Chimeric Antigen Receptor T Cell Therapy for Pancreatic Cancer: A Review of Current Evidence.

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of malignant and non-malignant disorders. CARs are synthetic transmembrane receptors expressed on genetically modified immune effector cells, including T cells, natural killer (NK) cells, or macrophages, which are able to recognize specific surface antigens on target cells and eliminate them. CAR-modified immune cells mediate cytotoxic antitumor effects via numerous mechanisms, including the perforin and granzyme pathway, Fas and Fas Ligand (FasL) pathway, and cytokine secretion. High hopes are associated with the prospective use of the CAR-T strategy against solid cancers, especially the ones resistant to standard oncological therapies, such as pancreatic cancer (PC). Herein, we summarize the current pre-clinical and clinical studies evaluating potential tumor-associated antigens (TAA), CAR-T cell toxicities, and their efficacy in PC.

Regulatory T cells contribute to the immunosuppressive phenotype of neutrophils in a mouse model of chronic lymphocytic leukemia.

BACKGROUND: Impaired neutrophil activity is an important issue in chronic lymphocytic leukemia (CLL), as it contributes to a dysfunctional immune response leading to life-threatening infections in patients. Some features typical of CLL neutrophils, e.g., the B-cell-supportive secretion profile, have already been described. However, most of these studies were performed on cells isolated from peripheral blood. It is still unclear which molecular factors and cell types are involved in shaping neutrophil function and phenotype in the CLL microenvironment. Since regulatory T cells (Treg) play an important role in CLL progression and influence the activity of neutrophils, we investigated the crosstalk between Treg and neutrophils in the spleen using a murine model of CLL. METHODS: In this work, we used an Eµ-TCL1 mouse model of human CLL. For our in vivo and ex vivo experiments, we inoculated wild-type mice with TCL1 leukemic cells isolated from Eµ-TCL1 transgenic mice and then monitored disease progression by detecting leukemic cells in peripheral blood. We analyzed both the phenotype and activity of neutrophils isolated from the spleens of TCL1 leukemia-bearing mice. To investigate the interrelation between Treg and neutrophils in the leukemia microenvironment, we performed experiments using TCL1-injected DEREG mice with Treg depletion or RAG2KO mice with adoptively transferred TCL1 cells alone or together with Treg. RESULTS: The obtained results underline the plasticity of the neutrophil phenotype, observed under the influence of leukemic cells alone and depending on the presence of Treg. In particular, Treg affect the expression of CD62L and IL-4 receptor in neutrophils, both of which are crucial for the function of these cells. Additionally, we show that Treg depletion and IL-10 neutralization induce changes in the leukemia microenvironment, partially restoring the "healthy" phenotype of neutrophils. CONCLUSIONS: Altogether, the results indicate that the crosstalk between Treg and neutrophils in CLL may play an important role in CLL progression by interfering with the immune response.

Mapping the chemotactic landscape in NK cells reveals subset-specific synergistic migratory responses to dual chemokine receptor ligation.

BACKGROUND: Natural killer (NK) cells have a unique capability of spontaneous cytotoxicity against malignant cells and hold promise for off-the-shelf cell therapy against cancer. One of the key challenges in the field is to improve NK cell homing to solid tumors. METHODS: To gain a deeper understanding of the cellular mechanisms regulating trafficking of NK cells into the tumor, we used high-dimensional flow cytometry, mass cytometry, and single-cell RNA-sequencing combined with functional assays, creating a comprehensive map of human NK cell migration phenotypes. FINDINGS: We found that the chemokine receptor repertoire of peripheral blood NK cells changes in a coordinated manner becoming progressively more diversified during NK cell differentiation and correlating tightly with the migratory response of the distinct NK cell subsets. Simultaneous ligation of CXCR1/2 and CX3CR1, synergistically potentiated the migratory response of NK cells. Analysis of 9471 solid cancers from publicly available TCGA/TARGET repositories revealed dominant chemokine patterns that varied across tumor types but with no tumor group expressing ligands for more than one chemokine receptor present on mature NK cells. INTERPRETATION: The finding that chemokine stimulation can elicit a synergistic migratory response in NK cells combined with the identified lack of naturally occurring pairs of chemokines-chemokine receptors in human cancers may explain the systematic exclusion of NK cells from the tumor microenvironment and provides a basis for engineering next-generation NK cell therapies against malignancies. FUNDING: The Polish Ministry of Science and Higher Education, the National Science Centre, Poland, The Norwegian Cancer Society, the Norwegian Research Council, the South-Eastern Norway Regional Health Authority, The Swedish Cancer Society, the Swedish Children's Cancer Foundation, The Swedish Research Council, The Center of Excellence: Precision Immunotherapy Alliance, Knut and Alice Wallenberg Foundation and National Cancer Institute.

Exclusion of older adults from clinical trials in cancer-related pain.

Pain is one of the most common symptoms in cancer patients including older adults. The objective of this study was to evaluate the enrollment criteria that can limit the inclusion of older adults in clinical trials concerning cancer-related pain (CRP). The study included 356 trials registered with ClinicalTrials.gov. Our primary outcome measures were the proportion of trials that excluded patients based on upper age limits (80 years of age or less), strict organ-specific exclusion criteria, broad and imprecise criteria, and inadequate performance score. One hundred and twenty-six trials (35.4%) had upper age limits. Strict exclusion criteria were used in 95 (26.7%) trials. Broad and imprecise exclusion criteria were listed in 57 (16.2%) trials. Low performance score was used as an exclusion criterion in 4 trials (1.1%). Overall, in 241 trials (67.7%) there was either an upper age limit or at least one strict or broad and imprecise exclusion criterion, or a criterion involving the performance status. The odds of excluding older adults were significantly higher in certain neoplasm types, study objectives, intervention types, and center locations. In conclusion, considerable proportion of recent clinical trials concerning CRP either explicitly exclude older adults or create high risk of such exclusion which substantially limits the evidence base for the treatment of such patients in clinical practice. Sponsors and investigators should consider careful modification of the enrollment criteria to improve the inclusion of older individuals who make up the major proportion of cancer patients population.

PRDX-1 Supports the Survival and Antitumor Activity of Primary and CAR-Modified NK Cells under Oxidative Stress.

Oxidative stress, caused by the imbalance between reactive species generation and the dysfunctional capacity of antioxidant defenses, is one of the characteristic features of cancer. Here, we quantified hydrogen peroxide in the tumor microenvironment (TME) and demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo, when compared with blood or normal subcutaneous fluid. Therefore, we investigated the effects of increased hydrogen peroxide concentration on immune cell functions. NK cells were more susceptible to hydrogen peroxide than T cells or B cells, and by comparing T, B, and NK cells' sensitivities to redox stress and their antioxidant capacities, we identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. We observed that priming with IL15 protected NK cells' functions in the presence of high hydrogen peroxide and simultaneously upregulated PRDX1 expression. However, the effect of IL15 on PRDX1 expression was transient and strictly dependent on the presence of the cytokine. Therefore, we genetically modified NK cells to stably overexpress PRDX1, which led to increased survival and NK cell activity in redox stress conditions. Finally, we generated PD-L1-CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells under oxidative stress. These results demonstrate that hydrogen peroxide, at concentrations detected in the TME, suppresses NK cell function and that genetic modification strategies can improve CAR NK cells' resistance and potency against solid tumors.

Osteopontin Gene Polymorphisms rs1126616 C>T and rs1126772 A>G are Associated with Atopic Dermatitis in Polish Population.

PURPOSE: Atopic dermatitis (AD) is a chronic, relapsing inflammatory disease, caused by environmental and genetic factors, which lead to immunological abnormalities. Osteopontin (OPN), also named secreted phosphoprotein 1 (SPP1), is a protein involved in the pathogenesis of numerous autoimmune and inflammatory conditions. However, its role in AD has not been fully elucidated. Therefore, we aim to gain an insight into the role of OPN in AD pathogenesis through investigating its gene single nucleotide polymorphisms (SNPs) and their possible associations with disease clinical features. PATIENTS AND METHODS: A total of 182 Caucasian participants (45 AD patients and 137 gender- and age-matched controls) were studied. Genomic DNA was isolated from peripheral blood samples. Genotyping for the rs1126616 C>T, rs1126772 A>G, rs9138 A>C, and rs3841116 T>G SNPs was performed by real time polymerase chain reaction (RT-PCR). RESULTS: The frequency of the minor TT genotype and the T allele of rs1126616 C>T was higher in AD patients compared to controls (P = 0.019, OD = 4.86, 95% CI = 1.46-16.20, and P = 0.047, OR = 1.77, 95% CI = 1.04-3.00, respectively) and was associated with the higher prevalence of asthma (P = 0.017, OR = 3.73, 95% CI = 0.71-19.67, and P = 0.004, OR = 3.96, 95% CI = 1.53-10.25, respectively). Likewise, the minor GG genotype and the G allele of rs1126772 A>G were more frequent in AD patients (P = 0.026, OR = 3.27, 95% CI = 1.29-8.33, and P = 0.013, OR = 1.94, 95% CI = 1.18-3.21, respectively) and were associated with the increased incidence of asthma (P = 0.016, OR = 5.06, 95% CI = 1.14-22.49, and P = 0.002, OR = 4.40, 95% CI = 1.71-11.35, respectively). Furthermore, haplotype frequency estimation determined the four-loci haplotype TGCT, as a significant risk factor for AD compared to controls (P = 0.031, OR = 9.48, 95% CI = 1.23-71.91). CONCLUSION: Our results suggest that the variation in the OPN gene might be associated with AD and increased incidence of asthma in Caucasians. Further studies should be conducted to look into the possible role of OPN as a biomarker for AD.

Bioinformatic Analysis Reveals Central Role for Tumor-Infiltrating Immune Cells in Uveal Melanoma Progression.

Tumor-infiltrating immune cells are capable of effective cancer surveillance, and their abundance is linked to better prognosis in numerous tumor types. However, in uveal melanoma (UM), extensive immune infiltrate is associated with poor survival. This study aims to decipher the role of different tumor-infiltrating cell subsets in UM in order to identify potential targets for future immunotherapeutic treatment. We have chosen the TCGA-UVM cohort as a training dataset and GSE22138 as a testing dataset by mining publicly available databases. The abundance of 22 immune cell types was estimated using CIBERSORTx. Then, to determine the significance of tumor-infiltrating cell subsets in UM, we built a multicell type prognostic signature, which was validated in the testing cohort. The created signature was built upon the negative prognostic role of CD8+ T cells and M0 macrophages and the positive role of neutrophils. Based on the created signature score, we divided the patients into low- and high-risk groups. Kaplan-Meier, Cox, and ROC analyses demonstrated superior performance of our risk score compared to either clinical or pathologic characteristics of both cohorts. Further, we found the molecular pathways associated with cancer immunoevasion and metastasis to be enriched in the high-risk group, explaining both the lack of adequate immune surveillance despite increased infiltration of CD8+ T cells as well as the higher metastatic potential. Genes associated with tryptophan metabolism (IDO1 and KYNU) and metalloproteinases were among the most differentially expressed between the high- and low-risk groups. Our correlation analyses interpreted in context of published in vitro data strongly suggest the central role of CD8+ T cells in shifting the UM tumor microenvironment towards suppressive and metastasis-promoting. Therefore, we propose further investigations of IDO1 and metalloproteinases as novel targets for immunotherapy in lymphocyte-rich metastatic UM patients.

NF-κB activation in retinal microglia is involved in the inflammatory and neovascularization signaling in laser-induced choroidal neovascularization in mice.

PURPOSE: To evaluate Nuclear Factor NF-κB (NF-κB) signaling on microglia activation, migration, and angiogenesis in laser-induced choroidal neovascularization (CNV). METHODS: Nine-week-old C57BL/6 male mice were randomly assigned to IMD-0354 treated or untreated groups (5 mice, 10 eyes per group). CNV was induced with a 532-nm laser. Laser spots (power 250 mW, spot size 100 µm, time of exposure 50 ms) were created in each eye using a slit-lamp delivery system. Selective inhibitor of nuclear factor kappa-B kinase subunit beta (IKK2) inhibitor IMD-0354 (10 µg) was delivered subconjunctivally; vehicle-treated mice were the control. The treatment effect on CNV development was assessed at five days post-CNV induction in vivo in C57BL/6 and Cx3cr1gfp/wt mice by fluorescent angiography, fundus imaging, and ex vivo by retinal flatmounts immunostaining and Western blot analysis of RPE/Choroidal/Scleral complexes (RCSC) lysates. In vitro evaluations of IMD-0354 effects were performed in the BV-2 microglial cell line using lipopolysaccharide (LPS) stimulation. RESULTS: IMD-0354 caused a significant reduction in the fluorescein leakage and size of the laser spot, as well as a reduction in microglial cell migration and suppression of phospho-IκBα, Vascular endothelial growth factor (VEGF-A), and Prostaglandin-endoperoxide synthase 2 (COX-2). In vivo and ex vivo observations demonstrated reduced lesion size in mice, CD68, and Allograft inflammatory factor 1 (IBA-1) positive microglia cells migration to the laser injury site in IMD-0354 treated eyes. The data further corroborate with GFP-positive cells infiltration of the CNV site in Cx3cr1wt/gfp mice. In vitro IMD-0354 (10-25 ng/ml) treatment reduced NF-κB activation, expression of COX-2, caused decreased Actin-F presence and organization, resulting in reduced BV-2 cells migration capacity. CONCLUSION: The present data indicate that NF-κB activation in microglia and it's migration capacity is involved in the development of laser CNV in mice. Its suppression by NF-κB inhibition might be a promising therapeutic strategy for wet AMD.

Prospects for NK Cell Therapy of Sarcoma.

Natural killer (NK) cells are innate lymphoid cells with potent antitumor activity. One of the most NK cell cytotoxicity-sensitive tumor types is sarcoma, an aggressive mesenchyme-derived neoplasm. While a combination of radical surgery and radio- and chemotherapy can successfully control local disease, patients with advanced sarcomas remain refractory to current treatment regimens, calling for novel therapeutic strategies. There is accumulating evidence for NK cell-mediated immunosurveillance of sarcoma cells during all stages of the disease, highlighting the potential of using NK cells as a therapeutic tool. However, sarcomas display multiple immunoevasion mechanisms that can suppress NK cell function leading to an uncontrolled tumor outgrowth. Here, we review the current evidence for NK cells' role in immune surveillance of sarcoma during disease initiation, promotion, progression, and metastasis, as well as the molecular mechanisms behind sarcoma-mediated NK cell suppression. Further, we apply this basic understanding of NK-sarcoma crosstalk in order to identify and summarize the most promising candidates for NK cell-based sarcoma immunotherapy.

The Tumor Microenvironment-A Metabolic Obstacle to NK Cells' Activity.

NK cells have unique capabilities of recognition and destruction of tumor cells, without the requirement for prior immunization of the host. Maintaining tolerance to healthy cells makes them an attractive therapeutic tool for almost all types of cancer. Unfortunately, metabolic changes associated with malignant transformation and tumor progression lead to immunosuppression within the tumor microenvironment, which in turn limits the efficacy of various immunotherapies. In this review, we provide a brief description of the metabolic changes characteristic for the tumor microenvironment. Both tumor and tumor-associated cells produce and secrete factors that directly or indirectly prevent NK cell cytotoxicity. Here, we depict the molecular mechanisms responsible for the inhibition of immune effector cells by metabolic factors. Finally, we summarize the strategies to enhance NK cell function for the treatment of tumors.

Time-dependent LXR/RXR pathway modulation characterizes capillary remodeling in inflammatory corneal neovascularization.

Inflammation in the normally immune-privileged cornea can initiate a pathologic angiogenic response causing vision-threatening corneal neovascularization. Inflammatory pathways, however, are numerous, complex and are activated in a time-dependent manner. Effective resolution of inflammation and associated angiogenesis in the cornea requires knowledge of these pathways and their time dependence, which has, to date, remained largely unexplored. Here, using a model of endogenous resolution of inflammation-induced corneal angiogenesis, we investigate the time dependence of inflammatory genes in effecting capillary regression and the return of corneal transparency. Endogenous capillary regression was characterized by a progressive thinning and remodeling of angiogenic capillaries and inflammatory cell retreat in vivo in the rat cornea. By whole-genome longitudinal microarray analysis, early suppression of VEGF ligand-receptor signaling and inflammatory pathways preceded an unexpected later-phase preferential activation of LXR/RXR, PPARα/RXRα and STAT3 canonical pathways, with a concurrent attenuation of LPS/IL-1 inhibition of RXR function and Wnt/ß-catenin signaling pathways. Potent downstream inflammatory cytokines such as Cxcl5, IL-1ß, IL-6 and Ccl2 were concomitantly downregulated during the remodeling phase. Upstream regulators of the inflammatory pathways included Socs3, Sparc and ApoE. A complex and coordinated time-dependent interplay between pro- and anti-inflammatory signaling pathways highlights a potential anti-inflammatory role of LXR/RXR, PPARα/RXRα and STAT3 signaling pathways in resolving inflammatory corneal angiogenesis.

Selective IKK2 inhibitor IMD0354 disrupts NF-κB signaling to suppress corneal inflammation and angiogenesis.

Corneal neovascularization is a sight-threatening condition caused by angiogenesis in the normally avascular cornea. Neovascularization of the cornea is often associated with an inflammatory response, thus targeting VEGF-A alone yields only a limited efficacy. The NF-κB signaling pathway plays important roles in inflammation and angiogenesis. Here, we study consequences of the inhibition of NF-κB activation through selective blockade of the IKK complex IκB kinase ß (IKK2) using the compound IMD0354, focusing on the effects of inflammation and pathological angiogenesis in the cornea. In vitro, IMD0354 treatment diminished HUVEC migration and tube formation without an increase in cell death and arrested rat aortic ring sprouting. In HUVEC, the IMD0354 treatment caused a dose-dependent reduction in VEGF-A expression, suppressed TNFα-stimulated expression of chemokines CCL2 and CXCL5, and diminished actin filament fibers and cell filopodia formation. In developing zebrafish embryos, IMD0354 treatment reduced expression of Vegf-a and disrupted retinal angiogenesis. In inflammation-induced angiogenesis in the rat cornea, systemic selective IKK2 inhibition decreased inflammatory cell invasion, suppressed CCL2, CXCL5, Cxcr2, and TNF-α expression and exhibited anti-angiogenic effects such as reduced limbal vessel dilation, reduced VEGF-A expression and reduced angiogenic sprouting, without noticeable toxic effect. In summary, targeting NF-κB by selective IKK2 inhibition dampened the inflammatory and angiogenic responses in vivo by modulating the endothelial cell expression profile and motility, thus indicating an important role of NF-κB signaling in the development of pathologic corneal neovascularization.