Chromosome instability, a hallmark of lung cancer, is a driving mechanism for hexavalent chromium Cr(VI) carcinogenesis in humans. Cr(VI) induces structural and numerical chromosome instability in ...human lung cells by inducing DNA double-strand breaks and inhibiting homologous recombination repair and causing spindle assembly checkpoint (SAC) bypass and centrosome amplification. Great whales are long-lived species with long-term exposures to Cr(VI) and accumulate Cr in their tissue, but exhibit a low incidence of cancer. Data show Cr(VI) induces fewer chromosome aberrations in whale cells after acute Cr(VI) exposure suggesting whale cells can evade Cr(VI)-induced chromosome instability. However, it is unknown if whales can evade Cr(VI)-induced chromosome instability. Thus, we tested the hypothesis that whale cells resist Cr(VI)-induced loss of homologous recombination repair activity and increased SAC bypass and centrosome amplification. We found Cr(VI) induces similar amounts of DNA double-strand breaks after acute (24 h) and prolonged (120 h) exposures in whale lung cells, but does not inhibit homologous recombination repair, SAC bypass, or centrosome amplification, and does not induce chromosome instability. These data indicate whale lung cells resist Cr(VI)-induced chromosome instability, the major driver for Cr(VI) carcinogenesis at a cellular level, consistent with observations that whales are resistant to cancer.
Cancer immunotherapy is a highly successful and rapidly evolving treatment modality that works by augmenting the body's own immune system. While various immune stimulation strategies such as ...PD-1/PD-L1 or CTLA-4 checkpoint blockade result in robust responses, even in patients with advanced cancers, the overall response rate is low. While immune checkpoint inhibitors are known to enhance cytotoxic T cells' antitumor response, current evidence suggests that immune responses independent of cytotoxic T cells, such as Natural Killer (NK) cells, play crucial role in the efficacy of immunotherapeutic interventions. NK cells hold a distinct role in potentiating the innate immune response and activating the adaptive immune system. This review highlights the importance of the early actions of the NK cell response and the pivotal role NK cells hold in priming the immune system and setting the stage for successful response to cancer immunotherapy. Yet, in many patients the NK cell compartment is compromised thus lowering the chances of successful outcomes of many immunotherapies. An overview of mechanisms that can drive NK cell dysfunction and hinder immunotherapy success is provided. Rather than relying on the likely dysfunctional endogenous NK cells to work with immunotherapies, adoptive allogeneic NK cell therapies provide a viable solution to increase response to immunotherapies. This review highlights the advances made in development of NK cell therapeutics for clinical application with evidence supporting their combinatorial application with other immune-oncology approaches to improve outcomes of immunotherapies.
Abstract
Lung cancer is the leading cause of cancer death; however, the mechanisms of lung carcinogens are poorly understood. Metals, including hexavalent chromium Cr(VI), induce chromosome ...instability, an early event in lung cancer. Failure of homologous recombination repair is a key mechanism for chromosome instability. Particulate Cr(VI) causes DNA double-strand breaks and prolonged exposure impairs homologous recombination targeting a key effector protein in this pathway, RAD51. Reduced RAD51 protein is a key endpoint of particulate Cr(VI) exposure. It is currently unknown how Cr(VI) reduces RAD51 protein. E2F1 is the predominant transcription factor for RAD51. This study sought to identify if E2F1 modulates the RAD51 response to particulate Cr(VI). Particulate Cr(VI) reduced RAD51 protein and mRNA levels but had a minimal effect on RAD51 half-life. E2F1 protein and mRNA were also inhibited by particulate Cr(VI) exposure. To connect these two outcomes, we tested if modulating E2F1 affects RAD51 outcomes after particulate Cr(VI) exposure. E2F1 knockdown inhibited RAD51 nuclear foci formation after acute particulate Cr(VI) exposure. These data indicate reduced RAD51 protein levels after prolonged particulate Cr(VI) exposure are predominantly due to inhibited expression. Particulate Cr(VI) also inhibits E2F1 expression. However, although loss of E2F1 does not modulate RAD51 expression after particulate Cr(VI) exposure, RAD51 nuclear foci formation is inhibited. These findings suggest E2F1 is important for RAD51 localization to double-strand breaks, but not expression after particulate Cr(VI) exposure in human lung cells.
There is a great interest in developing natural killer (NK) cells as adoptive cancer immunotherapy. For off-the-shelf approaches and to conduct multicenter clinical trials, cryopreserved NK cells are ...the preferred product. However, recent studies reported that cryopreservation of NK cells results in loss of cell motility and, as a consequence, cytotoxicity which limits the clinical utility of such products. This study assessed the impact of cryopreservation on the recovery and function of PM21-particle expanded NK cells (PM21-NK cells) as well as their antitumor activity
using 2D and 3D cancer models and
in ovarian cancer models, including patient-derived xenografts (PDX). Viable PM21-NK cells were consistently recovered from cryopreservation and overnight rest with a mean recovery of 73 ± 22% (N = 19). Thawed and rested NK cells maintained the expression of activating receptors when compared to expansion-matched fresh NK cells. Cryopreserved NK cells that were thawed and rested showed no decrease in cytotoxicity when co-incubated with tumor cells at varying effector-to-target (NK:T) ratios compared to expansion-matched fresh NK cells. Moreover, no differences in cytotoxicity were observed between expansion-matched cryopreserved and fresh NK cells in 3D models of tumor killing. These were analyzed by kinetic, live-cell imaging assays co-incubating NK cells with tumor spheroids. When exposed to tumor cells, or upon cytokine stimulation, cryopreserved NK cells that were thawed and rested showed no significant differences in surface expression of degranulation marker CD107a or intracellular expression of TNFα and IFNγ.
antitumor activity was also assessed by measuring the extension of survival of SKOV-3-bearing NSG mice treated with fresh
cryopreserved NK cells. Cryopreserved NK cells caused a statistically significant survival extension of SKOV-3-bearing NSG mice that was comparable to that observed with fresh NK cells. Additionally, treatment of NSG mice bearing PDX tumor with cryopreserved PM21-NK cells resulted in nearly doubling of survival compared to untreated mice. These data suggest that PM21-NK cells can be cryopreserved and recovered efficiently without appreciable loss of viability or activity while retaining effector function both
and
. These findings support the use of cryopreserved PM21-NK cells as a cancer immunotherapy treatment.
Evaluating health risks of environmental contaminants can be better achieved by considering toxic impacts across species. Hexavalent chromium Cr(VI) is a marine pollutant and global environmental ...contaminant. While Cr(VI) has been identified as a human lung carcinogen, health effects in marine species are poorly understood. Little is known about how Cr(VI) might impact humans and marine species differently. This study used a One Environmental Health Approach to compare the cytotoxicity and genotoxicity of particulate Cr(VI) in human and leatherback sea turtle (Dermochelys coriacea) lung fibroblasts. Leatherbacks may experience prolonged exposures to environmental contaminants and provide insight to how environmental exposures affect health across species. Since humans and leatherbacks may experience prolonged exposure to Cr(VI), and prolonged Cr(VI) exposure leads to carcinogenesis in humans, in this study we considered both acute and prolonged exposures. We found particulate Cr(VI) induced cytotoxicity in leatherback cells comparable to human cell data supporting current research that shows Cr(VI) impacts health across species. To better understand mechanisms of Cr(VI) toxicity we assessed the genotoxic effects of particulate Cr(VI) in human and leatherback cells. Particulate Cr(VI) induced similar genotoxicity in both cell lines, however, human cells arrested at lower concentrations than leatherback cells. We also measured intracellular Cr ion concentrations and found after prolonged exposure human cells accumulated more Cr than leatherback cells. These data indicate Cr(VI) is a health concern for humans and leatherbacks. The data also suggest humans and leatherbacks respond to chemical exposure differently, possibly leading to the discovery of species-specific protective mechanisms.
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•Cr(VI) is cytotoxic and genotoxic to leatherback sea turtle lung cells.•Leatherback and human cells respond differently to Cr(VI).•Cr(VI) exposure is a health concern for humans and leatherback sea turtles.
NK cell therapeutics have gained significant attention as a potential cancer treatment. Towards therapeutic use, NK cells need to be activated and expanded to attain high potency and large quantities ...for an effective dosage. This is typically done by ex vivo stimulation with cytokines to enhance functionality or expansion for 10-14 days to increase both their activity and quantity. Attaining a robust methodology to produce large doses of potent NK cells for an off-the-shelf product is highly desirable. Notably, past reports have shown that stimulating NK cells with IL-12, IL-15, and IL-18 endows them with memory-like properties, better anti-tumor activity, and persistence. While this approach produces NK cells with clinically favorable characteristics supported by encouraging early results for the treatment of hematological malignancies, its limited scalability, variability in initial doses, and the necessity for patient-specific production hinder its broader application. In this study, stimulation of NK cells with PM21-particles derived from K562-41BBL-mbIL21 cells was combined with memory-like induction using cytokines IL-12, IL-15, and IL-18 to produce NK cells with enhanced anti-tumor function. The use of cytokines combined with PM21-particles (cytokine and particle, CAP) significantly enhanced NK cell expansion, achieving a remarkable 8,200-fold in 14 days. Mechanistically, this significant improvement over expansion with PM21-particles alone was due to the upregulation of receptors for key stimulating ligands (4-1BBL and IL-2), resulting in a synergy that drives substantial NK cell growth, showcasing the potential for more effective therapeutic applications. The therapeutic potential of CAP-NK cells was demonstrated by the enhanced metabolic fitness, persistence, and anti-tumor function both
and
. Finally, CAP-NK cells were amenable to current technologies used in developing therapeutic NK cell products, including CRISPR/Cas9-based techniques to generate a triple-gene knockout or a gene knock-in. Taken together, these data demonstrate that the addition of cytokines enhanced the already effective method of ex vivo generation of therapeutic NK cells with PM21-particles, yielding a superior NK cell product for manufacturing efficiency and potential therapeutic applications.
Treatments targeting TIGIT have gained a lot of attention due to strong preclinical and early clinical results, particularly with anti-PD-(L)1 therapeutics. However, this combination has failed to ...meet progression-free survival endpoints in phase III trials. Most of our understanding of TIGIT comes from studies of T cell function. Yet, this inhibitory receptor is often upregulated to the same, or higher, extent on NK cells in cancers. Studies in murine models have demonstrated that TIGIT inhibits NK cells and promotes exhaustion, with its effects on tumor control also being dependent on NK cells. However, there are limited studies assessing the role of TIGIT on the function of human NK cells (hNK), particularly in lung cancer. Most studies used NK cell lines or tested TIGIT blockade to reactivate exhausted cells obtained from cancer patients. For therapeutic advancement, a better understanding of TIGIT in the context of activated hNK cells is crucial, which is different than exhausted NK cells, and critical in the context of adoptive NK cell therapeutics that may be combined with TIGIT blockade. In this study, the effect of TIGIT blockade on the anti-tumor activities of human ex vivo-expanded NK cells was evaluated in vitro in the context of lung cancer. TIGIT expression was higher on activated and/or expanded NK cells compared to resting NK cells. More TIGIT
NK cells expressed major activating receptors and exerted anti-tumor response as compared to TIGIT
cells, indicating that NK cells with greater anti-tumor function express more TIGIT. However, long-term TIGIT engagement upon exposure to PVR
tumors downregulated the cytotoxic function of expanded NK cells while the inclusion of TIGIT blockade increased cytotoxicity, restored the effector functions against PVR-positive targets, and upregulated immune inflammation-related gene sets. These combined results indicate that TIGIT blockade can preserve the activation state of NK cells during exposure to PVR
tumors. These results support the notion that a functional NK cell compartment is critical for anti-tumor response and anti-TIGIT/adoptive NK cell combinations have the potential to improve outcomes.
Hexavalent chromium Cr(VI) is a human lung carcinogen with widespread exposure risks. Cr(VI) causes DNA double strand breaks that if unrepaired, progress into chromosomal instability (CIN), a key ...driving outcome in Cr(VI)-induced tumors. The ability of Cr(VI) to cause DNA breaks and inhibit repair is poorly understood in human lung epithelial cells, which are extremely relevant since pathology data show Cr(VI)-induced tumors originate from bronchial epithelial cells. In the present study, we considered immortalized and primary human bronchial epithelial cells. Cells were treated with zinc chromate at concentrations ranging 0.05 to 0.4μg/cm2 for acute (24 h) and prolonged (120 h) exposures. DNA double strand breaks (DSBs) were measured by neutral comet assay and the status of homologous recombination repair, the main pathway to fix Cr(VI)-induced DSBs, was measured by RAD51 foci formation with immunofluorescence, RAD51 localization with confocal microscopy and sister chromatid exchanges. We found acute and prolonged Cr(VI) exposure induced DSBs. Acute exposure induced homologous recombination repair, but prolonged exposure inhibited it resulting in chromosome instability in immortalized and primary human bronchial epithelial cells.
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•Cr(VI) is genotoxic to primary and immortalized human bronchial epithelial cells.•Acute Cr(VI) induces DNA breaks and their repair in human lung epithelial cells.•Prolonged Cr(VI) exposure induces DNA breaks in human bronchial epithelial cells.•Prolonged Cr(VI) exposure inhibits repair in human bronchial epithelial cells.•Particulate Cr(VI) induces chromosome instability in human bronchial epithelial cells.
Abstract Hexavalent chromium Cr(VI) is an established human lung carcinogen, but the carcinogenesis mechanism is poorly understood. Chromosome instability, a hallmark of lung cancer, is considered a ...major driver of Cr(VI)-induced lung cancer. Unrepaired DNA double-strand breaks are the underlying cause, and homologous recombination repair is the primary mechanism preventing Cr(VI)-induced DNA breaks from causing chromosome instability. Cell culture studies show acute Cr(VI) exposure causes DNA double-strand breaks and increases homologous recombination repair activity. However, the ability of Cr(VI)-induced DNA breaks and repair impact has only been reported in cell culture studies. Therefore, we investigated whether acute Cr(VI) exposure could induce breaks and homologous recombination repair in rat lungs. Male and female Wistar rats were acutely exposed to either zinc chromate particles in a saline solution or saline alone by oropharyngeal aspiration. This exposure route resulted in increased Cr levels in each lobe of the lung. We found Cr(VI) induced DNA double-strand breaks in a concentration-dependent manner, with females being more susceptible than males, and induced homologous recombination repair at similar levels in both sexes. Thus, these data show this driving mechanism discovered in cell culture indeed translates to lung tissue in vivo.
Hexavalent chromium Cr(VI) is a well-known and widespread environmental contaminant associated with a variety of adverse health effects, in particular lung cancer. The primary route of exposure in ...humans is through inhalation. Particulate forms of Cr(VI) are the most potent but in vivo studies are difficult. Intratracheal instillation requires highly trained surgical procedures which also limits the number of repeated exposures possible and thus requires high doses. Inhalation studies can deliver lower more chronic doses but are expensive and generate dangerous aerosols. We evaluated an oropharyngeal aspiration exposure route for zinc chromate particles in Wistar rats. Animals were treated once per week for 90 days. We found chromium accumulated in the lungs, blood, and reproductive tissues of all treated animals. Additionally, we found inflammatory indicators in the lung were elevated and circulating lymphocytes had increased chromosomal damage. These results show oropharyngeal aspiration provides a practicable exposure route for chronic and sub-chronic exposures of Cr(VI) particles.
•Oropharyngeal aspiration of zinc chromate particles leads to increased chromium levels in multiple tissues.•Sub-chronic exposure of zinc chromate particles in rats increased macrophage infiltration in the lungs.•Sub-chronic exposure of zinc chromate particles in rats increased chromosome instability in lymphocytes.
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