Anti-tumor effects of NK cells and anti-PD-L1 antibody with antibody-dependent cellular cytotoxicity in PD-L1-positive cancer cell lines

Background Although programmed cell death-1/programmed death-ligand 1 (PD-L1) inhibitors show remarkable antitumor activity, a large portion of patients with cancer, even those with high PD-L1-expressing tumors, do not respond to their effects. Most PD-L1 inhibitors contain modified fragment crystallizable region (Fc) receptor binding sites to prevent antibody-dependent cellular cytotoxicity (ADCC) against PD-L1-expressing non-tumor cells. However, natural killer (NK) cells have specific antitumor activity in the presence of tumor-targeting antibody through ADCC, which could enhance NK cell-induced cytotoxicity. We evaluated the antitumor efficacy of ADCC via anti-PD-L1 monoclonal antibodies (mAbs) and NK cells against several PD-L1-positive cancer cell lines. Methods Various cancer cell lines were used as target cell lines. Surface PD-L1 expression was analyzed by flow cytometry. IMC-001 and anti-hPD-L1-hIgG1 were tested as anti-PD-L1 mAbs with ADCC and atezolizumab as an anti-PD-L1 mAb without ADCC. NK cell cytotoxicity was measured by(51)Cr-release assay and CD107a degranulation assay. Also, live cell imaging was performed to evaluate cytotoxicity in a single-cell level. NK-92-CD16 (CD16-transduced NK-92 cell line) and peripheral blood mononuclear cells from healthy donors, respectively, were used as an effector cell. Fc gamma RIIIa (CD16a)-V158F genotyping was performed for healthy donors. Results We demonstrated that the cytotoxicity of NK-92-CD16 cells toward PD-L1-positive cancer cell lines was significantly enhanced in the presence of anti-PD-L1 mAb with ADCC. We also noted a significant increase in primary human NK cell cytotoxicity against PD-L1-positive human cancer cells when cocultured with anti-PD-L1 mAb with ADCC. Moreover, NK cells expressing aFCGR3Ahigh-affinity genotype displayed higher anti-PD-L1 mAb-mediated ADCC lysis of tumor cells than donors with a low-affinity genotype. Conclusion These results suggest that NK cells induce an ADCC response in combination with anti-PD-L1 mAbs, which helps promote ADCC antitumor activity against PD-L1-positive tumors. This study provides support for NK cell immunotherapy against high PD-L1-expressing tumors in combination with ADCC through anti-PD-L1 mAbs.

Combined blockade of polo-like kinase and pan-RAF is effective against NRAS-mutant non-small cell lung cancer cells

NRAS mutation is rarely observed in non-small cell lung cancer (NSCLC) patients, and there are no approved treatments for NRAS-mutant NSCLC. Here, we evaluated the effect of pan-RAF inhibitors on human NRAS-mutant NSCLC cell lines and performed high-throughput screening using human kinome small interfering (si) RNA or CRISPR/Cas9 libraries to identify new targets for combination NSCLC treatment. Our results indicate that human NRAS-mutant NSCLC cells are moderately sensitive to pan-RAF inhibitors. High-throughput kinome screenings further showed that G2/M arrest, particularly following knockdown of polo-like kinase 1 (PLK1), can inhibit the growth of human NRAS-mutant NSCLC cells and those treated with the type II pan-RAF inhibitor LXH254. In addition, treatment with volasertib plus LXH254, resulting in dual blockade of PLK1 and pan-RAF, was found to be more effective than LXH254 monotherapy for inhibiting long-term cell viability, suggesting that this combination therapeutic strategy may lead to promising results in the clinic

Effects of HA and NA glycosylation pattern changes on the transmission of avian influenza A(H7N9) virus in guinea pigs

AbstractAvian influenza H7N9 virus has posed a concern of potential human-to-human transmission by resulting in seasonal virus-like human infection cases. To address the issue of sustained human infection with the H7N9 virus, here we investigated the effects of hemagglutinin (HA) and neuraminidase (NA) N-linked glycosylation (NLG) patterns on influenza virus transmission in a guinea pig model. Based on the NLG signatures identified in the HA and NA genetic sequences of H7N9 viruses, we generated NLG mutant viruses using either HA or NA gene of a H7N9 virus, A/Anhui/01/2013, by reverse genetics on the 2009 pandemic H1N1 virus backbone. For the H7 HA NLG mutant viruses, NLG pattern changes appeared to reduce viral transmissibility in guinea pigs. Intriguingly, however, the NLG changes in the N9 NA protein, such as a removal from residue 42 or 66 or an addition at residue 266, increased transmissibility of the mutant viruses by more than 33%, 50%, and 16%, respectively, compared with a parental N9 virus. Given the effects of HA-NA NLG changes with regard to viral transmission, we then generated the HA-NA NLG mutant viruses harboring the H7 HA of double NLG addition and the N9 NA of various NLG patterns. As seen in the HA NLG mutants above, the double NLG-added H7 HA decreased viral transmissibility. However, when the NA NLG changes occurred by a removal of residue 66 and an addition at 266 were additionally accompanied, the HA-NA NLG mutant virus recovered the transmissibility of its parental virus. These demonstrate the effects of specific HA-NA NLG changes on the H7N9 virus transmission by highlighting the importance of a HA-NA functional balance

Highly tunable low frequency metamaterial cavity for vibration localization

Metamaterial cavity has gathered much attention recently due to its capability of localizing vibration energy. Despite the active research, however, there are still big technical challenges not solved yet. Especially, there has been no approach to maximize the wave localization performance of metamaterial cavity; therefore, there has been a possibility that obtained cavity mode does not show sufficiently high performance. Also, there is a tunability issue that whole metamaterials should be re-designed to tune the cavity frequency. Here, we present the metamaterial cavity system that can control its cavity mode frequency from 589 to 2184 Hz by adjusting the cavity length from 140 to 60 mm without re-designing the whole metamaterial based on the broad bandgap. Also, the performance of the obtained cavity mode can be improved by adjusting the length of the side beam attached to the metamaterial; the displacements are amplified more than 18???110 times. Consequently, one may easily obtain the highly localized vibration energy at the desired frequency by adjusting two geometric parameters based on the proposed metamaterial cavity system. Numerical and experimental supports are provided to validate our new metamaterial cavity system. This metamaterial cavity system is expected to provide a guideline for localizing vibration energy in various applications, such as energy harvesting, sensing or vibration dissipatio

Clinical Application of Next-Generation Sequencing-Based Panel to BRAF Wild-Type Advanced Melanoma Identifies Key Oncogenic Alterations and Therapeutic Strategies

Molecular profiling with next-generation sequencing (NGS) has been applied in multiple solid cancers to discover potential therapeutic targets. Here, we describe the results of a clinical NGS panel in patients with advanced melanoma. Thirty-six tumor tissues from patients with BRAF wild-type melanoma at Seoul National University Hospital (SNUH; Seoul, Republic of Korea) were collected and deep-sequenced using the SNUH FIRST-Cancer NGS panel to assess single-nucleotide variants, small insertions/deletions, copy number variations, and structural variations to estimate tumor mutation burden (TMB). We discovered 106 oncogenic alterations and most of the patients (n = 33, 92%) harbored at least one oncogenic alteration, including 2 patients who were initially diagnosed as BRAF V600E-negative but were later confirmed to be positive. Altogether, 36 samples were classified into RAS/BRAF/NF1-mutant (n = 14, 39%) or triple wild-type (n = 22, 61%) melanoma subtypes. The estimated median TMB was 8.2 mutations per Mb, ranging from 0 to 146.67 mutations per Mb. Of the 36 patients, 25 (70%) had actionable alterations with currently developed drugs, and 7 (19.4%) were enrolled in dinical trials with an RAF inhibitor, multiple receptor tyrosine kinase inhibitor, and anti-programmed cell death-1 (PD-1) antibody. TMB tended to associate with progression-free survival (PFS) of treatment with anti-PD-1/PDL-1 antibody (HR, 0.96; 95% confidence interval, 0.92-1.00; P = 0.07). High-TMB (>= 13) group was associated with longer PFS than the low-TMB group (median 34.0 vs. 11.0 weeks, P = 0.04). Overall, the dinical use of a NGS panel in patients with advanced melanoma shows association with clinical outcomes and several therapeutic strategies.

Tumor immune profiles noninvasively estimated by FDG PET with deep learning correlate with immunotherapy response in lung adenocarcinoma

Rationale: The clinical application of biomarkers reflecting tumor immune microenvironment is hurdled by the invasiveness of obtaining tissues despite its importance in immunotherapy. We developed a deep learning-based biomarker which noninvasively estimates a tumor immune profile with fluorodeoxyglucose positron emission tomography (FDG-PET) in lung adenocarcinoma (LUAD). Methods: A deep learning model to predict cytolytic activity score (CytAct) using semi-automatically segmented tumors on FDG-PET trained by a publicly available dataset paired with tissue RNA sequencing (n = 93). This model was validated in two independent cohorts of LUAD: SNUH (n = 43) and The Cancer Genome Atlas (TCGA) cohort (n = 16). The model was applied to the immune checkpoint blockade (ICB) cohort, which consists of patients with metastatic LUAD who underwent ICB treatment (n = 29). Results: The predicted CytAct showed a positive correlation with CytAct of RNA sequencing in validation cohorts (Spearman rho = 0.32, p = 0.04 in SNUH cohort; spearman rho = 0.47, p = 0.07 in TCGA cohort). In ICB cohort, the higher predicted CytAct of individual lesion was associated with more decrement in tumor size after ICB treatment (Spearman rho = -0.54, p < 0.001). Higher minimum predicted CytAct in each patient associated with significantly prolonged progression free survival and overall survival (Hazard ratio 0.25, p = 0.001 and 0.18, p = 0.004, respectively). In patients with multiple lesions, ICB responders had significantly lower variance of predicted CytActs (p = 0.005). Conclusion: The deep learning model that predicts CytAct using FDG-PET of LUAD was validated in independent cohorts. Our approach may be used to noninvasively assess an immune profile and predict outcomes of LUAD patients treated with ICB.

Anti-metastatic effect of GV1001 on prostate cancer cells; roles of GnRHR-mediated Gαs-cAMP pathway and AR-YAP1 axis

Background Gonadotropin-releasing hormone receptor (GnRHR) transmits its signal via two major Gα-proteins, primarily Gαq and Gαi. However, the precise mechanism underlying the functions of Gαs signal in prostate cancer cells is still unclear. We have previously identified that GV1001, a fragment of the human telomerase reverse transcriptase, functions as a biased GnRHR ligand to selectively stimulate the Gαs/cAMP pathway. Here, we tried to reveal the potential mechanisms of which GV1001-stimulated Gαs-cAMP signaling pathway reduces the migration and metastasis of prostate cancer (PCa) cells. Methods The expression of epithelial-mesenchymal transition (EMT)-related genes was measured by western-blotting and spheroid formation on ultra-low attachment plate was detected after GV1001 treatment. In vivo Spleen-liver metastasis mouse model was used to explore the inhibitory effect of GV1001 on metastatic ability of PCa and the transwell migration assay was performed to identify whether GV1001 had a suppressive effect on cell migration in vitro. In order to demonstrate the interaction between androgen receptor (AR) and YAP1, co-immunoprecipitation (co-IP), immunofluorescence (IF) staining, chromatin immunoprecipitation (ChIP) were performed in LNCaP cells with and without GV1001 treatment. Results GV1001 inhibited expression of EMT-related genes and spheroid formation. GV1001 also suppressed in vivo spleen-liver metastasis of LNCaP cells as well as cell migration in vitro. GV1001 enhanced the phosphorylation of AR and transcription activity of androgen response element reporter gene through cAMP/protein kinase A pathway. Moreover, GV1001 increased Ser-127 phosphorylation of YAP1 and its ubiquitination, and subsequently decreased the levels of AR-YAP1 binding in the promoter region of the CTGF gene. In contrast, both protein and mRNA levels of NKX3.1 known for tumor suppressor gene and AR-coregulator were upregulated by GV1001 in LNCaP cells. YAP1 knockout using CRISPR/Cas9 significantly suppressed the migration ability of LNCaP cells, and GV1001 did not affect the cell migration of YAP1-deficient LNCaP cells. On the contrary, cell migration was more potentiated in LNCaP cells overexpressing YAP5SA, a constitutively active form of YAP1, which was not changed by GV1001 treatment. Conclusions Overall, this study reveals an essential role of AR-YAP1 in the regulation of PCa cell migration, and provides evidence that GV1001 could be a novel GnRHR ligand to inhibit metastasis of PCa via the Gαs/cAMP pathway.This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Kim HS, 2018M3A9C8021792, Kang KW, 2021R1A2C2093196)

Overestimation of own body weights in female university students: associations with lifestyles, weight control behaviors and depression

The study aimed to analyze the lifestyles, weight control behavior, dietary habits, and depression of female university students. The subjects were 532 students from 8 universities located in 4 provinces in Korea. According to percent ideal body weight, 33 (6.4%), 181 (34.0%), 283 (53.2%), 22 (4.1%) and 13 (2.5%) were severely underweight, underweight, normal, overweight and obese, respectively, based on self-reported height and weight. As much as 64.1% and only 2.4%, respectively, overestimated and underestimated their body weight status. Six overweight subjects were excluded from overestimation group for the purpose of this study, resulting in overestimation group consisting of only underweight and normal weight subjects. Compared to those from the normal perception group, significantly more subjects from the overestimation group were currently smoking (P = 0.017) and drank more often than once a week (P = 0.015), without any significant differences in dietary habits. Despite similar BMIs, subjects who overestimated their own weight statuses had significantly higher weight dissatisfaction (P = 0.000), obesity stress (P = 0.000), obsession to lose weight (P = 0.007) and depression (P = 0.018). Also, more of them wanted to lose weight (P = 0.000), checked their body weights more often than once a week (P = 0.025) and had dieting experiences using 'reducing meal size' (P = 0.012), 'reducing snacks' (P = 0.042) and 'taking prescribed pills' (P = 0.032), and presented 'for a wider range of clothes selection' as the reason for weight loss (P = 0.039), although none was actually overweight or obese. Unlike the case with overestimating one's own weight, being overweight was associated with less drinking (P = 0.035) and exercising more often (P = 0.001) and for longer (P = 0.001) and healthier reasons for weight control (P = 0.002), despite no differences in frequency of weighing and depression. The results showed that weight overestimation, independent of weight status, is associated with risky lifestyles, weight control behaviors, and mental conditions. Preventive interventions should focus not only on obesity, but also on body weight overestimation

The efficacy of immune checkpoint inhibitors in anaplastic lymphoma kinase-positive non-small cell lung cancer

Background Despite recent advances in treating non-small cell lung cancer (NSCLC) with immune checkpoint inhibitors (ICIs), their role in ALK-positive NSCLC patients is unclear. We investigated the efficacy of ICIs in patients with ALK-positive NSCLC. Methods Between 2011 and 2018, a total of 14 ALK-positive NSCLC patients treated with ICIs were evaluated retrospectively. Clinicopathologic features including age, PD-L1 expression, and treatment outcomes were analyzed. RNA expression level and cytolytic activity by ALK positivity were analyzed using The Cancer Genome Atlas (TCGA) and National Cancer Center Research Institute (NCCRI) data sets. Results A total of 13 patients (92.9%) received ALK inhibitors. Patients received a median of three (range 2-8) courses of therapy. The study included nine patients (64.3%) who were PD-L1-high (>50%) and four (28.6%) who were PD-L1-low (<50%). The objective response rate was 14.3% (2/14). The median progression-free survival time was 2.18 months (95% confidence interval [CI] 1.13 months-not reached [NR]). The median overall survival time was 5.67 months (95% CI 3.00 months-NR). RNA expression levels of CD274 were similar between the ALK-positive and negative groups in both TCGA and NCCRI datasets. RNA levels of CD8A in both TCGA and NCCRI data sets were nonsignificantly lower in the ALK-positive group. Cytolytic activity scores including interferon-gamma-related response were lower in the ALK-positive group in the NCCRI but not TCGA dataset. Conclusions Despite high PD-L1-positive rates, ICIs show limited efficacy in ALK-positive NSCLC. Decreased interferon-gamma-related response may underlie these findings.

Prognostic utility of body composition parameters based on computed tomography analysis of advanced non-small cell lung cancer treated with immune checkpoint inhibitors

Objective The purpose of this study was to evaluate the prognostic impact of body composition parameters based on computed tomography (CT) in patients with non-small cell lung cancer (NSCLC) who received ICI treatment. Methods This retrospective study analyzed the data from advanced NSCLC patients treated with ICI therapy between 2013 and 2019. We included patients with NSCLC who underwent baseline CT scans. The exclusion criteria included patients who received three or more lines of chemotherapy, those with insufficient clinical information, or those without treatment response evaluation. Results A total of 136 patients were enrolled. Among the volumetric body composition parameters, patients in the highest quartiles (Q2–4) of the visceral fat index (VFI) exhibited a higher response rate to ICI therapy than those in the lowest quartile (Q1) of VFI (Q1 vs. Q2–4: 18.2% vs. 43.1%, p = 0.012). Patients with a VFI in Q2–4 had significantly prolonged progression-free survival (PFS) and overall survival (OS) (PFS, Q1 vs. Q2–4: 3.0 months vs. 6.4 months, p = 0.043; OS, Q1 vs. Q2–4: 5.6 months vs. 16.3 months, p = 0.004). Kaplan–Meier analysis based on the VFI and visceral fat Hounsfield unit (HU) revealed that patients with VFI in Q1 and HU in Q2–4 had the worst prognosis. Conclusions Visceral fat volume is significantly associated with treatment outcomes in ICI-treated patients with NSCLC. Moreover, fat quality may impact the treatment outcomes. This finding underscores the potential significance of both fat compartments and fat quality as prognostic indicators. Critical relevance statement Visceral fat volume is significantly associated with treatment outcomes in ICI-treated patients with non-small cell lung cancer. Moreover, fat quality may impact the treatment outcomes. This finding underscores the potential significance of both fat compartments and fat quality as prognostic indicators. Graphical Abstract Key points • We found that visceral fat volume positively correlated with treatment response and survival in patients with non-small cell lung cancer receiving immune checkpoint inhibitors. • Additionally, a trend toward a negative correlation between visceral fat attenuation and survival was observed. • The findings highlight the prognostic utility of fat compartments and fat quality