 and Arg1 expression across all macrophage types. Differential gene expression analysis highlighted pathway alterations, including upregulation of oxidative phosphorylation and MYC target V1 in Arg1hi macrophages, and activation of pro-inflammatory pathways in MHCIIhi and tissue-resident macrophages.ConclusionOur findings suggest that caerin 1.1 and 1.9, combined with immunotherapy, effectively modulate the tumour microenvironment in primary and secondary tumours, leading to reduced tumour growth and enhanced systemic immunity. Further investigation into these mechanisms could pave the way for improved combination therapies in advanced melanoma treatment.

Expression of c-erb-B2 oncoprotein as a neoantigen strategy to repurpose anti-neu antibody therapy in a model of melanoma.

In this study, we tested a novel approach of "repurposing" a biomarker typically associated with breast cancer for use in melanoma. HER2/neu is a well characterized biomarker in breast cancer for which effective anti-HER2/neu therapies are readily available. We constructed a lentivirus encoding c-erb-B2, an animal (rat) homolog to HER2/neu. This was used to transfect B16 melanoma in vitro for use in an orthotopic preclinical mouse model, which resulted in expression of rat c-erb-B2 as a neoantigen target for anti-c-erb-B2 monoclonal antibody (7.16.4). The c-erb-B2-expressing melanoma was designated B16/neu. 7.16.4 produced statistically significant in vivo anti-tumor responses against B16/neu. This effect was mediated by NK-cell antibody-dependent cell-mediated cytotoxicity. To further model human melanoma (which expresses < 5% HER2/neu), our c-erb-B2 encoding lentivirus was used to inoculate naïve (wild-type) B16 tumors in vivo, resulting in successful c-erb-B2 expression. When combined with 7.16.4, anti-tumor responses were again demonstrated where approximately 40% of mice treated with c-erb-B2 lentivirus and 7.16.4 achieved complete clinical response and long-term survival. For the first time, we demonstrated a novel strategy to repurpose c-erb-B2 as a neoantigen target for melanoma. Our findings are particularly significant in the contemporary setting where newer anti-HER2/neu antibody-drug therapies have shown increased efficacy.

Abstract A031: Unhelpful CD4 help: Removal of conventional CD4+ T cells promotes priming of tumor antigen-specific CD8 T cells in the context of CD4-depletion therapy

Abstract Preclinical and clinical studies have used antibodies to deplete CD4-expressing cells to promote anti-tumor immunity. Specifically, a monoclonal anti-CD4 antibody (anti-CD4) has been employed to remove FOXP3+ CD4+ regulatory T cells (Tregs), which are known to impede anti-tumor immunity. Our previous studies demonstrated that administering anti-CD4 during B16 melanoma tumor growth in mice leads to the generation of tumor Ag-specific CD8 T cells that disseminate and form memory following surgical tumor excision. However, the importance of co-depleting other CD4-expressing cell compartments for anti-CD4 treatment efficacy are unknown. We utilized flow cytometry and single-cell RNA sequencing (scRNAseq) to evaluate the primary response of tumor Ag-specific CD8 T cells (pmel) in B16 melanoma tumor bearing mice treated with anti-CD4. Responses were compared to those induced by anti-PD1 and anti-CTLA4 dual immune checkpoint blockade (ICB). To determine the importance of homeostatic space in priming, homeostatic expansion and priming were decoupled, and neutralizing antibodies were administered. Treg-depleted (Foxp3-DTR) mice were used for comparison, and Batf3-/- mice and IL-2R blocking antibodies were used to determine requirements for CD8 T cell priming. Finally, scRNAseq was conducted on CD4 T cells from human melanoma tumors previously treated with ICB to characterize transcriptional properties of intratumoral CD4 T cells. Compared to ICB, anti-CD4 treatment elicited a more robust, disseminated, and persistent primary tumor Ag-specific CD8 T cell response. ScRNAseq revealed that anti-CD4 treatment induced more stem-like (TCF1+) memory precursor cells, whereas ICB induced more differentiated effector (Gzmb+) cells. Removal of homeostatic space or blockade of IL-7R and IL-15 during priming did not diminish pmel responses in anti-CD4 treated mice, indicating that homeostatic space was not required. Importantly, pmel cells were shown to produce their own IL-2, and pmel priming was significantly lower after IL-2 receptor blockade, suggesting dependence on autocrine IL-2 signals. Using DT-treated FOXP3-DTR mice, we found that targeted Treg depletion was insufficient for the pmel priming that disseminated and formed memory, indicating that total CD4 T cell depletion was required for this systemic response. Depletion of total CD4 T cells uniquely increased cDC1 proportions in tumor-draining LNs as well as their expression of CD86. However, treatment of tumor-bearing RAG-/- mice with anti-CD4 confirmed that depletion of CD4-expressing APC compartments does not contribute to anti-CD4 treatment efficacy. ScRNAseq of CD4 T cells from human melanoma tumors showed dominance of Tr1-like cells and other subsets without transcriptional evidence of helper differentiation, despite prior ICB. In mice, combination of anti-CD4 with dual ICB restored the systemic response of tumor-specific CD8 T cells. This study underscores the importance of eliminating conventional CD4 T cells in tumor-bearing hosts to elicit systemic primary and memory CD8 T cell responses. Citation Format: Christo P. C. Dragnev, Delaney E. Ramirez, Tyler G. Searles, Nathaniel Spicer, TIffany Chen, J. Louise Lines, Aaron R. Hawkes, Wilson L. Davis III, Asmaa Mohamed, Keisuke Shirai, Joesph D. Phillips, Pamela C. Rosato, Yina H. Huang, Mary Jo Turk. Unhelpful CD4 help: Removal of conventional CD4+ T cells promotes priming of tumor antigen-specific CD8 T cells in the context of CD4-depletion therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A031.

Doramectin Induces Apoptosis in B16 Melanoma Cells.

Metastatic melanoma resists current pharmacological regimens that act through apoptosis. This indicates that therapies acting via non-apoptotic cell-death pathways could be pursued. Doramectin has shown promising results in another cancer of neural crest origin, neuroblastoma, through the inhibition of growth via autophagy. Our research hypothesis is that doramectin induces autophagy in B16F10 melanoma cells. Cells were treated with doramectin (15 uM) or a combination of both doramectin and a cell-death inhibitor, compared to untreated control cells (media), and then analyzed with MTT analysis. Likewise, MDC analysis was completed to detect autophagy involvement with doramectin treatment. Flow cytometry and TUNEL Assay were conducted to observe cell death-related effects. MTT analysis of doramectin-treated cells displayed a decrease in cell growth compared to control. Apoptotic morphology was prominent in melanoma cells treated with doramectin. Increased autophagy was not detected by fluorometric microscopic analysis. Flow cytometry analysis of doramectin-treated cells showed apoptosis as a major mode of cell death with some necrosis. Doramectin induces a novel cell-death mechanism in melanoma compared to other forms of cancer and should be studied as an effective anti-cancer agent for melanoma treatment.

PP2A negatively regulates NK cell T-bet expression and anti-tumor effector function.

The transcription factor T-bet is essential for the anti-tumor effector function of NK cells, but the mechanism regulating its expression in NK cells remains unclear. In this study, we aimed to identify an NK cell intrinsic regulator that controls T-bet expression. Using T-bet-luciferase reporter assay screening, we identified a protein phosphatase inhibitor as a potential activator of T-bet expression. A series of PP2A-specific inhibitors (PP2Ai) or PP2A siRNA induced the expression of T-bet. In PP2Ai-treated mice, the expressions of T-bet and its downstream effector molecules, granzyme B and IFN-γ, were also upregulated in NK cells. Mechanistically, PP2Ai increased the phosphorylation of mTOR and ribosomal protein S6 in NK cells, and mTOR inhibitor canceled the effects of PP2Ai in NK cells. Importantly, NK cells isolated from PP2Ai-treated mice showed higher cytotoxicity and IFN-γ production; therefore, they increased the anti-tumor effector function of NK cells. Accordingly, PP2Ai treatment inhibited lung metastasis of B16 melanoma by NK cell- and mTOR-dependent mechanisms. These results suggest that PP2A negatively regulates NK cell T-bet expression and effector function by an mTOR-dependent mechanism.

Anti-tumor effects of recombinant human cyclophilin A combined with immune checkpoint inhibitors in the experimental model of melanoma B16 in vivo

Introduction. Immune checkpoint inhibitors have an exceptional position in cancer immunotherapy. Currently, anti-CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) and anti-PD-1/PD-L1 (PD-1 – programmed cell death 1, PD-L1 – programmed death 1 ligand 1) therapies are most widely applied in clinical practice. Still, immune checkpoint inhibitors therapy is not always successful, and multiple studies have indicated that it should be combined with other immunotherapeutic strategies, including cytokines. Secreted cyclophilin A (CypA) could be of particular interest in this respect. Previously, we showed that recombinant human CypA (rhCypA) had pleiotropic immunostimulatory activity and anti-tumor effects. Studies of rhCypA as an anti-cancer factor pointed to its potential use in cancer chemoimmunotherapy and combination immunotherapy.Aim. To evaluate anti-tumor effects of combined immunotherapy using rhCypA and immune checkpoint inhibitors in the mouse model of melanoma B16 in vivo.Materials and methods. C57BL/6 mice were subcutaneously transplanted with melanoma B16. On days 6 and 9 posttumor transplantation, monoclonal antibodies to PD-1, PD-L1 and programmed cell death 1 ligand 2 (PD-L2), CTLA-4, lymphocyte-activation gene 3 (LAG-3), or CD276 were intravenously injected into mice at a dose of 100 μg/mouse. RhCypA was injected s/c on days 6–10 post-tumor transplantation at a dose of 100 μg/mouse. The therapeutic effects of combined immunotherapy were evaluated by melanoma B16 growth dynamics and the survival of tumor-bearing mice.Results. In combination with anti-CTLA-4 monoclonal antibodies, rhCypA had the most distinct and prolonged synergic anti-tumor effects until day 19 post-immunotherapy, with an increase in animal lifespan of 70 %. When used with anti-LAG-3 monoclonal antibodies, rhCypA exhibited a synergic therapeutic effect by day 12 post-therapy. Combination of rhCypA with anti-PD-L1 or anti-CD276 monoclonal antibodies had short-term synergic effects until day 5 after therapy. Recombinant human CypA impeded the anti-tumor effects of dual anti-PD-1 + anti-LAG-3 therapy.Conclusion. Our findings pointed out that rhCypA could significantly improve therapeutic effects of individual immune checkpoint inhibitors. Therefore, rhCypA could be potentially proposed as a component of combined anti-tumor immunotherapy.

Predictive Production of a New Highly Soluble Glucoside, Corylin-7-O-β-Glucoside with Potent Anti-inflammatory and Anti-melanoma Activities.

Computational tools can now facilitate screening precursors and selecting suitable biotransformation enzymes for producing new bioactive compounds. This study applied the data-mining approach to screen for candidate precursors of glycosyltransferases to produce new glucosides from 412 commercial natural compounds. Among five candidates, experimental results showed that only corylin could be glycosylated by the bacterial glycosyltransferase, BsUGT489. Analysis of interaction potential between candidates and glycosyltransferase by molecular docking tools also found that corylin was the only compatible substrate. The new glucoside was purified and confirmed to be corylin-7-O-β-glucoside. The aqueous solubility of corylin-7-O-β-glucoside was 14.2 times more than its precursor aglycone, corylin. Corylin-7-O-β-glucoside retained anti-inflammatory activity in lipopolysaccharide-induced nitric oxide production of murine macrophage RAW 264.7 cells, with an IC50 value of 121.1 ± 9.5µM. Further, corylin-7-O-β-glucoside exhibited more potent anti-melanoma activity against murine B16 and human A2058 melanoma cells than corylin. Together, predictive studies facilitate the production of a new glucoside, corylin-7-O-β-glucoside, which is highly soluble and possesses anti-inflammatory and anti-melanoma activities and therefore has promising future applications in pharmacology.

KDM4B Histone Demethylase Inhibition Attenuates Tumorigenicity of Malignant Melanoma Cells by Overriding the p53-Mediated Tumor Suppressor Pathway.

Despite significant advances in the treatment of cutaneous melanoma (hereaftermelanoma), the prognosis remains less favorable due to therapeutic resistance, which is presumably linked to epigenetic dysregulation. We hypothesized that the histone lysine demethylase KDM4B could play a pivotal role in controlling therapy-resistant melanoma. To validate our hypothesis, we retrieved RNA sequencing data from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA)program and observed upregulation of KDM4B in both primary and metastatic melanoma, which was associated with poor survival. To explore its role, we used murine B16, human SK-MEL-5, and G-361 melanoma cells as in vitro models of melanoma. We found that KDM4B inhibition using NCGC00244536 increased global levels of H3K9me3 and downregulated the expressions of cell cycle progression-related genes Cdk1, Cdk4, Ccnb1, and Ccnd1. Moreover, genetic ablation of KDM4B or its chemical inhibition using NCGC00244536 reduced p53 production by upregulating MDM2, which enhances the proteolytic degradation of p53. Interestingly, despite the reduction of p53, these interventions augmented apoptosis and senescence-induced cell death by activating pathways downstream of p53, as evidenced by reduced levels of pro-survival Bcl-2 and Bcl-xL proteins and increased production of pro-apoptotic cleaved caspase-3, caspase-7, Bax, and the senescence inducer Cdkn1a. Compared to the FDA-approved anti-melanoma agent dacarbazine, NCGC00244536 exhibited more pronounced cytotoxic and antiproliferative effects in melanoma cells. Importantly, NCGC00244536 demonstrated minimal cytotoxicity to low Kdm4b-expressing mouse embryonic fibroblasts. In conclusion, our findings suggest that KDM4B inhibition can override the antitumor effect of p53, and potentially serve as a therapeutic strategy for melanoma.

Targeting ZC3H11A elicits immunogenic cancer cell death through augmentation of antigen presentation and interferon response

Zinc finger CCCH containing 11A (ZC3H11A) is a stress-induced protein that is upregulated in various conditions, such as heat shock and virus infection. It has also been reported to be upregulated in certain cancers. The aim of this study was to evaluate the feasibility of targeting ZC3H11A as a therapeutic approach for cancer treatment, using nuclease-resistant, affinity-enhanced antisense oligonucleotide (ASO). An ASO targeting ZC3H11A was validated and evaluated in vitro and in the B16 melanoma model in vivo. Antigen presentation, interferon response, cell proliferation and apoptosis were transcriptionally affected. These findings were validated on the protein level by upregulation of major histocompatibility complex I (MHC-I), an increased secretion of interferon-β (IFN-β), and induction of apoptosis observed as upregulation of caspases and annexin V. Immunogenic features of the induced apoptosis were evidenced by surface exposure of calreticulin (CRT) and the secretion of ATP leading to enhanced dendritic cell (DC) phagocytosis, maturation, and activation. Treatment with the ZC3H11A-targeted ASO had limited efficacy in vivo while constitutive lentiviral shRNA knockdown of ZC3H11A in murine B16 melanoma cells, and human Hela cells led to reduced tumor growth with prolonged survival of mice, validating ZC3H11A as a relevant target for cancer therapy.

Metabolic modulation of melanoma enhances the therapeutic potential of immune checkpoint inhibitors.

Lactate is a pivotal molecule with diverse functions in the metabolic reprogramming of cancer cells. Beyond its role in metabolism, lactate exerts a modulatory effect within the tumor microenvironment; it is utilized by stromal cells and has been implicated in the suppression of the immune response against the tumor. Using in vitro assays (including flow cytometry, live-cell imaging and metabolic analyses), the impact of lactate dehydrogenase inhibitors (LDHIs) on melanoma cells were assessed. The therapeutic potential of LDHIs with immune checkpoint inhibitors (ICIs) were tested in vivo in murine models of melanoma tumors. A potent anti-proliferative effect (via both cell cycle alterations and enhanced apoptosis) of LDHIs, Oxamate (Oxa) and methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate (NHI-2), was found upon treatment of melanoma cell lines. Using a combination of Oxa and NHI-2, a synergistic effect to inhibit proliferation, glycolysis, and ATP production was observed. Metabolic analysis revealed significant alteration in glycolysis and oxidative phosphorylation, while metabolite profiling emphasized consequential effects on lactate metabolism and induced energy depletion by LDHIs. Detection of increased RANTES and MCP-1, with Oxa and NHI-2 treatment, prompted the consideration of combining LDHIs with ICIs. In vivo studies using a murine B78 melanoma tumor model revealed a significant improvement in treatment efficacy when LDHIs were combined with ICIs. These findings propose the potential of targeting lactate metabolism to enhance the efficacy of ICI treatments in patients with melanoma.

Immunohistochemistry for assessing toxicity and mechanism of action of anticancer drugs during preclinical trials. From theory to practice

Aim. To identify the most suitable pathogenetic mechanisms for in-depth study of the antitumor and antimetastatic effects of tested hybrid organotin compounds using the immunohistochemical approach.Materials and Methods. Here, we tested bis(3,5-di-tert-butyl-4-hydroxyphenylthiolate) dimethyltin (laboratory code Me-3), belonging to the class of hybrid organotin compounds, on 30 female C57Bl/6 mice using a universal model of transplantable tumors with spontaneous metastasis (B16 melanoma). 48 hours after tumor cell transplantation, we intraperitoneally administered Me-3 once daily to female C57Bl/6 mice for 10 days at a total dose (TD) of 375 mg/kg. For histological analysis, we used the primary tumor node of B16 melanoma. Immunophenotyping of B16 melanoma tissue samples was carried out using the polyclonal antibodies to transforming growth factor beta 1 (TGFβ-1), vascular endothelial growth factor A (VEGFA), Bcl2-associated X Protein (Bcl-2), cluster of differentiation 34 (CD34).Results. After the exposure to Me-3, we found a reduced immunohistochemical signal to TGF-β1 and Bcl-2 3 in the tumor tissue. Low doses of Me-3 have also impacted angiogenesis.Conclusion. Me-3 has a pro-apoptotic and anti-angiogenetic effects on B16 melanoma cells in C57Bl/6 mice.

Self-Tumor Antigens in Solid Tumors Turned into Vaccines by α-gal Micelle Immunotherapy.

A major reason for the failure of the immune system to detect tumor antigens (TAs) is the insufficient uptake, processing, and presentation of TAs by antigen-presenting cells (APCs). The immunogenicity of TAs in the individual patient can be markedly increased by the in situ targeting of tumor cells for robust uptake by APCs, without the need to identify and characterize the TAs. This is feasible by the intra-tumoral injection of α-gal micelles comprised of glycolipids presenting the carbohydrate-antigen "α-gal epitope" (Galα1-3Galβ1-4GlcNAc-R). Humans produce a natural antibody called "anti-Gal" (constituting ~1% of immunoglobulins), which binds to α-gal epitopes. Tumor-injected α-gal micelles spontaneously insert into tumor cell membranes, so that multiple α-gal epitopes are presented on tumor cells. Anti-Gal binding to these epitopes activates the complement system, resulting in the killing of tumor cells, and the recruitment of multiple APCs (dendritic cells and macrophages) into treated tumors by the chemotactic complement cleavage peptides C5a and C3a. In this process of converting the treated tumor into a personalized TA vaccine, the recruited APC phagocytose anti-Gal opsonized tumor cells and cell membranes, process the internalized TAs and transport them to regional lymph-nodes. TA peptides presented on APCs activate TA-specific T cells to proliferate and destroy the metastatic tumor cells presenting the TAs. Studies in anti-Gal-producing mice demonstrated the induction of effective protection against distant metastases of the highly tumorigenic B16 melanoma following injection of natural and synthetic α-gal micelles into primary tumors. This treatment was further found to synergize with checkpoint inhibitor therapy by the anti-PD1 antibody. Phase-1 clinical trials indicated that α-gal micelle immunotherapy is safe and can induce the infiltration of CD4+ and CD8+ T cells into untreated distant metastases. It is suggested that, in addition to converting treated metastases into an autologous TA vaccine, this treatment should be considered as a neoadjuvant therapy, administering α-gal micelles into primary tumors immediately following their detection. Such an immunotherapy will convert tumors into a personalized anti-TA vaccine for the period prior to their resection.

Harnessing Spatiotemporal-Specific Tumorous Exosome Dynamics: Ultra-Sensitive Lanthanide Luminescence Detection Strategy Enabled by Exosomal Membrane Engineering for Melanoma Immunotherapy Monitoring.

Focused on the newly secreted tumorous exosomes during melanoma immunotherapy, this work has pioneered an ultra-sensitive spatiotemporal-specific exosome detection strategy, leveraging advanced exosomal membrane engineering techniques. The proposed strategy harnesses the power of amplified lanthanide luminescence signals on these exosomes, enabling precise and real-time monitoring of the efficacy of melanoma immunotherapy. The methodology comprises two pivotal steps. Initially, Ac4ManNAz-associated metabolic labeling is employed to evolve azide groups onto the membranes of newly secreted exosomes with remarkable selectivity. These azide groups serve as versatile clickable artificial tags, enabling the precise identification of melanoma exosomes emerging during immunotherapy. Subsequently, lanthanide-nanoparticle-functionalized polymer chains are controllably grafted onto the exosome surfaces through click chemistry and in situ Fenton-RAFT polymerization, serving as robust signal amplifiers. When integrated with time-resolved fluorescence detection, this strategy yields detection signals with an exceptionally high signal-to-noise ratio, enabling ultra-sensitive detection of PD-L1 antigen expression levels on the spatiotemporal-specific exosomes. The detection strategy boasts a wide linear concentration range spanning from 1.7 × 104 to 1.7 × 109 particles/mL, with a remarkable theoretical detection limit of 1.28 × 103 particles/mL. The remarkable enhancements in detection sensitivity and accuracy facilitate the evaluation of the efficacy of immunotherapeutic interventions in the mouse B16 melanoma model, notably revealing a substantial disparity in PD-L1 levels between immunotherapy-treated and untreated groups (P < 0.01) and further emphasizing the cumulative therapeutic effect that intensifies with repeated treatments (P < 0.001).

Microdissection tools to generate organoids for modeling the tumor immune microenvironment

Patient-derived tumor organoids have emerged as promising models for predicting personalized drug responses in cancer therapy, but they typically lack immune components. Preserving the in vivo association between tumor cells and endogenous immune cells is critical for accurate testing of cancer immunotherapies. Mechanical dissection of tumor specimens into tumor fragments, as opposed to enzymatic digestion into single cells, is essential for maintaining these native tumor-immune cell spatial relationships. However, conventional mechanical dissection relying on manual mincing is time-consuming and irreproducible. This study describes two microdissection devices, the µDicer and µGrater, to facilitate the generation of intact tumor fragments from mouse B16 melanoma, a common model of human melanoma. The µDicer- and µGrater-cut tumor fragments were used to generate air‒liquid interface (ALI) organoids that copreserve tumor cells with infiltrating immune subsets without artificial reconstitution. The µDicer, consisting of a hexagonal array of silicon microblades, was employed to investigate the effect of organoid size. The viability of ALI organoid immune cells appeared insensitive to organoid sizes exceeding ~400 µm but diminished in organoids ~200 µm in size. The µGrater, consisting of an array of submillimeter holes in stainless steel, was employed to accelerate dissection. For the samples studied, the µGrater was 4.5 times faster than manual mincing. Compared with those generated by manual mincing, ALI organoids generated by the µGrater demonstrated similar viability, immune cell composition, and responses to anti-PD-1 immunotherapy. With further optimization, the µGrater holds potential for integration into clinical workflows to support the advancement of personalized cancer immunotherapy.

Fatty acid derivatization and cyclization of the immunomodulatory peptide RP-182 targeting CD206high macrophages improves anti-tumor activity.

As tumor-associated macrophages (TAMs) exercise a plethora of pro-tumor and immune evasive functions, novel strategies targeting TAMs to inhibit tumor progression have emerged within the current arena of cancer immunotherapy. Activation of the mannose receptor 1 (Mrc1; CD206) is a recent approach that recognizes immune suppressive CD206high M2-like TAMs as a drug target. Ligation of CD206 both induces reprogramming of CD206high TAMs towards a pro-inflammatory phenotype and selectively triggers apoptosis in these cells. CD206-activating therapeutics are currently limited to the linear, 10mer peptide RP-182, 1, which is not a drug candidate. Here we sought to identify a better suitable candidate for future clinical development by synthesizing and evaluating a series of RP-182 analogues. Surprisingly, fatty acid derivative 1a (RP-182-PEG3-K(palmitic acid)) not only showed improved stability but also increased affinity to the CD206 receptor through enhanced interaction with a hydrophobic binding motif of CD206. Peptide 1a showed superior in vitro activity in cell-based assays of macrophage activation which was restricted to CD206high M2-polarized macrophages. Improvement of responses was disproportionally skewed towards improved induction of phagocytosis including cancer cell phagocytosis. 1a reprogrammed the immune landscape in genetically engineered murine KPC pancreatic tumors towards increased innate immune surveillance and improved tumor control, and effectively suppressed tumor growth of murine B16 melanoma allografts.

THE COMPARATIVE STUDY OF THE EFFECT OF LOW-INTENSITY BROADBAND AND LOW-INTENSITY PULSED ULTRASOUND ON B16 MELANOMA CELLS IN VITRO

Aim. To evaluate the effect of low-intensity broadband ultrasound (UMUS) and low-intensity pulsed ultrasound (LIPUS) on B16 melanoma cells in vitro. Methods. The research was conducted on B16 melanoma cells under two modes of low-intensity ultrasonic sounding of cells, for which an SDG 2082 X Siglent generator (USA) with an ultrasonic emitter diameter of 20 mm was used; with a frequency for UMUS of 1-7 MHz, intensity of 30 mW/cm, pulse duration of 50 μs, and for LIPUS with a frequency of 1.5 MHz, intensity of 30 mW/cm, pulse duration of 200 μs. Both modes of cell sounding were carried out with threefold irradiation, once a day after which the distribution of cells by cycle phases, the level of apoptosis and proliferative activity was determined by the method of ductal cytofluorimetry. Results. Under the influence of UMUS ultrasonic sounding, the inhibition of the proliferation of melanoma cells by 1.6 times (P&lt;0.05), a 2-fold decrease in the content of aneuploid cells, compared to the control, was found, however, cytostatic and proapoptotic effects were not recorded. Under the influence of LIPUS sounding, an anti-proliferative effect, an increase in the content of dead and apoptotic cells and a decrease in the DI-index compared to the corresponding control were found. Conclusions. Under the action of low-intensity broadband ultrasound (UMUS) and low-intensity pulsed ultrasound (LIPUS), a cytotoxic/cytostatic and pro-apoptotic effect and a decrease in the content of the aneuploid cell population were revealed.