Spatial analysis of stromal signatures identifies invasive front carcinoma-associated fibroblasts as suppressors of anti-tumor immune response in esophageal cancer.

BACKGROUND: Increasing evidence indicates that the tumor microenvironment (TME) is a crucial determinant of cancer progression. However, the clinical and pathobiological significance of stromal signatures in the TME, as a complex dynamic entity, is still unclear in esophageal squamous cell carcinoma (ESCC). METHODS: Herein, we used single-cell transcriptome sequencing data, imaging mass cytometry (IMC) and multiplex immunofluorescence staining to characterize the stromal signatures in ESCC and evaluate their prognostic values in this aggressive disease. An automated quantitative pathology imaging system determined the locations of the lamina propria, stroma, and invasive front. Subsequently, IMC spatial analyses further uncovered spatial interaction and distribution. Additionally, bioinformatics analysis was performed to explore the TME remodeling mechanism in ESCC. To define a new molecular prognostic model, we calculated the risk score of each patient based on their TME signatures and pTNM stages. RESULTS: We demonstrate that the presence of fibroblasts at the tumor invasive front was associated with the invasive depth and poor prognosis. Furthermore, the amount of α-smooth muscle actin (α-SMA)+ fibroblasts at the tumor invasive front positively correlated with the number of macrophages (MØs), but negatively correlated with that of tumor-infiltrating granzyme B+ immune cells, and CD4+ and CD8+ T cells. Spatial analyses uncovered a significant spatial interaction between α-SMA+ fibroblasts and CD163+ MØs in the TME, which resulted in spatially exclusive interactions to anti-tumor immune cells. We further validated the laminin and collagen signaling network contributions to TME remodeling. Moreover, compared with pTNM staging, a molecular prognostic model, based on expression of α-SMA+ fibroblasts at the invasive front, and CD163+ MØs, showed higher accuracy in predicting survival or recurrence in ESCC patients. Regression analysis confirmed this model is an independent predictor for survival, which also identifies a high-risk group of ESCC patients that can benefit from adjuvant therapy. CONCLUSIONS: Our newly defined biomarker signature may serve as a complement for current clinical risk stratification approaches and provide potential therapeutic targets for reversing the fibroblast-mediated immunosuppressive microenvironment.

Promotion of rs3746804 (p. L267P) polymorphism to intracellular SLC52A3a trafficking and riboflavin transportation in esophageal cancer cells.

Riboflavin is an essential micronutrient for normal cellular growth and function. Lack of dietary riboflavin is associated with an increased risk for esophageal squamous cell carcinoma (ESCC). Previous studies have identified that the human riboflavin transporter SLC52A3a isoform (encoded by SLC52A3) plays a prominent role in esophageal cancer cell riboflavin transportation. Furthermore, SLC52A3 gene single nucleotide polymorphisms rs3746804 (T>C, L267P) and rs3746803 (C >T, T278M) are associated with ESCC risk. However, whether SLC52A3a (p.L267P) and (p.T278M) act in riboflavin transportation in esophageal cancer cell remains inconclusive. Here, we constructed the full-length SLC52A3a protein fused to green fluorescent protein (GFP-SLC52A3a-WT and mutants L267P, T278M, and L267P/T278M). It was confirmed by immunofluorescence-based confocal microscopy that SLC52A3a-WT, L267P, T278M, and L267P/T278M expressed in cell membrane, as well as in a variety of intracellular punctate structures. The live cell confocal imaging showed that SLC52A3a-L267P and L267P/T278M increased the intracellular trafficking of SLC52A3a in ESCC cells. Fluorescence recovery after photobleaching of GFP-tagged SLC52A3a meant that intracellular trafficking of SLC52A3a-L267P and L267P/T278M was rapid dynamics process, leading to its stronger ability to transport riboflavin. Taken together, the above results indicated that the rs3746804 (p.L267P) polymorphism promoted intracellular trafficking of SLC52A3a and riboflavin transportation in ESCC cells.

Lithium-induced overexpression of ß-catenin delays murine palatal shelf elevation by Cdc-42 mediated F-actin remodeling in mesenchymal cells.

BACKGROUND: Lithium chloride (LiCl) is widely used for the treatment of manic and other psychotic disorders, but the administration of lithium can result in several congenital defects in the fetus, including cleft palate (Meng, Wang, Torensma, Jw & Bian, 2015) (Szabo, 1970). However, the mechanism of Lithium's action as a developmental toxicant in palatogenesis is not well known. METHODS: In this study, hematoxylin-eosin and immunofluorescence staining were employed to evaluate the phenotypes and the expression of related markers in the LiCl-treated mice model. The palatal mesenchymal cells were cultured in vitro, and stimulated with LiCl or SKL2000, and co-treated with CASIN. ß-catenin protein and other cytoskeleton associated markers were evaluated by Western blotting. RESULTS: We found that Lithium disrupted palate elevation by increasing the expression of ß-catenin in C57BL/6J mice with the high incidence of cleft palate (62.5%). LiCl disturbed the F-actin responsible for cytoskeletal remodeling in mesenchymal cells, which proved to be essential in generating the elevating force during palatal elevation. Additionally, our Western blotting analysis revealed that the overexpression of ß-catenin resulted in up-regulation of Cdc42, which mediated the downstream F-actin synthesis. CONCLUSIONS: We concluded the LiCl-induced ß-catenin overexpression delayed murine palatal shelf elevation by disturbing Cdc42 mediated F-actin cytoskeleton synthesis in the palatal mesenchyme.

A novel colorimetric and "turn-off" fluorescent probe based on catalyzed hydrolysis reaction for detection of Cu2+ in real water and in living cells.

A novel and highly selective fluorescent 1,8-naphthalimide-based probe, 3, was designed and synthesized for rapid Cu2+ detection in a CH3CN-H2O (3:1, v/v, pH = 7.4) solution by means of a distinct hydrolysis mechanism via its Cu2+-promoting feature. Upon treatment with Cu2+, the fluorescence response of probe 3 at 550 nm abruptly decreased, which was visible to the naked eye, and this response was accompanied by a clear change of the color of the solution; the color changed from the original yellow color to colorless. This color change occurred due to the Cu2+-promoted hydrolysis of 3, which yielded a fluorescence-quenched product. It is inspiring that probe 3 exhibited excellent sensitivity, a short response time and strong anti-interference recognition. Compared with the allowable amount of Cu2+ (∼20 µM) in drinking water, the detection limit of 3 for Cu2+ is calculated to be 9.15 nM, which is much lower than the amount defined by standards. The probe can be successfully applied for the determination of Cu2+ in real aqueous samples. Furthermore, probe 3 can be used as a fluorescent sensor to detect Cu2+ in biological environments, demonstrating its low toxicity to organisms and good cell permeability in live cell imaging.

Dietary riboflavin deficiency promotes N-nitrosomethylbenzylamine-induced esophageal tumorigenesis in rats by inducing chronic inflammation.

Epidemiological studies in high-incidence areas of esophageal cancer in China suggest that environmental carcinogen N-nitrosomethylbenzylamine (NMBA) and riboflavin (RBF) deficiency may be the main risk factors for esophageal cancer. However, it is not clear that the combination induces cancer. Here, experiment (Exp) 1 evaluated the effects of NMBA and RBF deficiency individually or in combination on esophageal tumorigenesis. Male F344 rats were randomly assigned to 4 groups into a 2 (no NMBA vs. NMBA) × 2 (normal RBF vs. RBF-deficient) factorial design, including normal RBF (6 mg/kg, R6), RBF-deficient (0 mg/kg, R0), normal RBF combined with NMBA (R6N), and RBF-deficient combined with NMBA (R0N) groups. The Exp 2 explored the effects of RBF deficiency at different doses combined with NMBA (0.6 mg/kg, R0.6N; 0.06 mg/kg, R0.06N) on esophageal tumorigenesis. Results showed that R0N enhanced the incidence of esophageal intraepithelial neoplasia (EIN, 53.3%, P = 0.06), including carcinoma in situ, whereas R6N mainly induced the occurrence of esophageal benign hyperplasia (38.9%) and EIN (16.7%). RBF deficiency promotes EIN in a dose-dependent manner, and R0.06N significantly increases the incidence of EIN (57.9%, P < 0.05). Gene expression profiling demonstrated that inflammatory cytokines were highly expressed in R0N EIN tissues, whereas R6N EIN tissues had a proliferation and differentiation gene signature (fold-change > 1.5). Furthermore, RBF deficiency aggravated oxidative DNA damage (8-OHdG) and double-strand breaks (γH2AX) (P < 0.05). Our results suggest that RBF deficiency causes chronic inflammation-associated genomic instability contributes to NMBA-induced esophageal tumorigenesis.

A New Fluorescent Chemosensor for Cobalt(II) Ions in Living Cells Based on 1,8-Naphthalimide.

In this work, a highly selective fluorescent chemosensor N-(2-(2-butyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)hydrazine-1-carbonothioyl)benzamide (L) was prepared and characterized. An assay to detect the presence of cobalt(II) ions was developed by utilizing turn-on fluorescence enhancement with visual colorimetric response. Upon treatment with Co2+, a remarkable fluorescence enhancement located at 450 nm was visible to naked eyes accompanied with a distinct color change (from pink to colorless) in a CH3CN/HEPES (4/1, v/v, pH = 7.4) solution due to the formation of a 1:1 complex at room temperature. In addition, the linear concentration range for Co2+ was 0-25 µM with the limit of detection down to 0.26 µM. Thus, a highly sensitive fluorescent method based on chelation-assisted fluorescence enhancement was developed for the trace-level detection of Co2+. The sensor was found to be highly selective toward Co2+ ions with a large number of coexisting ions. Furthermore, the L probe can serve as a fluorescent sensor for Co2+ detecting in biological environments, demonstrating its low toxic properties to organisms and good cell permeability in live cell imaging.

A novel dithiourea-appended naphthalimide "on-off" fluorescent probe for detecting Hg2+ and Ag+ and its application in cell imaging.

An effective dithiourea-appended 1,8-naphthalimide fluorescent probe was designed and synthesized. This probe could recognize Hg2+ and Ag+ sensitively and selectively in neutral and alkaline conditions. Moreover, the probe detected Hg2+ alone at pH between 2 and 6. The sensing ability of the probe was explored by UV-vis, fluorescence, FTIR and 1H NMR spectroscopy. The probe was quenched by Hg2+ and Ag+ with 1:1 binding ratios in MeCN/H2O (4/1, v/v) mixed solution with binding constants of 3.76 × 104 L mol-1 and 2.47 × 104 L mol-1, respectively. The linear concentration ranges for Hg2+ and Ag+ were 0-17 µmol L-1 and 0-24 µmol L-1 with detection limits of 0.83 µmol L-1 and 1.20 µmol L-1, respectively, which allowed for the quantitative determination of Hg2+ and Ag+. The new probe, 3a, was successfully applied to the fluorescence imaging of Hg2+ and Ag+ in HepG2 cells, demonstrating its potential application in biological science. Moreover, 3a was used to measure Hg2+ and Ag+ in tap water, drinking water and ultrapure water samples. The recoveries of Hg2+ and Ag+ in water samples were 96-99% and 98-103%, respectively. Therefore, the proposed method showed promising perspectives for its application, aimed at detecting Hg2+ and Ag+ in fluorescence imaging and real water samples.

A highly sensitive and selective fluorescent probe for determination of Cu (II) and application in live cell imaging.

A highly sensitive fluorescent probe, 2­butyl­6­(2­ethylidenehydrazinyl)­1H­benzo[de]isoquinoline­1,3(2H)­dione (P) has been designed and synthesized to detect Cu2+ in CH3CN-HEPES (4:1, v/v, pH = 7.4) solution. This probe functions via a distinctive hydrolysis mechanism through Cu2+-promoted accompany by extinction of the fluorescent and other competing metal ions did not showed any interference. The limit of detection toward Cu2+ was calculated of 320 nM. Probe P was not only successfully used for the determination of trace level Cu2+ in the CH3CN-HEPES (4:1, v/v, pH = 7.4) solution, but also valid for fluorescence imaging of Cu2+ in lysosomes of 293T cells and it was applied in real water samples. This work indicated that P would be of great application prospect in environmental monitoring and medical diagnosis.

Synthesis and Fluorescent Property Study of Novel 1,8-Naphthalimide-Based Chemosensors.

A series of novel mono- and di-substituted N-n-butyl-1,8-naphthalimide derivatives were synthesized simultaneously via a three-step reaction. The single crystal structure of N-n-butyl-4-[N',N'-bis(2',4'-dichlorobenzoyl)ethylamino]-1,8-naphthalimide (3f) was determined. The UV-vis and fluorescence properties of compound 3f were investigated. The 3f showed highly selective and sensitive fluorescence changes response towards Pb2+. A titration of monomer with Pb2+ ion was performed. When Pb2+ ion concentration increased from 0 to 10 eq., the fluorescent intensity of 3f decreased from 199.97 to 48.21. The pH effect on 3f showed that it is stable in a wide range of pH. The results indicated that 3f might be a probe molecule for Pb2+.

SLC52A3 expression is activated by NF-κB p65/Rel-B and serves as a prognostic biomarker in esophageal cancer.

The human riboflavin transporter-3 (encoded by SLC52A3) plays a prominent role in riboflavin absorption. Interestingly, abnormal expression patterns of SLC52A3 in multiple types of human cancers have been recently noted. However, the molecular mechanisms underlying its dysregulation remain unclear. In this study, we find that SLC52A3 has two transcript variants that differ in the transcriptional start site, and encode different proteins: SLC52A3a and SLC52A3b. Importantly, aberrant expressions of SLC52A3 are associated with stepwise development of esophageal squamous cell carcinoma (ESCC) as well as the survival rates of ESCC patients. Functionally, SLC52A3a, but not SLC52A3b, strongly promotes the proliferation and colony formation of ESCC cells. Furthermore, SLC52A3 5'-flanking regions contain NF-κB p65/Rel-B-binding sites, which are crucial for mediating SLC52A3 transcriptional activity in ESCC cells. Chromatin immunoprecipitation and electrophoretic mobility shift assay reveal that p65/Rel-B bind to 5'-flanking regions of SLC52A3. Accordingly, NF-κB signaling upregulates SLC52A3 transcription upon TNFα stimulation. Taken together, these results elucidate the mechanisms underlying SLC52A3 overexpression in ESCC. More importantly, our findings identify SLC52A3 as both a predictive and prognostic biomarker for this deadly cancer.

Biflavanones with anti-proliferative activity against eight human solid tumor cell lines from Stellera chamaejasme.

Six 3,3″-biflavanones, including a new compound isochamaejasmenin C (1), were isolated from EtOH extracts of the roots of Stellera chamaejasme L. Their structures were elucidated on the basis of spectroscopic methods, including HR-ESI-MS and 2D NMR techniques. The absolute configurations on 2, 3, 3″, and 2″ of compounds 1, 3, and 6, which represent three geometry types of these compounds, were determined by TDDFT quantum chemical calculations of their ECD spectra. All compounds were evaluated for their cytotoxicities against eight human solid tumor cell lines and compounds 2, 4, and 6 showed strong anti-proliferative effects against all these cell lines with IC50 values ranging from 1.08 to 15.97 µM, which were in the same range as the positive control paclitaxel.