Multiplex Immunofluorescence: A Powerful Tool in Cancer Immunotherapy.

Traditional immunohistochemistry (IHC) has already become an essential method of diagnosis and therapy in cancer management. However, this antibody-based technique is limited to detecting a single marker per tissue section. Since immunotherapy has revolutionized the antineoplastic therapy, developing new immunohistochemistry strategies to detect multiple markers simultaneously to better understand tumor environment and predict or assess response to immunotherapy is necessary and urgent. Multiplex immunohistochemistry (mIHC)/multiplex immunofluorescence (mIF), such as multiplex chromogenic IHC and multiplex fluorescent immunohistochemistry (mfIHC), is a new and emerging technology to label multiple biomarkers in a single pathological section. The mfIHC shows a higher performance in cancer immunotherapy. This review summarizes the technologies, which are applied for mfIHC, and discusses how they are employed for immunotherapy research.

Near Infrared Photoimmunotherapy: A Review of Recent Progress and Their Target Molecules for Cancer Therapy.

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed molecular targeted cancer treatment, which selectively kills cancer cells or immune-regulatory cells and induces therapeutic host immune responses by administrating a cancer targeting moiety conjugated with IRdye700. The local exposure to near-infrared (NIR) light causes a photo-induced ligand release reaction, which causes damage to the target cell, resulting in immunogenic cell death (ICD) with little or no side effect to the surrounding normal cells. Moreover, NIR-PIT can generate an immune response in distant metastases and inhibit further cancer attack by combing cancer cells targeting NIR-PIT and immune regulatory cells targeting NIR-PIT or other cancer treatment modalities. Several recent improvements in NIR-PIT have been explored such as catheter-driven NIR light delivery, real-time monitoring of cancer, and the development of new target molecule, leading to NIR-PIT being considered as a promising cancer therapy. In this review, we discuss the progress of NIR-PIT, their mechanism and design strategies for cancer treatment. Furthermore, the overall possible targeting molecules for NIR-PIT with their application for cancer treatment are briefly summarised.

EpCAM- and EGFR-Specific Antibody Drug Conjugates for Triple-Negative Breast Cancer Treatment.

Triple-negative breast cancer (TNBC) is a group of heterogeneous and refractory breast cancers with the absence of estrogen receptor (ER), progesterone receptor (PgR) and epidermal growth factor receptor 2 (HER2). Over the past decade, antibody drug conjugates (ADCs) have ushered in a new era of targeting therapy. Since the epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM) are over expressed on triple-negative breast cancer, we developed novel ADCs by conjugating benzylguanine (BG)-modified monomethyl auristatin E (MMAE) to EpCAM- and EGFR-specific SNAP-tagged single chain antibody fragments (scFvs). Rapid and efficient conjugation was achieved by SNAP-tag technology. The binding and internalization properties of scFv-SNAP fusion proteins were confirmed by flow cytometry and fluorescence microscopy. The dose-dependent cytotoxicity was evaluated in cell lines expressing different levels of EGFR and EpCAM. Both ADCs showed specific cytotoxicity to EGFR or EpCAM positive cell lines via inducing apoptosis at a nanomolar concentration. Our study demonstrated that EGFR specific scFv-425-SNAP-BG-MMAE and EpCAM-specific scFv-EpCAM-SNAP-BG-MMAE could be promising ADCs for the treatment of TNBC.

Atractylenolide III ameliorates spinal cord injury in rats by modulating microglial/macrophage polarization.

BACKGROUND: Inflammatory reactions induced by spinal cord injury (SCI) are essential for recovery after SCI. Atractylenolide III (ATL-III) is a natural monomeric herbal bioactive compound that is mainly derived in Atractylodes macrocephala Koidz and has anti-inflammatory and neuroprotective effects. OBJECTIVE: Here, we speculated that ATL-III may ameliorate SCI by modulating microglial/macrophage polarization. In the present research, we focused on investigating the role of ATL-III on SCI in rats and explored the potential mechanism. METHODS: The protective and anti-inflammatory effects of ATL-III on neuronal cells were examined in a rat SCI model and lipopolysaccharide (LPS)-stimulated BV2 microglial line. The spinal cord lesion area, myelin integrity, and surviving neurons were assessed by specific staining. Locomotor function was evaluated by the Basso, Beattie, and Bresnahan (BBB) scale, grid walk test, and footprint test. The activation and polarization of microglia/macrophages were assessed by immunohistofluorescence and flow cytometry. The expression of corresponding inflammatory factors from M1/M2 and the activation of relevant signaling pathways were assessed by Western blotting. RESULTS: ATL-III effectively improved histological and functional recovery in SCI rats. Furthermore, ATL-III promoted the transformation of M1 into M2 and attenuated the activation of microglia/macrophages, further suppressing the expression of corresponding inflammatory mediators. This effect may be partly mediated by inhibition of neuroinflammation through the NF-κB, JNK MAPK, p38 MAPK, and Akt pathways. CONCLUSION: This study reveals a novel effect of ATL-III in the regulation of microglial/macrophage polarization and provides initial evidence that ATL-III has potential therapeutic benefits in SCI rats.

Effect of morroniside on the transcriptome profiles of rat in injured spinal cords.

We have previously reported that morroniside promoted motor activity after spinal cord injury (SCI) in rats. However, the mechanism by which morroniside induces recovery of injured spinal cord (SC) remains unknown. In the current study, RNA sequencing (RNA-seq) was employed to evaluate changes of gene expressions at the transcriptional level of the injured spinal cords in morroniside-administrated rats. Principal component analysis, analysis of enriched Gene Ontology (GO), enrichment analyses Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and other bioinformatics analyses were executed to distinguish differentially expressed genes (DEGs). The results of RNA-seq confirmed the anti-inflammatory and anti-apoptotic effects of morroniside on injured SC tissues, and provided the basis for additional research of the mechanisms involving the protective effects of morroniside on SCI.

Toward Homogenous Antibody Drug Conjugates Using Enzyme-Based Conjugation Approaches.

In the last few decades, antibody-based diagnostic and therapeutic applications have been well established in medicine and have revolutionized cancer managements by improving tumor detection and treatment. Antibodies are unique medical elements due to their powerful properties of being able to recognize specific antigens and their therapeutic mechanisms such as blocking specific pathways, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity. Furthermore, modification techniques have paved the way for improving antibody properties and to develop new classes of antibody-conjugate-based diagnostic and therapeutic agents. These techniques allow arming antibodies with various effector molecules. However, these techniques are utilizing the most frequently used amino acid residues for bioconjugation, such as cysteine and lysine. These bioconjugation approaches generate heterogeneous products with different functional and safety profiles. This is mainly due to the abundance of lysine and cysteine side chains. To overcome these limitations, different site-direct conjugation methods have been applied to arm the antibodies with therapeutic or diagnostics molecules to generate unified antibody conjugates with tailored properties. This review summarizes some of the enzyme-based site-specific conjugation approaches.

Humidity Sensors with Shielding Electrode Under Interdigitated Electrode.

Recently, humidity sensors have been investigated extensively due to their broad applications in chip fabrication, health care, agriculture, amongst others. We propose a capacitive humidity sensor with a shielding electrode under the interdigitated electrode (SIDE) based on polyimide (PI). Thanks to the shielding electrode, this humidity sensor combines the high sensitivity of parallel plate capacitive sensors and the fast response of interdigitated electrode capacitive sensors. We use COMSOL Multiphysics to design and optimize the SIDE structure. The experimental data show very good agreement with the simulation. The sensitivity of the SIDE sensor is 0.0063% ± 0.0002% RH. Its response/recovery time is 20 s/22 s. The maximum capacitance drift under different relative humidity is 1.28% RH.

Effect of Hyperin and Icariin on steroid hormone secretion in rat ovarian granulosa cells.

AIM OF THE STUDY: This study was designed to investigate the effect of different concentrations of Hyperin and Icariin (ICA)on proliferation and the secretion of estrogen (E2), and progesterone (P) in granulosa cells, and to explore the effect of Hyperin and Icariin on the expression of CYP17 and CYP19. MATERIALS AND METHODS: Rat ovary granulosa cells were cultured in vitro and treated with different concentrations of Hyperin and Icariin. The proliferation of ovarian granulosa cells was measured with the MTT assay. The concentration of estradiol was measured with a magnetic particle-based enzyme-linked immunosorbent assay (ELISA) kit. The CYP17 and CYP19 mRNA expression was detected by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). The CYP17 and CYP19 protein expression was determined with Western blotting. RESULTS: Hyperin (50 µg/l) and Icariin (10 µg/l) significantly increased proliferation of ovarian granulosa cells and secretion of estrogen and progesterone. Hyperin and Icariin stimulated the mRNA and protein expression of CYP17 and CYP19. CONCLUSIONS: These results showed that Hyperin and Icariin can promote the secretion of E2 and P through up-regulation of CYP17 and CYP19. Frequently used Chinese herbs like Cuscuta Chinensis Lam and Epimedium Brevicornu maxim, which contain Hyperin and Icariin, could improve the ovarian endocrine function through these effects.

DNA demethylation pattern of in-vitro fertilized and cloned porcine pronuclear stage embryos.

Recent studies in mice showed that the Ten-eleven translocation Enzymes (TET) family is involved in the active DNA demethylation. The isotype TET-3 is responsible for the conversion of 5mc (5-methylcytosine) to 5hmc (5-hydroxymethylcytosine) at the pronuclear stages of mouse embryo. This study was performed to investigate the pattern of methylation change and the role of TET family in the demethylation process of porcine in-vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) derived embryo. Bisulfite-sequencing PCR (BSP) and DNA glucosylation and digestion before quantitative PCR (qGluMS-PCR) were done to evaluate the exact change of methylation during porcine pronuclear stages. The results showed that the amount of 5hmc detected increased whereas 5mc decreased in IVF embryo from pronuclear stage 2 (PN2) to pronuclear stage 5 (PN5). In addition, Immunofluorescent staining showed that the 5hmc signal, also detected in oocytes, significantly increased in both pronucleus from fertilization to PN2. The amount of 5hmc continued to rise in male pronucleus but decreased to a very low level in female pronucleus from PN2 to PN5. The above results indicate that female pronucleus might undergo active demethylation only at early pronuclear stages. On the other hand, male pronucleus might undergo active demethylation throughout all pronuclear stages. The expression of three TET isotypes (TET-1, TET-2, TET-3) were tested and TET-3 was found to be the highest expressed isotype. High TET-3 concentrations observed mainly in male pronucleus using immunofluorescent staining, implying that TET-3 might be the main enzyme which catalyzes the conversion of 5mc to 5hmc. In contrast, no TET-3 signal was detected in female pronucleus through the pronuclear stages. The demethylation pattern of SCNT embryos resembled that of the male pronucleus of IVF embryos, suggesting that active demethylation might happen in porcine cloned embryo.

[The influence of interfered circadian rhythm on pregnancy and neonatal rats].

The aim of this study was to observe the influence of interfered circadian rhythm on pregnancy of rats and growth of neonatal rats, and to explore the relationship between the interfered circadian rhythm and the changes of melatonin and progesterone. Continuous light was used to inhibit melatonin secretion and therefore the interfered circadian rhythm animal model was obtained. The influence of interfered circadian rhythm on delivery of pregnant rats was observed. Serum was collected from rats during different stages of pregnancy to measure the concentrations of melatonin and progesterone. In order to observe the embryo resorption rate, half of pregnant rats were randomly selected to undergo a laparotomy, and the remainder was used to observe delivery and assess the growth of neonatal rats after delivery. The results showed that the interfered circadian rhythm induced adverse effects on pregnancy outcomes, including an increase of embryo resorption rate and a decrease in the number of live births; inhibited the secretion of melatonin along with decreased serum progesterone level; prolonged the stage of labor, but not the duration of pregnancy; and disturbed the fetal intrauterine growth and the growth of neonatal rats. The results suggest that interfered circadian rhythm condition made by continuous light could make adverse effects on both pregnant rats and neonatal rats. The results of our study may provide a way to modulate pregnant women's circadian rhythm and a possibility of application of melatonin on pregnant women.

TGF-ß1 protein expression in non-small cell lung cancers is correlated with prognosis.

To investigate the expression intensity and prognostic significance of TGF-ß1 protein in non-small cell lung cancer (NSCLC), immunohistochemistry was carried out in 194 cases of NSCLC and 24 cases of normal lung tissues by SP methods. The PU (positive unit) value was used to assess the TGF-ß1 protein expression in systematically selected fields under the microscope with Leica Q500MC image analysis. We found that the TGF-ß1 PU value was nearly two-fold higher in NSCLC than in normal lung tissues (p=0.000), being associated with TNM stages (p=0.000) and lymph node metastases (p=0.000), but not to patient age, gender, smoking history, tumor differentiation, histological subtype and tumor location (P>0.05). Univariate analysis indicated that patients with high TGF-ß1 protein expression and lymph node metastases demonstrated a poor prognosis (both p=0.000, ). Multivariate analysis showed that TGF-ß1 protein expression (RR = 2.565, p=0.002) and lymph node metastases (RR=1.874, p= 0.030) were also independent prognostic factors. Thus, TGF-ß1 protein expression may be correlated to oncogenesis and serve as an independent prognostic biomarker for NSCLC.

Expression of Tiam1 predicts lymph node metastasis and poor survival of lung adenocarcinoma patients.

BACKGROUND: To assess the value of Tiam1 in predicting lymph node metastasis and survival after curative resection in patients with lung adenocarcinoma. METHODS: Immunohistochemical staining for Tiam1 was performed on 98 adenocarcinoma and 30 normal lung tissues. The association of Tiam1 protein expression with the clinicopathological characteristics and the prognosis of lung adenocarcinoma were subsequently assessed. RESULTS: Immunohistochemical analysis showed that 60 of 98 (61.22%) adenocarcinoma tissues showed high expression of Tiam1, and high Tiam1 expression was significantly associated with advanced TNM stage (P < 0.0005) and lymph node status (P < 0.0005) of lung adenocarcinoma. Moreover, the lung adenocarcinoma patients with low Tiam1 expression had higher overall survival than patients with high Tiam1 expression (log rank value = 10.805, P = 0.001). High expression of Tiam1 predicted poor overall survival of patients in stages I + II (P = 0.006). Furthermore, multivariate analysis indicated that high expression of Tiam1 protein was an independent prognostic factor for overall survival (P = 0.011) in patients with lung adenocarcinoma. CONCLUSION: These findings suggest for the first time that Tiam1 expression may be beneficial in predicting lymph node metastasis and survival of patients with lung adenocarcinoma. A future study will investigate whether Tiam1 can serve as a novel therapeutic target in lung adenocarcinoma. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1377798917111123.

Expression of PBK/TOPK in cervical cancer and cervical intraepithelial neoplasia.

UNLABELLED: objectives: To evaluate the expression of PBK/TOPK (PDZ-binding kinase/T-LAK cell-originated protein kinase) and its clinical significance in cervical cancer and cervical intraepithelial neoplasia. METHODS: PBK/TOPK expression was detected in 28 cases of low-grade cervical intraepithelial neoplasia (CINI), 62 cases of high-grade intraepithelial neoplasia and 80 cases of cervical cancer by immunohistochemistry (IHC). Then, the correlation between PBK/TOPK expression and clinicopathological features was quantitatively analyzed by measuring the positive unit (PU). RESULTS: PBK/TOPK expression was significantly greater in cervical cancer than that in high-grade intraepithelial neoplasia and CINI (P < 0.05). Meanwhile, PBK/TOPK expression in high-grade intraepithelial neoplasia was significantly higher compared with that in CINI (P < 0.05). In addition, PBK/TOPK expression in cervical cancer significantly correlated with histological type, differentiation, lymph node metastasis, vaginal and cervical invasion, TNM stage and tumor size (P < 0.05). CONCLUSION: PBK/TOPK expression is closely associated with cervical cancer and cervical intraepithelial neoplasia, which may be served as a useful target for tumor diagnosis and immunotherapy.

Expression of HAb18G in non-small lung cancer and characterization of activation, migration, proliferation, and apoptosis in A549 cells following siRNA-induced downregulation of HAb18G.

HAb18G, a novel cancer biomarker, has been shown to be involved in the progression of malignancy by regulating expression of vascular endothelial growth factor (VEGF) and matrixmetalloproteinases (MMPs). The goal of this study was to evaluate the role of HAb18G in the biology of NSCLC and to determine its potential as a therapeutic target. HAb18G protein expression was detected by immunohistochemistry in 150 NSCLC tissues. The results showed that HAb18G protein expression was associated with tumor diameter, lymph node status, tumor stage, and poor prognosis (P < 0.05). Multivariate analysis showed that HAb18G overexpression was an independent prognostic factor (HR, 3.713; 95 % CI, 1.114-12.373; P = 0.033). Transient infection of A549 lung cancer cells with small interfering RNA (SiRNA) against HAb18G efficiently inhibited the expression of HAb18G in A549 lung cancer cells at both mRNA and protein levels. Downregulation of HAb18G not only reduced MMP-2, MMP-9, and VEGF at mRNA and protein levels in A549 cells, but also inhibited fibroblasts to secrete MMP-2 and MMP-9 at mRNA level. Additionally, downregulation of HAb18G mRNA resulted in decreased migration, proliferation, and increased apoptosis of A549 in vitro. Our findings suggest that HAb18G overexpression plays an important role in progression of NSCLC and HAb18G may be a potential target of NSCLC therapy.