Immune-related adverse events and outcomes among pan-cancer patients receiving immune checkpoint inhibitors: A monocentric real-world observational study.

BACKGROUND: Real-world evidence on immune-related adverse events (irAEs) are relatively insufficient. Herein patterns and outcomes of irAEs after administration of anti-programmed cell death 1 (PD-1) and its legend 1 (PD-L1) antibodies were investigated. METHODS: Patients treated with anti-PD-1/PD-L1 drugs from January 2018 to September 2021 at Huadong Hospital, Fudan University were included. Common Terminology Criteria for Adverse Events (CTCAE) was used for irAEs evaluation. The primary endpoints were the clinical description of irAEs. RESULTS: Two hundred and forty-one solid tumor patients were included, with lung cancer as the most common tumor type (56%). 187 (77.6%) patients presented any kind of irAEs. The median time to any irAE onset was 28 (95% CI 24-32) days. Skin toxicities are the most common irAEs (46.1%) and the irAEs (36.5%) occurred earliest after immune-checkpoint inhibitors. The most frequently occurred all-grade irAEs were rash (23.7%), myelosuppression (20.7%), and hepatic injury (19.5%). 23 (9.5%) patients died of severe irAEs, which consists of 10 patients with pneumonitis, four colitis, four myocarditis, and one each for gastritis, pulmonary embolism, myelosuppression, hypophysitis, and encephalitis. Patients with any irAE onset had significantly longer progression-free survival (PFS) (p = 0.013) and overall survival (OS) (p = 0.007), respectively, than patients without irAEs. In addition, patients with skin toxicities (p = 0.012) or blood toxicities (p = 0.015) had achieved a longer PFS, than those without corresponding toxitities, respectively. CONCLUSION: Most irAEs are mild and manageable, while some irAEs can present at later time or can be life-threatening, especially pneumonitis as we observed. Patients with any irAE onset may achieve a better prognosis than those without irAEs, and presentation of skin or blood toxicities will indicate a better PFS.

Protocol for detecting substrates in living cells by targeted molecular probes through hyperpolarized 129Xe MRI.

Due to limited detection sensitivity and contrast limitation, imaging substrates with 129Xe MRI in living cells is still a challenge. Here, we present an effective protocol to detect and image substrates in human lung cancer cells A549 with hyperpolarized 129Xe MRI. This protocol was optimized for a cryptophane-based probe sensitive to biothiols and can be expanded to other Xe-based probes to detect potential biomarkers in other mammalian cells. For complete details on the use and execution of this protocol, please refer to Zeng et al. (2021).

MicroRNA-18 facilitates the stemness of gastric cancer by downregulating HMGB3 though targeting Meis2.

The recurrence and metastasis of gastric cancer are related to the stemness of gastric cancer cells. Researches have shown that miR-18 level is negatively correlated to the occurrence and development of certain cancer types. However, the effects of miR-18 on the stemness of gastric cancer remain uncertain. In this research, gastric cancer cell lines with stable overexpression of miR-18 were constructed through lentivirus infection. CCK-8 assay, RT-qPCR, Western blot, flow cytometry, and in vivo tumorigenesis assays were performed to evaluate the effects of miR-18 on the stemness of gastric cancer cells. Moreover, luciferase reporter assays found that Meis2 was the target of miR-18. Furthermore, we also found that the low-expressed oncogene HMGB3 is involved in this miR-18/Meis2 axis to further promote the stemness of gastric cancer cells. These findings suggest that the miR-18/Meis2/HMGB3 axis may be potential prognostic indicators for patients with gastric cancer.

Preoperative combination score of neutrophils, monocytes, and lymphocytes as a predictor for locally advanced rectal cancer.

OBJECTIVE: The aim of this study was to investigate the prognostic value of baseline peripheral blood neutrophils, monocytes, and lymphocytes on locally advanced rectal cancer (LARC) patients. METHODS: Clinicopathologic data of 317 LARC patients during July 2010 and October 2016 were retrospectively gathered. X-tile software was used to acquire the optimal cutoff values of neutrophils, monocytes, and lymphocytes. Peripheral blood immune score (PBIS) system was proposed and built based on neutrophils, monocytes, and lymphocytes. The Cox model was used to analyze the associations between clinicopathological characteristics and potential outcomes. C-index was used to assess model performance. A nomogram was constructed to predict prognosis, and a calibration plot was used to verify the accuracy of the nomogram prediction model. RESULTS: Cutoff values of neutrophils, lymphocytes, and monocytes were 4.46 (× 109/L), 1.66 (× 109/L), and 0.39 (× 109/L), respectively. PBIS was related to sex (P < 0.001), tumor length (P = 0.003), and tumor thickness (P = 0.014). Multivariate Cox regression analysis revealed that PBIS (HR = 0.707, 95% CI: 0.549-0.912, P = 0.008) was an independent predictor of DFS. High PBIS (HR = 0.697, 95% CI: 0.492-0.988, P = 0.043) and high lymphocyte count (HR = 0.511, 95%CI: 0.273-0.958, P = 0.036) were favorable factors of OS. Both C-index (0.74, 95% CI: 0.549-0.912) and the calibration plot showed good prediction ability of the nomogram for DFS. CONCLUSION: PBIS, composed of baseline peripheral blood neutrophils, monocytes, and lymphocytes, is an independent predictor of the prognosis of LARC. Combination of PBIS and ypTNM stage may be a promising marker to guide adjuvant therapy after the operation.

Delicately Designed Cancer Cell Membrane-Camouflaged Nanoparticles for Targeted 19F MR/PA/FL Imaging-Guided Photothermal Therapy.

Our exploration of multimodal nanoprobes aims to combine photoacoustic (PA) imaging, 19F magnetic resonance (MR), and fluorescence (FL) imaging, which offers complementary advantages such as high spatial resolution, unlimited penetration, and high sensitivity to enable more refined images for accurate tumor diagnoses. In this research, perfluorocarbons (PFCs) and indocyanine green (ICG) are encapsulated by poly(lactic-co-glycolic acid) (PLGA) for intravital 19F MR/FL/PA tri-modal imaging-guided photothermal therapy. Then, it is coated with an A549 cancer cell membrane (AM) to fabricate versatile theranostic nanoprobes (AM-PP@ICGNPs). After systemic administration, FLI reveals time-dependent tumor homing of NPs with high sensitivity, 19F MRI provides tumor localization of NPs without background signal interference, and PAI illustrates the detailed distribution of NPs inside the tumor with high spatial resolution. What is more, AM-PP@ICGNPs accumulated in the tumor area exhibit a prominent photothermal effect (48.4 °C) under near infrared (NIR) laser irradiation and realize an enhanced antitumor response in vivo. These benefits, in combination with the excellent biocompatibility, make AM-PP@ICGNPs a potential theranostic nanoagent for accurate tumor localization and ultimately achieve superior cancer therapy.

Silica nanoparticle coated perfluorooctyl bromide for ultrasensitive MRI.

MRI with hyperpolarized 129Xe can achieve low-concentration detection. Herein, nanoparticle-coated perfluorooctyl bromide (PFOB) was developed as a 129Xe MRI contrast agent with a moderate exchange rate, sufficient stability and feasible surface modification. The αvß3 integrin overexpressed by non-small-cell lung cancer A549 cells was successfully detected by 129Xe MRI with high specificity through adequate surface modifications.

A Small Molecular Multifunctional Tool for pH Detection, Fluorescence Imaging, and Photodynamic Therapy.

A smart multitool platform for theranostics would be useful for monitoring the administration of therapies in vivo. However, the integration of multiple functions into a single small-molecule platform remains a challenge. In this study, we developed a multifunctional probe based on a small-molecule platform. The properties of this probe were investigated via hyperpolarized 129Xe NMR/MRI, fluorescence imaging in cells and in vivo, and photodynamic therapy (PDT) in tumor mouse models. This multifunctional probe shows good pH response across a broad range of pH values. It also exhibits excellent fluorescence in vivo for mapping its biodistribution. Additionally, it produces enough 1O2 radicals for in vivo PDT. The combination of these functionalities into a single small-molecule platform, rather than a bulky nanoconstruct, offers unique possibilities for molecular imaging and therapy.

Wnt/ß-catenin signaling cascade: A promising target for glioma therapy.

Glioma is one of the most treatment-refractory intracranial tumors, and the aberrant expressed Wnt/ß-catenin pathway is closely associated with glioma malignancy. In this regard, Wnt/ß-catenin signaling has been reported to play an essential role in cellular proliferation, migration, invasion, and angiogenesis, therefore contributing to glioma progression. However, the underlying mechanisms of Wnt/ß-catenin signaling involvement in gliomagenesis remain unknown. Here, we present an overview of the Wnt components and then go on to summarize the current knowledge describing the multitude of roles of Wnt/ß-catenin in glioma, which are mediated by transcription factors, microRNAs, long noncoding RNAs, and so on. In the latter portion of the review, we elaborate the increasing apparent crosstalk of Wnt/ß-catenin pathway with PI3K/AKT signaling involved in these processes. Ultimately, compounds targeting the Wnt/ß-catenin are described in glioma. As better understanding of the regulatory mechanisms to glioma malignancies increases, Wnt/ß-catenin cascade may represent an area of developmental glioma therapeutics focus.

An intracellular diamine oxidase triggered hyperpolarized 129Xe magnetic resonance biosensor.

Here, a novel method was developed for suppressing 129Xe signals in cucurbit[6]uril (CB6) until the trigger is activated by a specific enzyme. Due to its noncovalent interactions with amino-groups and CB6, putrescine dihydrochloride (Put) was chosen for blocking interactions between 129Xe and CB6. Upon adding diamine oxidase (DAO), Put was released from CB6 and a 129Xe@CB6 Hyper-CEST signal emerged. This proposed 129Xe biosensor was then tested in small intestinal villus epithelial cells.

Comprehensive Bioinformatic Analysis Genes Associated to the Prognosis of Liposarcoma.

BACKGROUND Liposarcoma is the most common type of soft tissue sarcoma, but its molecular mechanism is poorly defined. This study aimed to identify genes crucial to the pathogenesis of liposarcoma and to explore their functions, related pathways, and prognostic value. MATERIAL AND METHODS Differentially expressed genes (DEGs) in the GSE59568 dataset were screened. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted to investigate the DEGs at the functional level. Protein-protein interaction (PPI) networks and module analysis were applied to identify hub genes from among the DEGs. The GSE30929 dataset was used to validate the relationship between hub genes and the distant recurrence-free survival (DRFS) of liposarcoma patients using Cox model analysis. RESULTS A total of 1111 DEGs were identified. GO and KEGG pathway analysis indicated that the DEGs were mainly associated with lipopolysaccharides and pathways in cancer. The PPI network and module analysis identified 10 hub genes from the DEG network. The Cox model identified 3 genes (NIP7, RPL10L, and MCM2) significantly associated with DRFS. The risk score calculated by the Cox model of the NIP7-RPL10L-MCM2 signature could largely predict the 1-, 3-, and 5-year DRFS of liposarcoma patients, and the prognostic value was even higher for subtypes of liposarcoma. CONCLUSIONS This study identified genes that might play critical roles in liposarcoma pathogenesis as well as a 3-gene-based signature that could be used as a candidate prognostic biomarker for patients with liposarcoma.

The Alzheimer's disease-protective CD33 splice variant mediates adaptive loss of function via diversion to an intracellular pool.

The immunomodulatory receptor Siglec-3/CD33 influences risk for late-onset Alzheimer's disease (LOAD), an apparently human-specific post-reproductive disease. CD33 generates two splice variants: a full-length CD33M transcript produced primarily by the "LOAD-risk" allele and a shorter CD33m isoform lacking the sialic acid-binding domain produced primarily from the "LOAD-protective" allele. An SNP that modulates CD33 splicing to favor CD33m is associated with enhanced microglial activity. Individuals expressing more protective isoform accumulate less brain ß-amyloid and have a lower LOAD risk. How the CD33m isoform increases ß-amyloid clearance remains unknown. We report that the protection by the CD33m isoform may not be conferred by what it does but, rather, from what it cannot do. Analysis of blood neutrophils and monocytes and a microglial cell line revealed that unlike CD33M, the CD33m isoform does not localize to cell surfaces; instead, it accumulates in peroxisomes. Cell stimulation and activation did not mobilize CD33m to the surface. Thus, the CD33m isoform may neither interact directly with amyloid plaques nor engage in cell-surface signaling. Rather, production and localization of CD33m in peroxisomes is a way of diminishing the amount of CD33M and enhancing ß-amyloid clearance. We confirmed intracellular localization by generating a CD33m-specific monoclonal antibody. Of note, CD33 is the only Siglec with a peroxisome-targeting sequence, and this motif emerged by convergent evolution in toothed whales, the only other mammals with a prolonged post-reproductive lifespan. The CD33 allele that protects post-reproductive individuals from LOAD may have evolved by adaptive loss-of-function, an example of the less-is-more hypothesis.

Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E.

CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation. Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer.

Biothiol Xenon MRI Sensor Based on Thiol-Addition Reaction.

Biothiols such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play an important role in regulating the vital functions of living organisms. Knowledge of their biodistribution in real-time could help diagnose a variety of conditions. However, existing methods of biothiol detection are invasive and require assays. Herein we report a molecular biosensor for biothiol detection using the nuclear spin resonance of (129)Xe. The (129)Xe biosensor consists of a cryptophane cage encapsulating a xenon atom and an acrylate group. The latter serves as a reactive site to covalently bond biothiols through a thiol-addition reaction. The biosensor enables discrimination of Cys from Hcy and GSH through the chemical shift and average reaction rate. This biosensor can be detected at a concentration of 10 µM in a single scan and it has been applied to detect biothiols in bovine serum solution. Our results indicate that this biosensor is a promising tool for the real-time imaging of biothiol distributions.

Proteomic study of benign and malignant pleural effusion.

BACKGROUND: Lung adenocarcinoma can easily cause malignant pleural effusion which was difficult to discriminate from benign pleural effusion. Now there was no biomarker with high sensitivity and specificity for the malignant pleural effusion. PURPOSE: This study used proteomics technology to acquire and analyze the protein profiles of the benign and malignant pleural effusion, to seek useful protein biomarkers with diagnostic value and to establish the diagnostic model. METHODS: We chose the weak cationic-exchanger magnetic bead (WCX-MB) to purify peptides in the pleural effusion, used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to obtain peptide expression profiles from the benign and malignant pleural effusion samples, established and validated the diagnostic model through a genetic algorithm (GA) and finally identified the most promising protein biomarker. RESULTS: A GA diagnostic model was established with spectra of 3930.9 and 2942.8 m/z in the training set including 25 malignant pleural effusion and 26 benign pleural effusion samples, yielding both 100 % sensitivity and 100 % specificity. The accuracy of diagnostic prediction was validated in the independent testing set with 58 malignant pleural effusion and 34 benign pleural effusion samples. Blind evaluation was as follows: the sensitivity was 89.6 %, specificity 88.2 %, PPV 92.8 %, NPV 83.3 % and accuracy 89.1 % in the independent testing set. The most promising peptide biomarker was identified successfully: Isoform 1 of caspase recruitment domain-containing protein 9 (CARD9), with 3930.9 m/z, was decreased in the malignant pleural effusion. CONCLUSIONS: This model is suitable to discriminate benign and malignant pleural effusion and CARD9 can be used as a new peptide biomarker.

Effect of cytotoxic T-lymphocyte antigen-4, TNF-alpha polymorphisms on osteosarcoma: evidences from a meta-analysis.

OBJECTIVE: Previous studies have investigated the role of cytotoxic T-lymphocyte antigen-4 (CTLA-4) and tumor necrosis factor-alpha (TNF-α) in carcinogenesis of osteosarcoma, but their results were inconsistent. We aimed to clarify the associations between CTLA-4, TNF-α polymorphism and osteosarcoma risk by using meta-analysis. METHODS: We searched relevant studies without language restriction in PubMed, EMbase, Cochrane Library, Google Scholar databases, Chinese National Knowledge Infrastructure (CNKI) and conference literature in humans published prior to March 2013. The strengths of the associations between genetic variants and osteosarcoma risk were estimated by odds ratio (OR) with 95% confidence interval (95% CI). RESULTS: A total of seven studies with 1,198 osteosarcoma patients and 1,493 controls were selected. Four studies were eligible for CTLA-4 (1,003 osteosarcoma and 1,162 controls), and three studies for TNF-α (195 osteosarcoma and 331 controls). Pooled results showed that rs231775 polymorphism of CTLA-4 was associated with osteosarcoma risk (GG vs. AA: OR=1.63, 95% CI=1.24-2.13; GG + GA vs. AA: OR=1.56, 95% CI=1.21-2.01; AA + GA vs. GG: OR=0.83, 95% CI=0.71-0.97; G vs. A: OR=1.21, 95% CI=1.08-1.36). No significant heterogeneity was observed across the studies. No significant associations were found between rs5742909 polymorphism of CTLA-4 or rs1800629 polymorphism of TNF-α and osteosarcoma risk. CONCLUSIONS: These results suggest that the rs231775 polymorphism of CTLA-4 may play an important role in carcinogenesis of osteosarcoma.

ProMMP-2: TIMP-1 complexes identified in plasma of healthy individuals.

Activation of MMPs in tissues is an important component of tissue injury. Based on earlier reports that (latent) proMMP-2 is incapable of forming a complex with TIMP-1, we reasoned that the identification of MMP-2:TIMP-1 complexes in blood might serve as a surrogate marker ("smoking gun") of MMP-2 activation in tissues. Using specific antibodies, we developed a sensitive and specific assay to detect MMP-2:TIMP-1 complexes. We were perplexed to find that approximate 40% of plasma specimens from healthy individuals had detectable levels of the MMP-2:TIMP-1 complexes. Employing recombinant TIMP-1 bound Sepharose beads and Western blots, we demonstrated binding between recombinant proMMP-2 and TIMP-1 proteins. Recombinant MMP-2 lacking the catalytic domain also bound to TIMP-1 coated beads. These data are consistent with TIMP-1 binding to the hemopexin or hinge domain of proMMP-2. The explanation for the presence of plasma proMMP-2:TIMP-1 complexes in selected healthy individuals remains to be determined. In contrast to our immunoassay and bead-binding experiments, proMMP-2 failed to bind to immobilized TIMP-1 employing surface plasmon resonance technology. Additional studies are needed to clarify these contrasting results.

Enzymatic properties of human kallikrein-related peptidase 12 (KLK12).

Human kallikrein-related peptidase 12 (KLK12) is a new member of the human tissue kallikrein family. Preliminary studies suggest that KLK12 is differentially expressed in breast cancer and may have potential use as a cancer biomarker. It has been predicted that KLK12 is a secreted serine protease. However, the enzymatic properties of this protein have not been reported so far. Here, we report the production of recombinant KLK12 and analyses of its enzymatic characteristics, including zymogen activation, substrate specificity, and regulation of its activity. KLK12 is secreted as an inactive pro-enzyme, which is able to autoactivate to gain enzymatic activity. Through screening of a panel of fluorogenic and chromogenic peptide substrates, we establish that active KLK12 possesses trypsin-like activity, cleaving peptide bonds after both arginine and lysine. Active KLK12 quickly loses its activity due to autodegradation, and its activity can also be rapidly inhibited by zinc ions and by alpha2-antiplasmin through covalent complex formation. Furthermore, we demonstrate that KLK12 is able to activate KLK11 zymogen in vitro. Our results indicate that KLK12 may participate in enzymatic cascades involving other kallikreins.

Inhibition profiles of human tissue kallikreins by serine protease inhibitors.

Accumulated evidence has shown that human tissue kallikreins (hKs), a group of 15 homologous secreted serine proteases, are novel cancer biomarkers. We report here the inhibition profiles of selected hKs, including hK5, hK7, hK8, hK11, hK12, hK13, and hK14, by several common serine protease inhibitors (serpins) found in plasma. The association constants for the binding of serpins to kallikreins were determined and compared. Protein C inhibitor was found to be the fastest-binding serpin for most of these hKs. alpha2-Antiplasmin, alpha1-antichymotrypsin, and alpha1-antitrypsin also showed rapid inhibition of certain hKs. Kallistatin exhibited fast inhibition only with hK7. Our data demonstrate that these hKs are specifically regulated by certain serpins and their distinct inhibition profiles will be valuable aids in various aspects of kallikrein research.

Mannan-binding lectin-associated serine protease 3 cleaves synthetic peptides and insulin-like growth factor-binding protein 5.

Mannan-binding lectin-associated serine proteases (MASPs) are secreted as single-chain precursors and processed into two disulfide bond-linked chains. MASP-3 and MASP-1, derived from the same gene, contain identical A chains, but entirely different catalytic domain-containing B chains. In contrast to MASP-1 and MASP-2, the proteinase activity of MASP-3 has not been described previously. We show here the proteolytic activity of the purified recombinant human MASP-3 catalytic domain toward peptides and protein substrates. Among the fluorogenic peptides tested, it specifically cleaved peptides with Arg at the P1 position. Among seven insulin-like growth factor-binding proteins, it selectively cleaved IGFBP-5, which is the first protein substrate identified for MASP-3. All three cleavage sites identified contained Arg or Lys at the P1 position and Pro at the P2 position. As compared to MASP-1 and MASP-2, MASP-3 has distinct substrate specificity and inhibitor profile. These results should be useful for further studies of the structure and function of human MASP-3.

Characterization of cathepsin L secreted by Sf21 insect cells.

Sf21 cells, derived from the Spodoptera frugiperda pupa, are commonly used for the heterologous expression of proteins. While purifying recombinant proteins from this system we encountered a protease, secreted at high levels by Sf21 cells, that readily degraded recombinant proteins and also tended to co-purify with histidine-tagged proteins from Ni(2+) affinity columns. Purification and characterization of the protease revealed that it has many properties consistent with cysteine proteases of the papain family, including autoactivation under reducing conditions and acidic pH, and inhibition by E-64. Amino acid sequence analysis showed that the Sf21 enzyme may be identical to a putative insect procathepsin L cloned from the cotton bollworm. The subsite specificity of the Sf21 cathepsin and its inhibition profile by cystatins are consistent with the protease being an insect homologue of cathepsin L. Monoclonal antibodies useful for the detection and purification of the insect cathepsin L were developed.