The association between clinical outcome and CD8+ lymphocytic infiltration in advanced stages of colorectal cancer differs by latent virus infection in tumour tissue.

AIMS: In the near future, an immunoscore based on the quantification of lymphocytic populations can be expected as a fundamental supplement of colorectal cancer (CRC) classification. This study explored whether latent viral infection has an influence on prognostically relevant host immunity in CRC. METHODS AND RESULTS: CD8+ lymphocytic infiltration in three tumour compartments of 121 CRC was compared with clinical data and occurrence of latent infection with herpes simplex virus (HSV1, HSV2), cytomegalovirus (CMV), human papillomavirus (HPV16 and HPV18) in the tumour tissue, which was determined by polymerase chain reaction (PCR). Intraepithelial CD8+ lymphocytic infiltration (IECD8+ ) showed a trend towards correlation with clinical stage (P = 0.073), significant differences between CRC with and without metastases (P = 0.001) and a significant correlation with overall survival (OS, P = 0.001). Each of these three clinical parameters showed a significant link to IECD8+ in the virus DNA-negative (P-values: 0.001-0.036), but no significant differences in the virus DNA-positive subgroup, which is consistent with a moderating effect of virus DNA on these associations. A significant correlation of CD8+ infiltration in the invasive margin (IMCD8+ ) with OS (P = 0.016) was also moderated by virus DNA. CONCLUSION: Our data suggest a possible influence of latent viral infection on the association between clinical outcome and CD8+ lymphocytic infiltration in CRC tissue. After confirmation of these results by large cohort studies, a potential interaction between microbial pathogens and host immunity in CRC and its impact on prognostic immunoscores and/or new therapeutic strategies should be investigated further.

Image-Based Identification and Genomic Analysis of Single Circulating Tumor Cells in High Grade Serous Ovarian Cancer Patients.

In Ovarian Cancer (OC), the analysis of single circulating tumor cells (sCTCs) might help to investigate genetic tumor evolution during the course of treatment. Since common CTC identification features failed to reliably detect CTCs in OC, we here present a workflow for their detection and genomic analysis. Blood of 13 high-grade serous primary OC patients was analyzed, using negative immunomagnetic enrichment, followed by immunofluorescence staining and imaging for Hoechst, ERCC1, CD45, CD11b and cytokeratin (CK) and sCTC sorting with the DEPArrayTM NxT. The whole genome of single cells was amplified and profiled for copy number variation (CNV). We detected: Type A-cells, epithelial (Hoechstpos, ERCC1pos, CD45neg, CD11bpos, CKpos); Type B-cells, potentially epithelial (Hoechstpos, ERCC1pos, CD45neg, CD11bpos, CKneg) and Type C-cells, potentially mesenchymal (Hoechstpos, ERCC1pos, CD45neg, CD11bneg, CKneg). In total, we identified five (38.5%) patients harboring sCTCs with an altered CN profile, which were mainly Type A-cells (80%). In addition to inter-and intra-patient genomic heterogeneity, high numbers of Type B- and C-cells were identified in every patient with their aberrant character only confirmed in 6.25% and 4.76% of cases. Further identification markers and studies in the course of treatment are under way to expand sCTC analysis for the identification of tumor evolution in OC.

6-Color/1-Target Immuno-SERS Microscopy on the Same Single Cancer Cell.

There is an urgent clinical need for multicolor imaging of single cancer cells (no ensemble averaging) for identifying heterogenous expression of predictive biomarkers. Specifically, the comprehensive characterization of single disseminated tumor cells (sDTCs) responsible for metastatic relapse is the key to personalized therapy for patients. Current bioimaging methods lack the necessary multicolor capacity and suffer from background/autofluorescence. Both these central limitations can be overcome by immuno-SERS microscopy using SERS nanotags conjugated to antibodies. Here, we demonstrate the proof of concept for 6-color iSERS microscopy on the same single cancer cell. Human epidermal growth factor receptor 2 (HER2), the most prominent breast cancer marker, is localized on the membrane of single SkBr-3 cells, which overexpress HER2 and are an accepted model for sDTCs in breast cancer. This work paves the way for future multicolor/multitarget imaging for characterizing heterogeneous protein expression at the single-cell level.

Localization of PD-L1 on single cancer cells by iSERS microscopy with Au/Au core/satellite nanoparticles.

Programmed cell death-ligand 1 (PD-L1) is an important predictive biomarker. The detection of PD-L1 can be crucial for patients with advanced cancer where the use of immunotherapy is considered. Here, we demonstrate the use of immuno-SERS microscopy (iSERS) for localizing PD-L1 on single cancer SkBr-3 cells. A central advantage of iSERS is that the disturbing autofluorescence from cells and tissues can be efficiently minimized by red to near-infrared laser excitation. In this study we employed Au/Au core/satellite nanoparticles as SERS nanotags because of their remarkable signal brightness and colloidal stability upon red laser excitation. False-color iSERS images of the positive and negative controls clearly reveal the specific localization of PD-L1 with SERS nanotag-labeled antibodies.

Effect of 158 herbal remedies on human TRPV1 and the two-pore domain potassium channels KCNK2, 3 and 9.

BACKGROUND AND AIM: Herbal medicines are used to treat a broad number of maladies. However, the pharmacological profile of most remedies is poorly understood. We investigated the effect of herbal remedies from kampo, traditional Chinese medicine (TCM) and other phytotherapies on human two-pore domain potassium channels (KCNK channels; TREK-1, TASK-1 and TASK-3) as well as the human TRPV1 channel. KCNK channels are responsible for the background potassium current of excitable cells, thus essential for the maintenance of the resting membrane potential. Hence, modulators of KCNK channels are of medical significance, e.g. for the treatment of sleep disorders and pain. The transient receptor potential channel TRPV1 is a pain detector for noxious heat. Agonists of this receptor are still used for the treatment of pain in ectopic applications. EXPERIMENTAL PROCEDURE: We evaluated the effect of 158 herbal remedies on these channels in a heterologous expression system (Xenopus laevis oocytes) using the two-electrode voltage-clamp technique with the aim of increasing the comprehension of their pharmacological profile. RESULTS AND CONCLUSION: Some remedies with modulating effects were identified such as Angelica pubescens (radix), which inhibit TASK-1 and TASK-3 channels. Furthermore, the modulatory effects of the most effective remedies on the two TASK family members TASK-1 and TASK-3 correlate positively, reflecting their close relation. For the TRPV1 channel Terminalia chebula and Alchemilla xanthochlora were identified as potentiators. This study identifies a variety of herbal remedies as modulators of human K2P and TRPV1 channels and gives new insights into the pharmacological profile of these herbal remedies.

The Effect of Pungent and Tingling Compounds from Piper nigrum L. on Background K+ Currents.

Black peppercorns (Piper nigrum L.) elicit a pungent and tingling oral impression. Their pungency is partially explained by the agonist activity of some of their active principles, especially piperine, on TRP channels. However, we recently showed that piperine, as well as other pungent compounds, also possess a marked effect on two-pore domain (KCNK, K2P) K+ channels. Members of this family play a key role in maintaining the resting membrane potential of excitable cells. Interestingly, tingling compounds have been shown to induce neuronal excitation by inhibiting KCNK channels. We addressed the question of whether it was plausible that KCNK channels could constitute a physiologically relevant target for the sensory active compounds present in black peppercorns. Because previous studies have demonstrated that mouse trigeminal neurons respond to several pungent compounds, to which humans are also sensitive, we used a primary culture of mouse trigeminal neurons to investigate whether the effect of piperine on these cell types could also be mediated by KCNK channels. We observed that even in the presence of classical TRP-antagonists, piperine was still able to activate a fraction of trigeminal neurons. Furthermore, our results showed that piperine is capable of inducing neuronal depolarization by a mechanism that does not require extracellular Na+ or Ca2+. This depolarization was mediated by the inhibition of a background K+ conductance, most likely corresponding to the KCNK channels of the TASK subfamily. We then performed a screening with 12 other pungent and/or tingling chemosensates isolated from black peppercorns. These compounds were evaluated on Xenopus laevis oocytes expressing the human orthologues of KCNK3, KNCK9 and KCNK18, which we previously showed to be inhibited by piperine. Remarkably, almost all of the isolated chemosensates inhibited the basal activity of hKCNK3, with 1-(octadeca-2E,4E,13/12Z-trienoyl)pyrrolidine acting as one of the most potent natural blockers for hKCNK3 found to date. Our results suggest that KCNK channels, especially KCNK3, are likely to play a complementary role to TRP channels in the complex orosensory impression elicited by black peppercorns, while they also help to expand the pharmacological knowledge of KCNK channels.