Protocol for genomic editing in human resting primary NK cells and NK-92 cells via CRISPR-Cas9 ribonucleoproteins.

Here, we present a protocol to perform CRISPR-Cas9 genome editing in human resting primary natural killer (NK) and NK-92 cells. We describe steps for guide RNA selection, ribonucleoprotein (RNP) complex formation, delivery via Nucleofection, and analysis of CRISPR edits to assess editing efficiencies. This protocol offers a tool for functional studies in NK cells, paving the way for potential applications in immunotherapy and beyond. We also discuss limitations such as off-target effects and cell-type-specific considerations.

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.

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.

Establishment of head and neck squamous cell carcinoma mouse models for cetuximab resistance and sensitivity.

Aim: Acquired resistance to the targeted agent cetuximab poses a significant challenge in finding effective anti-cancer treatments for head and neck squamous cell carcinoma (HNSCC). To accurately study novel combination treatments, suitable preclinical mouse models for cetuximab resistance are key yet currently limited. This study aimed to optimize an acquired cetuximab-resistant mouse model, with preservation of the innate immunity, ensuring intact antibody-dependent cellular cytotoxicity (ADCC) functionality. Methods: Cetuximab-sensitive and acquired-resistant HNSCC cell lines, generated in vitro, were subcutaneously engrafted in Rag2 knock-out (KO), BALB/c Nude and CB17 Scid mice with/without Matrigel or Geltrex. Once tumor growth was established, mice were intraperitoneally injected twice a week with cetuximab for a maximum of 3 weeks. In addition, immunohistochemistry was used to evaluate the tumor and its microenvironment. Results: Despite several adjustments in cell number, cell lines and the addition of Matrigel, Rag2 KO and BALB/C Nude mice proved to be unsuitable for xenografting our HNSCC cell lines. Durable tumor growth of resistant SC263-R cells could be induced in CB17 Scid mice. However, these cells had lost their resistance phenotype in vivo. Immunohistochemistry revealed a high infiltration of macrophages in cetuximab-treated SC263-R tumors. FaDu-S and FaDu-R cells successfully engrafted into CB17 Scid mice and maintained their sensitivity/resistance to cetuximab. Conclusion: We have established in vivo HNSCC mouse models with intact ADCC functionality for cetuximab resistance and sensitivity using the FaDu-R and FaDu-S cell lines, respectively. These models serve as valuable tools for investigating cetuximab resistance mechanisms and exploring novel drug combination strategies.

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.

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.

Auranofin and Cold Atmospheric Plasma Synergize to Trigger Distinct Cell Death Mechanisms and Immunogenic Responses in Glioblastoma.

Targeting the redox balance of malignant cells via the delivery of high oxidative stress unlocks a potential therapeutic strategy against glioblastoma (GBM). We investigated a novel reactive oxygen species (ROS)-inducing combination treatment strategy, by increasing exogenous ROS via cold atmospheric plasma and inhibiting the endogenous protective antioxidant system via auranofin (AF), a thioredoxin reductase 1 (TrxR) inhibitor. The sequential combination treatment of AF and cold atmospheric plasma-treated PBS (pPBS), or AF and direct plasma application, resulted in a synergistic response in 2D and 3D GBM cell cultures, respectively. Differences in the baseline protein levels related to the antioxidant systems explained the cell-line-dependent sensitivity towards the combination treatment. The highest decrease of TrxR activity and GSH levels was observed after combination treatment of AF and pPBS when compared to AF and pPBS monotherapies. This combination also led to the highest accumulation of intracellular ROS. We confirmed a ROS-mediated response to the combination of AF and pPBS, which was able to induce distinct cell death mechanisms. On the one hand, an increase in caspase-3/7 activity, with an increase in the proportion of annexin V positive cells, indicates the induction of apoptosis in the GBM cells. On the other hand, lipid peroxidation and inhibition of cell death through an iron chelator suggest the involvement of ferroptosis in the GBM cell lines. Both cell death mechanisms induced by the combination of AF and pPBS resulted in a significant increase in danger signals (ecto-calreticulin, ATP and HMGB1) and dendritic cell maturation, indicating a potential increase in immunogenicity, although the phagocytotic capacity of dendritic cells was inhibited by AF. In vivo, sequential combination treatment of AF and cold atmospheric plasma both reduced tumor growth kinetics and prolonged survival in GBM-bearing mice. Thus, our study provides a novel therapeutic strategy for GBM to enhance the efficacy of oxidative stress-inducing therapy through a combination of AF and cold atmospheric plasma.