Lack of functional TCR-epitope interaction is associated with herpes zoster through reduced downstream T cell activation.

The role of T cell receptor (TCR) diversity in infectious disease susceptibility is not well understood. We use a systems immunology approach on three cohorts of herpes zoster (HZ) patients and controls to investigate whether TCR diversity against varicella-zoster virus (VZV) influences the risk of HZ. We show that CD4+ T cell TCR diversity against VZV glycoprotein E (gE) and immediate early 63 protein (IE63) after 1-week culture is more restricted in HZ patients. Single-cell RNA and TCR sequencing of VZV-specific T cells shows that T cell activation pathways are significantly decreased after stimulation with VZV peptides in convalescent HZ patients. TCR clustering indicates that TCRs from HZ patients co-cluster more often together than TCRs from controls. Collectively, our results suggest that not only lower VZV-specific TCR diversity but also reduced functional TCR affinity for VZV-specific proteins in HZ patients leads to lower T cell activation and consequently affects the susceptibility for viral reactivation.

Auranofin repurposing for lung and pancreatic cancer: low CA12 expression as a marker of sensitivity in patient-derived organoids, with potentiated efficacy by AKT inhibition.

BACKGROUND: This study explores the repurposing of Auranofin (AF), an anti-rheumatic drug, for treating non-small cell lung cancer (NSCLC) adenocarcinoma and pancreatic ductal adenocarcinoma (PDAC). Drug repurposing in oncology offers a cost-effective and time-efficient approach to developing new cancer therapies. Our research focuses on evaluating AF's selective cytotoxicity against cancer cells, identifying RNAseq-based biomarkers to predict AF response, and finding the most effective co-therapeutic agents for combination with AF. METHODS: Our investigation employed a comprehensive drug screening of AF in combination with eleven anticancer agents in cancerous PDAC and NSCLC patient-derived organoids (n = 7), and non-cancerous pulmonary organoids (n = 2). Additionally, we conducted RNA sequencing to identify potential biomarkers for AF sensitivity and experimented with various drug combinations to optimize AF's therapeutic efficacy. RESULTS: The results revealed that AF demonstrates a preferential cytotoxic effect on NSCLC and PDAC cancer cells at clinically relevant concentrations below 1 µM, sparing normal epithelial cells. We identified Carbonic Anhydrase 12 (CA12) as a significant RNAseq-based biomarker, closely associated with the NF-κB survival signaling pathway, which is crucial in cancer cell response to oxidative stress. Our findings suggest that cancer cells with low CA12 expression are more susceptible to AF treatment. Furthermore, the combination of AF with the AKT inhibitor MK2206 was found to be particularly effective, exhibiting potent and selective cytotoxic synergy, especially in tumor organoid models classified as intermediate responders to AF, without adverse effects on healthy organoids. CONCLUSION: Our research offers valuable insights into the use of AF for treating NSCLC and PDAC. It highlights AF's cancer cell selectivity, establishes CA12 as a predictive biomarker for AF sensitivity, and underscores the enhanced efficacy of AF when combined with MK2206 and other therapeutics. These findings pave the way for further exploration of AF in cancer treatment, particularly in identifying patient populations most likely to benefit from its use and in optimizing combination therapies for improved patient outcomes.

Training of epitope-TCR prediction models with healthy donor-derived cancer-specific T cells.

Discovery of epitope-specific T-cell receptors (TCRs) for cancer therapies is a time consuming and expensive procedure that usually requires a large amount of patient cells. To maximize information from and minimize the need of precious samples in cancer research, prediction models have been developed to identify in silico epitope-specific TCRs. In this chapter, we provide a step-by-step protocol to train a prediction model using the user-friendly TCRex webtool for the nearly universal tumor-associated antigen Wilms' tumor 1 (WT1)-specific TCR repertoire. WT1 is a self-antigen overexpressed in numerous solid and hematological malignancies with a high clinical relevance. Training of computational models starts from a list of known epitope-specific TCRs which is often not available for new cancer epitopes. Therefore, we describe a workflow to assemble a training data set consisting of TCR sequences obtained from WT137-45-reactive CD8 T cell clones expanded and sorted from healthy donor peripheral blood mononuclear cells.

IL-15-secreting CAR natural killer cells directed toward the pan-cancer target CD70 eliminate both cancer cells and cancer-associated fibroblasts.

BACKGROUND: It remains challenging to obtain positive outcomes with chimeric antigen receptor (CAR)-engineered cell therapies in solid malignancies, like colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC). A major obstacle is the lack of targetable surface antigens that are not shared by healthy tissues. CD70 emerges as interesting target, due to its stringent expression pattern in healthy tissue and its apparent role in tumor progression in a considerable amount of malignancies. Moreover, CD70 is also expressed on cancer-associated fibroblasts (CAFs), another roadblock for treatment efficacy in CRC and PDAC. We explored the therapeutic potential of CD70 as target for CAR natural killer (NK) cell therapy in CRC, PDAC, focusing on tumor cells and CAFs, and lymphoma. METHODS: RNA-seq data and immunohistochemical analysis of patient samples were used to explore CD70 expression in CRC and PDAC patients. In addition, CD70-targeting CAR NK cells were developed to assess cytotoxic activity against CD70+ tumor cells and CAFs, and the effect of cytokine stimulation on their efficacy was evaluated. The in vitro functionality of CD70-CAR NK cells was investigated against a panel of tumor and CAF cell lines with varying CD70 expression. Lymphoma-bearing mice were used to validate in vivo potency of CD70-CAR NK cells. Lastly, to consider patient variability, CD70-CAR NK cells were tested on patient-derived organoids containing CAFs. RESULTS: In this study, we identified CD70 as a target for tumor cells and CAFs in CRC and PDAC patients. Functional evaluation of CD70-directed CAR NK cells indicated that IL-15 stimulation is essential to obtain effective elimination of CD70+ tumor cells and CAFs, and to improve tumor burden and survival of mice bearing CD70+ tumors. Mechanistically, IL-15 stimulation resulted in improved potency of CD70-CAR NK cells by upregulating CAR expression and increasing secretion of pro-inflammatory cytokines, in a mainly autocrine or intracellular manner. CONCLUSIONS: We disclose CD70 as an attractive target both in hematological and solid tumors. IL-15 armored CAR NK cells act as potent effectors to eliminate these CD70+ cells. They can target both tumor cells and CAFs in patients with CRC and PDAC, and potentially other desmoplastic solid tumors.

Single-organoid analysis reveals clinically relevant treatment-resistant and invasive subclones in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases, characterized by a treatment-resistant and invasive nature. In line with these inherent aggressive characteristics, only a subset of patients shows a clinical response to the standard of care therapies, thereby highlighting the need for a more personalized treatment approach. In this study, we comprehensively unraveled the intra-patient response heterogeneity and intrinsic aggressive nature of PDAC on bulk and single-organoid resolution. We leveraged a fully characterized PDAC organoid panel (N = 8) and matched our artificial intelligence-driven, live-cell organoid image analysis with retrospective clinical patient response. In line with the clinical outcomes, we identified patient-specific sensitivities to the standard of care therapies (gemcitabine-paclitaxel and FOLFIRINOX) using a growth rate-based and normalized drug response metric. Moreover, the single-organoid analysis was able to detect resistant as well as invasive PDAC organoid clones, which was orchestrates on a patient, therapy, drug, concentration and time-specific level. Furthermore, our in vitro organoid analysis indicated a correlation with the matched patient progression-free survival (PFS) compared to the current, conventional drug response readouts. This work not only provides valuable insights on the response complexity in PDAC, but it also highlights the potential applications (extendable to other tumor types) and clinical translatability of our approach in drug discovery and the emerging era of personalized medicine.

PD-1, PD-L1, IDO, CD70 and microsatellite instability as potential targets to prevent immune evasion in sarcomas.

Background: Soft tissue and bone sarcomas are rare entities, hence, standardized therapeutic strategies are difficult to assess. Materials & methods: Immunohistochemistry was performed on 68 sarcoma samples to assess the expression of PD-1, PD-L1, IDO and CD70 in different tumor compartments and molecular analysis was performed to assess microsatellite instability status. Results: PD-1/PD-L1, IDO and CD70 pathways are at play in the immune evasion of sarcomas in general. Soft tissue sarcomas more often show an inflamed phenotype compared with bone sarcomas. Specific histologic sarcoma types show high expression levels of different markers. Finally, this is the first presentation of a microsatellite instability-high Kaposi sarcoma. Discussion/conclusion: Immune evasion occurs in sarcomas. Specific histologic types might benefit from immunotherapy, for which further investigation is needed.

Sarcomas of the soft tissue and bone are rare cancers. When these cancers spread to other parts of the body, it is hard to find good treatments. Recently, doctors have been using a new type of treatment called immunotherapy to fight several types of cancer. Immunotherapy works by getting one's body's own defense cells to attack the cancer cells. Unfortunately, immunotherapy does not work well for sarcomas and we do not know why. This study was designed to determine if there are certain mechanisms in these tumors that help the cancer cells to hide from defense cells. Determining how to change these mechanisms could make immunotherapy a better treatment for sarcomas in the future.

Trial watch: chemotherapy-induced immunogenic cell death in oncology.

Immunogenic cell death (ICD) refers to an immunologically distinct process of regulated cell death that activates, rather than suppresses, innate and adaptive immune responses. Such responses culminate into T cell-driven immunity against antigens derived from dying cancer cells. The potency of ICD is dependent on the immunogenicity of dying cells as defined by the antigenicity of these cells and their ability to expose immunostimulatory molecules like damage-associated molecular patterns (DAMPs) and cytokines like type I interferons (IFNs). Moreover, it is crucial that the host's immune system can adequately detect the antigenicity and adjuvanticity of these dying cells. Over the years, several well-known chemotherapies have been validated as potent ICD inducers, including (but not limited to) anthracyclines, paclitaxels, and oxaliplatin. Such ICD-inducing chemotherapeutic drugs can serve as important combinatorial partners for anti-cancer immunotherapies against highly immuno-resistant tumors. In this Trial Watch, we describe current trends in the preclinical and clinical integration of ICD-inducing chemotherapy in the existing immuno-oncological paradigms.

Site-specific 68Ga-labeled nanobody for PET imaging of CD70 expression in preclinical tumor models.

BACKGROUND: CD70-CD27 is a costimulatory ligand-receptor pair in the tumor necrosis factor receptor family. With only limited expression in normal tissues, CD70 is constitutively expressed in a variety of solid tumors and hematologic malignancies, facilitating immunosuppression through CD27 signaling in the tumor microenvironment by enhanced survival of regulatory T cells, induction of T cell apoptosis, and T cell exhaustion. Consequently, CD70 is an increasingly recognized target for developing antibody-based therapies, but its expression patterns vary among different tumor types in spatial distribution, magnitude of expression and percentage of positive cells. In that regard, individual confirmation of CD70 expression at screening and during treatment could enhance the successful implementation of anti-CD70 therapies. Here, we developed a gallium-68 (68Ga) radiolabeled single-domain antibody-fragment targeting CD70 for in vivo positron emission tomography (PET) imaging. RESULTS: An anti-CD70 VHH construct containing a C-direct-tag with a free thiol was developed to enable site-specific conjugation to a NOTA bifunctional chelator for 68Ga radiolabeling. [68Ga]Ga-NOTA-anti-CD70 VHH was obtained in good radiochemical yield of 30.4 ± 1.7% and high radiochemical purity (> 94%). The radiolabeled VHH showed excellent in vitro and in vivo stability. Specific binding of [68Ga]Ga-NOTA-anti-CD70 VHH was observed on CD70high 786-O cells, showing significantly higher cell-associated activity when compared to the blocking condition (p < 0.0001) and CD70low NCl-H1975 cells (p < 0.0001). PET imaging showed specific radiotracer accumulation in CD70 expressing human tumor xenografts, which was efficiently blocked by prior injection of unlabeled anti-CD70 VHH (p = 0.0029). In addition, radiotracer uptake in CD70high tumors was significantly higher when compared with CD70low tumors (p < 0.0001). The distribution of the radioactivity in the tumors using autoradiography was spatially matched with immunohistochemistry analysis of CD70 expression. CONCLUSION: [68Ga]Ga-NOTA-anti-CD70 VHH showed excellent in vivo targeting of CD70 in human cancer xenografts. PET imaging using this radioimmunoconjugate holds promise as a non-invasive method to identify and longitudinally follow-up patients who will benefit most from anti-CD70 therapies.

Inactivation of SARS-CoV-2 and Other Enveloped and Non-Enveloped Viruses with Non-Thermal Plasma for Hospital Disinfection.

As recently highlighted by the SARS-CoV-2 pandemic, viruses have become an increasing burden for health, global economy, and environment. The control of transmission by contact with contaminated materials represents a major challenge, particularly in hospital environments. However, the current disinfection methods in hospital settings suffer from numerous drawbacks. As a result, several medical supplies that cannot be properly disinfected are not reused, leading to severe shortages and increasing amounts of waste, thus prompting the search for alternative solutions. In this work, we report that non-thermal plasma (NTP) can effectively inactivate SARS-CoV-2 from non-porous and porous materials commonly found in healthcare facilities. We demonstrated that 5 min treatment with a dielectric barrier discharge NTP can inactivate 100% of SARS-CoV-2 (Wuhan and Omicron strains) from plastic material. Using porcine respiratory coronavirus (surrogate for SARS-CoV-2) and coxsackievirus B3 (highly resistant non-enveloped virus), we tested the NTP virucidal activity on hospital materials and obtained complete inactivation after 5 and 10 min, respectively. We hypothesize that the produced reactive species and local acidification contribute to the overall virucidal effect of NTP. Our results demonstrate the potential of dielectric barrier discharge NTPs for the rapid, efficient, and low-cost disinfection of healthcare materials.

Auranofin Synergizes with the PARP Inhibitor Olaparib to Induce ROS-Mediated Cell Death in Mutant p53 Cancers.

Auranofin (AF) is a potent, off-patent thioredoxin reductase (TrxR) inhibitor that efficiently targets cancer via reactive oxygen species (ROS)- and DNA damage-mediated cell death. The goal of this study is to enhance the efficacy of AF as a cancer treatment by combining it with the poly(ADP-ribose) polymerase-1 (PARP) inhibitor olaparib (referred to as 'aurola'). Firstly, we investigated whether mutant p53 can sensitize non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) cancer cells to AF and olaparib treatment in p53 knock-in and knock-out models with varying p53 protein expression levels. Secondly, we determined the therapeutic range for synergistic cytotoxicity between AF and olaparib and elucidated the underlying molecular cell death mechanisms. Lastly, we evaluated the effectiveness of the combination strategy in a murine 344SQ 3D spheroid and syngeneic in vivo lung cancer model. We demonstrated that high concentrations of AF and olaparib synergistically induced cytotoxicity in NSCLC and PDAC cell lines with low levels of mutant p53 protein that were initially more resistant to AF. The aurola combination also led to the highest accumulation of ROS, which resulted in ROS-dependent cytotoxicity of mutant p53 NSCLC cells through distinct types of cell death, including caspase-3/7-dependent apoptosis, inhibited by Z-VAD-FMK, and lipid peroxidation-dependent ferroptosis, inhibited by ferrostatin-1 and alpha-tocopherol. High concentrations of both compounds were also needed to obtain a synergistic cytotoxic effect in 3D spheroids of the murine lung adenocarcinoma cell line 344SQ, which was interestingly absent in 2D. This cell line was used in a syngeneic mouse model in which the oral administration of aurola significantly delayed the growth of mutant p53 344SQ tumors in 129S2/SvPasCrl mice, while either agent alone had no effect. In addition, RNA sequencing results revealed that AF- and aurola-treated 344SQ tumors were negatively enriched for immune-related gene sets, which is in accordance with AF's anti-inflammatory function as an anti-rheumatic drug. Only 344SQ tumors treated with aurola showed the downregulation of genes related to the cell cycle, potentially explaining the growth inhibitory effect of aurola since no apoptosis-related gene sets were enriched. Overall, this novel combination strategy of oxidative stress induction (AF) with PARP inhibition (olaparib) could be a promising treatment for mutant p53 cancers, although high concentrations of both compounds need to be reached to obtain a substantial cytotoxic effect.

Targeting CD70 in combination with chemotherapy to enhance the anti-tumor immune effects in non-small cell lung cancer.

Despite the recent emergence of immune checkpoint inhibitors, clinical outcomes of metastatic NSCLC patients remain poor, pointing out the unmet need to develop novel therapies to enhance the anti-tumor immune response in NSCLC. In this regard, aberrant expression of the immune checkpoint molecule CD70 has been reported on many cancer types, including NSCLC. In this study, the cytotoxic and immune stimulatory potential of an antibody-based anti-CD70 (aCD70) therapy was explored as single agent and in combination with docetaxel and cisplatin in NSCLC in vitro and in vivo. Anti-CD70 therapy resulted in NK-mediated killing of NSCLC cells and increased production of pro-inflammatory cytokines by NK cells in vitro. The combination of chemotherapy and anti-CD70 therapy further enhanced NSCLC cell killing. Moreover, in vivo findings showed that the sequential treatment of chemo-immunotherapy resulted in a significant improved survival and delayed tumor growth compared to single agents in Lewis Lung carcinoma-bearing mice. The immunogenic potential of the chemotherapeutic regimen was further highlighted by increased numbers of dendritic cells in the tumor-draining lymph nodes in these tumor-bearing mice after treatment. The sequential combination therapy resulted in enhanced intratumoral infiltration of both T and NK cells, as well as an increase in the ratio of CD8+ T cells over Tregs. The superior effect of the sequential combination therapy on survival was further confirmed in a NCI-H1975-bearing humanized IL15-NSG-CD34+ mouse model. These novel preclinical data demonstrate the potential of combining chemotherapy and aCD70 therapy to enhance anti-tumor immune responses in NSCLC patients.

In vitro expansion of Wilms' tumor protein 1 epitope-specific primary T cells from healthy human peripheral blood mononuclear cells.

Wilms' tumor protein 1 (WT1) is a tumor-associated antigen overexpressed in various cancers. As a self-antigen, negative selection reduces the number of WT1-specific T cell receptors (TCRs). Here, we provide a protocol to generate WT137-45-specific TCRs using healthy human peripheral blood mononuclear cells. We describe the expansion of WT1-specific T cell clones by two consecutive in vitro stimulations with autologous WT137-45-pulsed dendritic cells and peripheral blood lymphocytes. We then detail the detection with human leukocyte antigen/WT137-45 tetramers.

Acquired non-thermal plasma resistance mediates a shift towards aerobic glycolysis and ferroptotic cell death in melanoma.

AIMS: To gain insights into the underlying mechanisms of NTP therapy sensitivity and resistance, using the first-ever NTP-resistant cell line derived from sensitive melanoma cells (A375). METHODS: Melanoma cells were exposed to NTP and re-cultured for 12 consecutive weeks before evaluation against the parental control cells. Whole transcriptome sequencing analysis was performed to identify differentially expressed genes and enriched molecular pathways. Glucose uptake, extracellular lactate, media acidification, and mitochondrial respiration was analyzed to determine metabolic changes. Cell death inhibitors were used to assess the NTP-induced cell death mechanisms, and apoptosis and ferroptosis was further validated via Annexin V, Caspase 3/7, and lipid peroxidation analysis. RESULTS: Cells continuously exposed to NTP became 10 times more resistant to NTP compared to the parental cell line of the same passage, based on their half-maximal inhibitory concentration (IC50). Sequencing and metabolic analysis indicated that NTP-resistant cells had a preference towards aerobic glycolysis, while cell death analysis revealed that NTP-resistant cells exhibited less apoptosis but were more vulnerable to lipid peroxidation and ferroptosis. CONCLUSIONS: A preference towards aerobic glycolysis and ferroptotic cell death are key physiological changes in NTP-resistance cells, which opens new avenues for further, in-depth research into other cancer types.

OrBITS: label-free and time-lapse monitoring of patient derived organoids for advanced drug screening.

BACKGROUND: Patient-derived organoids are invaluable for fundamental and translational cancer research and holds great promise for personalized medicine. However, the shortage of available analysis methods, which are often single-time point, severely impede the potential and routine use of organoids for basic research, clinical practise, and pharmaceutical and industrial applications. METHODS: Here, we developed a high-throughput compatible and automated live-cell image analysis software that allows for kinetic monitoring of organoids, named Organoid Brightfield Identification-based Therapy Screening (OrBITS), by combining computer vision with a convolutional network machine learning approach. The OrBITS deep learning analysis approach was validated against current standard assays for kinetic imaging and automated analysis of organoids. A drug screen of standard-of-care lung and pancreatic cancer treatments was also performed with the OrBITS platform and compared to the gold standard, CellTiter-Glo 3D assay. Finally, the optimal parameters and drug response metrics were identified to improve patient stratification. RESULTS: OrBITS allowed for the detection and tracking of organoids in routine extracellular matrix domes, advanced Gri3D®-96 well plates, and high-throughput 384-well microplates, solely based on brightfield imaging. The obtained organoid Count, Mean Area, and Total Area had a strong correlation with the nuclear staining, Hoechst, following pairwise comparison over a broad range of sizes. By incorporating a fluorescent cell death marker, intra-well normalization for organoid death could be achieved, which was tested with a 10-point titration of cisplatin and validated against the current gold standard ATP-assay, CellTiter-Glo 3D. Using this approach with OrBITS, screening of chemotherapeutics and targeted therapies revealed further insight into the mechanistic action of the drugs, a feature not achievable with the CellTiter-Glo 3D assay. Finally, we advise the use of the growth rate-based normalised drug response metric to improve accuracy and consistency of organoid drug response quantification. CONCLUSION: Our findings validate that OrBITS, as a scalable, automated live-cell image analysis software, would facilitate the use of patient-derived organoids for drug development and therapy screening. The developed wet-lab workflow and software also has broad application potential, from providing a launching point for further brightfield-based assay development to be used for fundamental research, to guiding clinical decisions for personalized medicine.

Challenges in neoantigen-directed therapeutics.

A fundamental prerequisite for the efficacy of cancer immunotherapy is the presence of functional, antigen-specific T cells within the tumor. Neoantigen-directed therapy is a promising strategy that aims at targeting the host's immune response against tumor-specific antigens, thereby eradicating cancer cells. Initial forays have been made in clinical environments utilizing vaccines and adoptive cell therapy; however, many challenges lie ahead. We provide an in-depth overview of the current state of the field with an emphasis on in silico neoantigen discovery and the clinical aspects that need to be addressed to unlock the full potential of this therapy.

Humoral and Cellular Immune Responses against SARS-CoV-2 after Third Dose BNT162b2 following Double-Dose Vaccination with BNT162b2 versus ChAdOx1 in Patients with Cancer.

PURPOSE: Patients with cancer display reduced humoral responses after double-dose COVID-19 vaccination, whereas their cellular response is more comparable with that in healthy individuals. Recent studies demonstrated that a third vaccination dose boosts these immune responses, both in healthy people and patients with cancer. Because of the availability of many different COVID-19 vaccines, many people have been boosted with a different vaccine from the one used for double-dose vaccination. Data on such alternative vaccination schedules are scarce. This prospective study compares a third dose of BNT162b2 after double-dose BNT162b2 (homologous) versus ChAdOx1 (heterologous) vaccination in patients with cancer. EXPERIMENTAL DESIGN: A total of 442 subjects (315 patients and 127 healthy) received a third dose of BNT162b2 (230 homologous vs. 212 heterologous). Vaccine-induced adverse events (AE) were captured up to 7 days after vaccination. Humoral immunity was assessed by SARS-CoV-2 anti-S1 IgG antibody levels and SARS-CoV-2 50% neutralization titers (NT50) against Wuhan and BA.1 Omicron strains. Cellular immunity was examined by analyzing CD4+ and CD8+ T-cell responses against SARS-CoV-2-specific S1 and S2 peptides. RESULTS: Local AEs were more common after heterologous boosting. SARS-CoV-2 anti-S1 IgG antibody levels did not differ significantly between homologous and heterologous boosted subjects [GMT 1,755.90 BAU/mL (95% CI, 1,276.95-2,414.48) vs. 1,495.82 BAU/mL (95% CI, 1,131.48-1,977.46)]. However, homologous-boosted subjects show significantly higher NT50 values against BA.1 Omicron. Subjects receiving heterologous boosting demonstrated increased spike-specific CD8+ T cells, including higher IFNγ and TNFα levels. CONCLUSIONS: In patients with cancer who received double-dose ChAdOx1, a third heterologous dose of BNT162b2 was able to close the gap in antibody response.

Optimization of the Solvent and In Vivo Administration Route of Auranofin in a Syngeneic Non-Small Cell Lung Cancer and Glioblastoma Mouse Model.

The antineoplastic activity of the thioredoxin reductase 1 (TrxR) inhibitor, auranofin (AF), has already been investigated in various cancer mouse models as a single drug, or in combination with other molecules. However, there are inconsistencies in the literature on the solvent, dose and administration route of AF treatment in vivo. Therefore, we investigated the solvent and administration route of AF in a syngeneic SB28 glioblastoma (GBM) C57BL/6J and a 344SQ non-small cell lung cancer 129S2/SvPasCrl (129) mouse model. Compared to daily intraperitoneal injections and subcutaneous delivery of AF via osmotic minipumps, oral gavage for 14 days was the most suitable administration route for high doses of AF (10-15 mg/kg) in both mouse models, showing no measurable weight loss or signs of toxicity. A solvent comprising 50% DMSO, 40% PEG300 and 10% ethanol improved the solubility of AF for oral administration in mice. In addition, we confirmed that AF was a potent TrxR inhibitor in SB28 GBM tumors at high doses. Taken together, our results and results in the literature indicate the therapeutic value of AF in several in vivo cancer models, and provide relevant information about AF's optimal administration route and solvent in two syngeneic cancer mouse models.

The prognostic impact of the immune signature in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that retain their poor prognosis despite recent advances in their standard of care. As the involvement of the immune system against HNSCC development is well-recognized, characterization of the immune signature and the complex interplay between HNSCC and the immune system could lead to the identification of novel therapeutic targets that are required now more than ever. In this study, we investigated RNA sequencing data of 530 HNSCC patients from The Cancer Genome Atlas (TCGA) for which the immune composition (CIBERSORT) was defined by the relative fractions of 10 immune-cell types and expression data of 45 immune checkpoint ligands were quantified. This initial investigation was followed by immunohistochemical (IHC) staining for a curated selection of immune cell types and checkpoint ligands markers in tissue samples of 50 advanced stage HNSCC patients. The outcome of both analyses was correlated with clinicopathological parameters and patient overall survival. Our results indicated that HNSCC tumors are in close contact with both cytotoxic and immunosuppressive immune cells. TCGA data showed prognostic relevance of dendritic cells, M2 macrophages and neutrophils, while IHC analysis associated T cells and natural killer cells with better/worse prognostic outcome. HNSCC tumors in our TCGA cohort showed differential RNA over- and underexpression of 28 immune inhibitory and activating checkpoint ligands compared to healthy tissue. Of these, CD73, CD276 and CD155 gene expression were negative prognostic factors, while CD40L, CEACAM1 and Gal-9 expression were associated with significantly better outcomes. Our IHC analyses confirmed the relevance of CD155 and CD276 protein expression, and in addition PD-L1 expression, as independent negative prognostic factors, while HLA-E overexpression was associated with better outcomes. Lastly, the co-presence of both (i) CD155 positive cells with intratumoral NK cells; and (ii) PD-L1 expression with regulatory T cell infiltration may hold prognostic value for these cohorts. Based on our data, we propose that CD155 and CD276 are promising novel targets for HNSCC, possibly in combination with the current standard of care or novel immunotherapies to come.

The pro- and anti-tumoral properties of gap junctions in cancer and their role in therapeutic strategies.

Gap junctions (GJs), essential structures for cell-cell communication, are made of two hemichannels (commonly called connexons), one on each adjacent cell. Found in almost all cells, GJs play a pivotal role in many physiological and cellular processes, and have even been linked to the progression of diseases, such as cancer. Modulation of GJs is under investigation as a therapeutic strategy to kill tumor cells. Furthermore, GJs have also been studied for their key role in activating anti-cancer immunity and propagating radiation- and oxidative stress-induced cell death to neighboring cells, a process known as the bystander effect. While, gap junction (GJ)-based therapeutic strategies are being developed, one major challenge has been the paradoxical role of GJs in both tumor progression and suppression, based on GJ composition, cancer factors, and tumoral context. Therefore, understanding the mechanisms of action, regulation, and the dual characteristics of GJs in cancer is critical for developing effective therapeutics. In this review, we provide an overview of the current understanding of GJs structure, function, and paradoxical pro- and anti-tumoral role in cancer. We also discuss the treatment strategies to target these GJs properties for anti-cancer responses, via modulation of GJ function.

Expression of SARS-CoV-2-Related Surface Proteins in Non-Small-Cell Lung Cancer Patients and the Influence of Standard of Care Therapy.

In this study, we aimed to study the expression of SARS-CoV-2-related surface proteins in non-small-cell lung cancer (NSCLC) cells and identify clinicopathological characteristics that are related to increased membranous (m)ACE2 protein expression and soluble (s)ACE2 levels, with a particular focus on standard of care (SOC) therapies. ACE2 (n = 107), TMPRSS2, and FURIN (n = 38) protein expression was determined by immunohistochemical (IHC) analysis in NSCLC patients. sACE2 levels (n = 64) were determined in the serum of lung cancer patients collected before, during, or after treatment with SOC therapies. Finally, the TCGA lung adenocarcinoma (LUAD) database was consulted to study the expression of ACE2 in EGFR- and KRAS-mutant samples and ACE2 expression was correlated with EGFR/HER, RAS, BRAF, ROS1, ALK, and MET mRNA expression. Membranous (m)ACE2 was found to be co-expressed with mFURIN and/or mTMPRSS2 in 16% of the NSCLC samples and limited to the adenocarcinoma subtype. TMPRSS2 showed predominantly atypical cytoplasmic expression. mACE2 and sACE2 were more frequently expressed in mutant EGFR patients, but not mutant-KRAS patients. A significant difference was observed in sACE2 for patients treated with targeted therapies, but not for chemo- and immunotherapy. In the TCGA LUAD cohort, ACE2 expression was significantly higher in EGFR-mutant patients and significantly lower in KRAS-mutant patients. Finally, ACE2 expression was positively correlated with ERBB2-4 and ROS1 expression and inversely correlated with KRAS, NRAS, HRAS, and MET mRNA expression. We identified a role for EGFR pathway activation in the expression of mACE2 in NSCLC cells, associated with increased sACE2 levels in patients. Therefore, it is of great interest to study SARS-CoV-2-infected EGFR-mutated NSCLC patients in greater depth in order to obtain a better understanding of how mACE2, sACE2, and SOC TKIs can affect the course of COVID-19.