CD81 and CD82 expressing tumor-infiltrating lymphocytes in the NSCLC tumor microenvironment play a crucial role in T-cell activation and cytokine production.

Introduction: To understand the immune system within the tumor microenvironment (TME) of non-small cell lung cancer (NSCLC), it is crucial to elucidate the characteristics of molecules associated with T cell activation. Methods: We conducted an in-depth analysis using single-cell RNA sequencing data obtained from tissue samples of 19 NSCLC patients. T cells were classified based on the Tumor Proportion Score (TPS) within the tumor region, and molecular markers associated with activation and exhaustion were analyzed in T cells from high TPS areas. Results: Notably, tetraspanins CD81 and CD82, belonging to the tetraspanin protein family, were found to be expressed in activated T cells, particularly in cytotoxic T cells. These tetraspanins showed strong correlations with activation and exhaustion markers. In vitro experiments confirmed increased expression of CD81 and CD82 in IL-2-stimulated T cells. T cells were categorized into CD81highCD82high and CD81lowCD82low groups based on their expression levels, with CD81highCD82high T cells exhibiting elevated activation markers such as CD25 and CD69 compared to CD81lowCD82low T cells. This trend was consistent across CD3+, CD8+, and CD4+ T cell subsets. Moreover, CD81highCD82high T cells, when stimulated with anti-CD3, demonstrated enhanced secretion of cytokines such as IFN-γ, TNF-α, and IL-2, along with an increase in the proportion of memory T cells. Bulk RNA sequencing results after sorting CD81highCD82high and CD81lowCD82low T cells consistently supported the roles of CD81 and CD82. Experiments with overexpressed CD81 and CD82 showed increased cytotoxicity against target cells. Discussion: These findings highlight the multifaceted roles of CD81 and CD82 in T cell activation, cytokine production, memory subset accumulation, and target cell cytolysis. Therefore, these findings suggest the potential of CD81 and CD82 as promising candidates for co-stimulatory molecules in immune therapeutic strategies for cancer treatment within the intricate TME.

The Value of Low Prostate Imaging-Reporting and Data System (PI-RADS) Scores in Preventing Unnecessary Prostate Biopsies.

Background and Objectives: Magnetic resonance imaging (MRI) and the Prostate Imaging-Reporting and Data System (PI-RADS) have become essential tools for prostate cancer evaluation. We evaluated the ability of PI-RADS scores in identifying significant prostate cancer, which would help avoid unnecessary prostate biopsies. Materials and Methods: Patients with prostate-specific antigen (PSA) levels ≤ 20 ng/mL, who underwent prostate MRI for evaluation from January 2018 to November 2019, were analyzed. Among them, 105 patients who received transrectal ultrasonography (TRUS)-guided biopsy were included. PSA, PI-RADS scores (low 1-2, high 3-5), biopsy results, and Gleason scores (GS) were evaluated. Biopsies with GS higher than 3 + 4 were considered as significant cancers and biopsies with no cancer or Gleason 3 + 3 were considered insignificant or no cancers. Results: Among the 105 patients, 45 patients had low PI-RADS and 60 had high PI-RADS scores. There were no patients with significant prostate cancer in the low PI-RADS groups. For the high PI-RADS group, 28 (46.7%) patients had significant cancer and 32 (53.3%) had insignificant or no cancer. The sensitivity and specificity of high PI-RADS to detect significant cancer was 100% and 58.4%, respectively. Positive predictive value was 46.7% and negative predictive value was 100%. Conclusions: Low PI-RADS scores on MRI did not show significant prostate cancer and surveillance should be considered in selected cases to prevent unnecessary invasive procedures and overdiagnosis.

LC3B upregulation by NANOG promotes immune resistance and stem-like property through hyperactivation of EGFR signaling in immune-refractory tumor cells.

Immune selection drives tumor cells to acquire refractory phenotypes. We previously demonstrated that cytotoxic T lymphocyte (CTL)-mediated immune pressure enriches NANOG+ tumor cells with stem-like and immune-refractory properties that make them resistant to CTLs. Here, we report that the emergence of refractory phenotypes is highly associated with an aberrant macroautophagic/autophagic state of the NANOG+ tumor cells and that the autophagic phenotype arises through transcriptional induction of MAP1LC3B/LC3B by NANOG. Furthermore, we found that upregulation of LC3B expression contributes to an increase in EGF secretion. The subsequent hyperactivation of EGFR-AKT signaling rendered NANOG+ tumor cells resistant to CTL killing. The NANOG-LC3B-p-EGFR axis was preserved across various types of human cancer and correlated negatively with the overall survival of cervical cancer patients. Inhibition of LC3B in immune-refractory tumor models rendered tumors susceptible to adoptive T-cell transfer, as well as PDCD1/PD-1 blockade, and led to successful, long-term control of the disease. Thus, our findings demonstrate a novel link among immune-resistance, stem-like phenotypes, and LC3B-mediated autophagic secretion in immune-refractory tumor cells, and implicate the LC3B-p-EGFR axis as a central molecular target for controlling NANOG+ immune-refractory cancer.Abbreviations: ACTB: actin beta; ATG7: autophagy related 7; BafA1: bafilomycin A1; CASP3: caspase 3; CFSE: carboxyfluorescein succinimidyl ester; ChIP: chromatin immunoprecipitation; CI: confidence interval; CIN: cervical intraepithelial neoplasia; CSC: cancer stem cell; CTL: cytotoxic T lymphocyte; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; FIGO: International Federation of Gynecology and Obstetrics; GFP: green fluorescent protein; GZMB: granzyme B; HG-CIN: high-grade CIN; IHC: immunohistochemistry; LG-CIN: low-grade CIN; LN: lymph node; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MCL1: myeloid cell leukemia sequence 1; MLANA/MART-1: melanoma antigen recognized by T cells 1; MUT: mutant; NANOG: Nanog homeobox; PDCD1/PD-1: programmed cell death 1; PMEL/gp100: premelanosome protein; RTK: receptor tyrosine kinase; TMA: tissue microarray; WT: wild type.

Case of the Index Patient Who Caused Tertiary Transmission of COVID-19 Infection in Korea: the Application of Lopinavir/Ritonavir for the Treatment of COVID-19 Infected Pneumonia Monitored by Quantitative RT-PCR.

Since mid-December of 2019, coronavirus disease 2019 (COVID-19) infection has been spreading from Wuhan, China. The confirmed COVID-19 patients in South Korea are those who came from or visited China. As secondary transmissions have occurred and the speed of transmission is accelerating, there are rising concerns about community infections. The 54-year old male is the third patient diagnosed with COVID-19 infection in Korea. He is a worker for a clothing business and had mild respiratory symptoms and intermittent fever in the beginning of hospitalization, and pneumonia symptoms on chest computerized tomography scan on day 6 of admission. This patient caused one case of secondary transmission and three cases of tertiary transmission. Hereby, we report the clinical findings of the index patient who was the first to cause tertiary transmission outside China. Interestingly, after lopinavir/ritonavir (Kaletra, AbbVie) was administered, ß-coronavirus viral loads significantly decreased and no or little coronavirus titers were observed.

HSP90A inhibition promotes anti-tumor immunity by reversing multi-modal resistance and stem-like property of immune-refractory tumors.

Cancer immunotherapy has emerged as a promising cancer treatment. However, the presence of immune-refractory tumor cells limits its clinical success by blocking amplification of anti-tumor immunity. Previously, we found that immune selection by immunotherapy drives the evolution of tumors toward multi-modal resistant and stem-like phenotypes via transcription induction of AKT co-activator TCL1A by NANOG. Here, we report a crucial role of HSP90A at the crossroads between NANOG-TCL1A axis and multi-aggressive properties of immune-edited tumor cells by identifying HSP90AA1 as a NANOG transcriptional target. Furthermore, we demonstrate that HSP90A potentiates AKT activation through TCL1A-stabilization, thereby contributing to the multi-aggressive properties in NANOGhigh tumor cells. Importantly, HSP90 inhibition sensitized immune-refractory tumor to adoptive T cell transfer as well as PD-1 blockade, and re-invigorated the immune cycle of tumor-reactive T cells. Our findings implicate that the HSP90A-TCL1A-AKT pathway ignited by NANOG is a central molecular axis and a potential target for immune-refractory tumor.

Defective Localization With Impaired Tumor Cytotoxicity Contributes to the Immune Escape of NK Cells in Pancreatic Cancer Patients.

Tumor-infiltrating lymphocytes (TILs), found in patients with advanced pancreatic ductal adenocarcinoma (PDAC), are shown to correlate with overall survival (OS) rate. Although majority of TILs consist of CD8+/CD4+ T cells, the presence of NK cells and their role in the pathogenesis of PDAC remains elusive. We performed comprehensive analyses of TIL, PBMC, and autologous tumor cells from 80 enrolled resectable PDAC patients to comprehend the NK cell defects within PDAC. Extremely low frequencies of NK cells (<0.5%) were found within PDAC tumors, which was attributable not to the low expression of tumor chemokines, but to the lack of chemokine receptor, CXCR2. Forced expression of CXCR2 in patients' NK cells rendered them capable of trafficking into PDAC. Furthermore, NK cells exhibited impaired cell-mediated killing of autologous PDAC cells, primarily due to insufficient ligation of NKG2D and DNAM-1, and failed to proliferate within the hypoxic tumor microenvironment. Importantly, these defects could be overcome by ex-vivo stimulation of NK cells from such patients. Importantly, when the proliferative capacity of NK cells in vitro was used to stratify patients on the basis of cell expansion, patients whose NK cells proliferated <250-fold experienced significantly lower DFS and OS than those with ≥250-fold. Ex-vivo activation of NK cells restored tumor trafficking and reactivity, hence provided a therapeutic modality while their fold expansion could be a potentially significant prognostic indicator of OS and DFS in such patients.

CVD-grown monolayer MoS2 in bioabsorbable electronics and biosensors.

Transient electronics represents an emerging technology whose defining feature is an ability to dissolve, disintegrate or otherwise physically disappear in a controlled manner. Envisioned applications include resorbable/degradable biomedical implants, hardware-secure memory devices, and zero-impact environmental sensors. 2D materials may have essential roles in these systems due to their unique mechanical, thermal, electrical, and optical properties. Here, we study the bioabsorption of CVD-grown monolayer MoS2, including long-term cytotoxicity and immunological biocompatibility evaluations in biofluids and tissues of live animal models. The results show that MoS2 undergoes hydrolysis slowly in aqueous solutions without adverse biological effects. We also present a class of MoS2-based bioabsorbable and multi-functional sensor for intracranial monitoring of pressure, temperature, strain, and motion in animal models. Such technology offers specific, clinically relevant roles in diagnostic/therapeutic functions during recovery from traumatic brain injury. Our findings support the broader use of 2D materials in transient electronics and qualitatively expand the design options in other areas.

Clozapine reduces Toll-like receptor 4/NF-κB-mediated inflammatory responses through inhibition of calcium/calmodulin-dependent Akt activation in microglia.

Clozapine is an atypical antipsychotic agent used in the treatment of schizophrenia and severe mood disorders. Accumulating evidence suggests that neuroinflammation is closely associated with the pathogenesis of various neurodegenerative diseases and psychiatric disorders. Clozapine exerts anti-inflammatory activity. However, the molecular mechanism underlying the anti-inflammatory activity of clozapine is poorly understood. In this study, we found that clozapine suppressed lipopolysaccharide (LPS)-induced phosphorylation of IκBα at Ser-32 and of p65/RelA at Ser-468, as well as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-dependent transcriptional activity in microglial cells. Clozapine downregulated LPS-induced Akt phosphorylation at Ser-473. Pharmacological Akt inhibitors ameliorated LPS-induced NF-κB activation. Removal of extracellular Ca2+ by EGTA or sequestration of intracellular Ca2+ by BAPTA-AM attenuated LPS-induced Akt phosphorylation. Treatment with calmodulin (CaM) antagonists and the CaM kinase inhibitor, KN-93, also prevented LPS-induced Akt and NF-κB activation, suggesting that Ca2+/CaM-dependent Akt activation is critical in LPS-induced NF-κB activation in microglia. These results suggest that clozapine exhibits anti-inflammatory activity through the inhibition of Ca2+/CaM/Akt-mediated NF-κB activation.

A novel strategy for highly efficient isolation and analysis of circulating tumor-specific cell-free DNA from lung cancer patients using a reusable conducting polymer nanostructure.

We have developed a reusable nanostructured polypyrrole nanochip and demonstrated its use in the electric field-mediated recovery of circulating cell-free DNA (cfDNA) from the plasma of lung cancer patients. Although cfDNA has been recognized and widely studied as a versatile and promising biomarker for the diagnosis and prognosis of cancers, the lack of efficient strategies to directly isolate cfDNA from the plasma has become a great hindrance to its potential clinical use. As a proof-of-concept study, we demonstrated a technique for the rapid and efficient isolation of cfDNA with high yield and purity. In particular, the synergistic effects of the electro-activity and the nanostructured features of the polypyrrole polymer enabled repeated retrieval of cfDNA using a single platform. Moreover, polypyrrole nanochip facilitated the amplification of tumor-specific DNA fragments from the plasma samples of patients with lung cancer characterized by mutations in exons 21 of the epidermal growth factor receptor gene (EGFR). Overall, the proposed polypyrrole nanochip has enormous potential for industrial and clinical applications with significantly enhanced efficiency in the recovery of tumor-associated circulating cfDNA. This may ultimately contribute to more robust and reliable evaluation of gene mutations in peripheral blood.

Efficient Capture and Isolation of Tumor-Related Circulating Cell-Free DNA from Cancer Patients Using Electroactive Conducting Polymer Nanowire Platforms.

Circulating cell-free DNA (cfDNA) is currently recognized as a key non-invasive biomarker for cancer diagnosis and progression and therapeutic efficacy monitoring. Because cfDNA has been detected in patients with diverse types of cancers, the use of efficient strategies to isolate cfDNA not only provides valuable insights into tumour biology, but also offers the potential for developing new cancer-specific targets. However, the challenges associated with conventional cfDNA extraction methods prevent their further clinical applications. Here, we developed a nanostructured conductive polymer platform for the efficient capture and release of circulating cfDNA and demonstrated its potential clinical utility using unprocessed plasma samples from patients with breast and lung cancers. Our results confirmed that the platform's enhanced efficiency allows tumor-specific circulating cfDNA to be recovered at high yield and purity.

Electroactive polypyrrole nanowire arrays: synergistic effect of cancer treatment by on-demand drug release and photothermal therapy.

An electroresponsive drug release system based on polypyrrole (Ppy) nanowires was developed to induce the local delivery of anticancer drug, doxorubicin (DOX), according to the applied electric field. DOX-conjugated Ppy nanowire (NW) (DOX/Ppy NW) array was initially prepared by electrochemical deposition of a mixture of pyrrole monomers and biotin as dopants in the anodic alumina oxide membrane as a sacrificial template. Morphological observation by scanning electron microscopy revealed free-standing and 3D nanotopographical features with large surface area and high density. In addition, we investigated the antitumor efficacy of DOX released from DOX/Ppy NW array in response to the external electric field using two kinds of cancer cell lines, human oral squamous carcinoma cells (KB cells) and human breast cancer cells (MCF7 cells). Meanwhile, strong photothermal effect as a result of a near-infrared absorbing ability of Ppy synergistically maximizes the chemotherapeutic efficacy. Our results suggested that the proposed multifunctional Ppy platform possessing several beneficial features is very promising for many therapeutic applications including cancer.

High-purity isolation and recovery of circulating tumor cells using conducting polymer-deposited microfluidic device.

We have developed a conductive nano-roughened microfluidic device and demonstrated its use as an electrically modulated capture and release system for studying rare circulating tumor cells (CTCs). The microchannel surfaces were covalently decorated with epithelial cancer-specific anti-EpCAM antibody by electrochemical deposition of biotin-doped polypyrrole (Ppy), followed by the assembly of streptavidin and biotinylated antibody. Our method utilizes the unique topographical features and excellent electrical activity of Ppy for i) surface-induced preferential recognition and release of CTCs, and ii) selective elimination of non-specifically immobilized white blood cells (WBCs), which are capable of high-purity isolation of CTCs. In addition, the direct incorporation of biotin molecules offers good flexibility, because it allows the modification of channel surfaces with diverse antibodies, in addition to anti-EpCAM, for enhanced detection of multiple types of CTCs. By engineering a series of electrical, chemical, and topographical cues, this simple yet efficient device provides a significant advantage to CTC detection technology as compared with other conventional methods.

An electroactive biotin-doped polypyrrole substrate that immobilizes and releases EpCAM-positive cancer cells.

The specific capture and remotely controlled release of the EpCAM-positive cancer cells from biotin-doped polypyrrole (Ppy) films in response to an electrical potential is presented. As Ppy allows the direct incorporation of biotin molecules during the electrochemical process, densely packed biotin molecules can serve as the binding sites for streptavidin-tagged biomolecular complexes. This study demonstrates not only the enhanced capture and enrichment of EpCAM-positive cancer cells but also "on-demand" release of the viable cells from conductive Ppy in an electrical-potential-dependent way. This novel approach is of great importance in a diverse range of applications, and in particular in cancer diagnostics and screening.

A new synthetic chalcone derivative, 2-hydroxy-3',5,5'-trimethoxychalcone (DK-139), suppresses the Toll-like receptor 4-mediated inflammatory response through inhibition of the Akt/NF-κB pathway in BV2 microglial cells.

Microglial cells are the resident innate immune cells that sense pathogens and tissue injury in the central nervous system (CNS). Microglial activation is critical for neuroinflammatory responses. The synthetic compound 2-hydroxy-3',5,5'-trimethoxychalcone (DK-139) is a novel chalcone-derived compound. In this study, we investigated the effects of DK-139 on Toll-like receptor 4 (TLR4)-mediated inflammatory responses in BV2 microglial cells. DK-139 inhibited lipopolysaccharide (LPS)-induced TLR4 activity, as determined using a cell-based assay. DK-139 blocked LPS-induced phosphorylation of IκB and p65/RelA NF-κB, resulting in inhibition of the nuclear translocation and trans-acting activity of NF-κB in BV2 microglial cells. We also found that DK-139 reduced the expression of NF-κB target genes, such as those for COX-2, iNOS, and IL-1ß, in LPS-stimulated BV2 microglial cells. Interestingly, DK-139 blocked LPS-induced Akt phosphorylation. Inhibition of Akt abrogated LPS-induced phosphorylation of p65/RelA, while overexpression of dominant- active p110CAAX enhanced p65/RelA phosphorylation as well as iNOS and COX2 expression. These results suggest that DK-139 exerts an anti-inflammatory effect on microglial cells by inhibiting the Akt/IκB kinase (IKK)/NF-κB signaling pathway.

The tricyclic antidepressant imipramine induces autophagic cell death in U-87MG glioma cells.

In this study, we investigated the antitumor effects of the tricyclic antidepressant 3-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N,N-dimethylpropan-1-amine (imipramine) on glioma cells. We found that exposure of U-87MG cells to imipramine resulted in the inhibition of PI3K/Akt/mTOR signaling, reduction of clonogenicity, and induction of cell death. Imipramine stimulated the formation of acidic vesicular organelles, the conversion of LC3-I to LC3-II, and the redistribution of LC3 to autophagosomes, suggesting that it stimulates the progression of autophagy. It did not, however, induce apoptosis. We further showed that knockdown of Beclin-1 using siRNA abrogated imipramine-induced cell death. These results suggest that imipramine exerts antitumor effects on PTEN-null U-87MG human glioma cells by inhibiting PI3K/Akt/mTOR signaling and by inducing autophagic cell death.