Programmed death-ligand 1 (PD-L1) expression on tumorcells (TC) or tumor-infiltrating immune cells (IC) correlated in several studies with PD-L1/programmed death-1 (PD-1) checkpoint inhibitor efficacy. Since June 2018, a positive PD-L1 status is required for atezolizumab or pembrolizumab treatment of patients with advanced or metastasized urothelial bladder cancer, who are ineligible for cisplatin-containing therapy. We examined technical comparability and inter-reader agreement of four clinically developed PD-L1 assays in locally advanced disease. Archived, formalin-fixed, paraffin-embedded sections from 30 patients (73.3% cystectomies, 26.7% transurethral resections) were stained by PD-L1 immunohistochemistry using VENTANA SP142, VENTANA SP263, DAKO 22C3, and DAKO 28-8 at two sites per manufacturers’ protocols and scored blinded at five sites for PD-L1 expression on IC (% per tumor area) and TC (%). Small, non-significant inter-assay differences were observed for IC. For TC, SP142 showed significantly lower staining percentages. Pairwise comparisons revealed − 0.3 to 1.6% differences in adjusted means between assays for IC, and for TC, − 10.5 to − 7.8% (SP142 versus others) and − 1.9 to 2.7% (other compari- sons). Inter-reader and inter-assay agreement was moderate to high for both IC and TC. Allocation to binary cutoffs (1%, 5%, 10%) showed substantial to high Kappa agreement scores (0.440–0.923) for IC and TC between assays for each reader. This first multicenter study, with five independent readers blinded with respect to the assay used, suggests that all four currently clinically relevant assays are analytically similar for evaluation of PD-L1-stained IC and three (SP263, 22C3, and 28-8) for PD-L1-stained TC. Inter-observer agreement for trained readers in scoring of both IC and TC positivity was generally high.
The inhibition of the proliferation of hyperdiploid Ehrlich ascites tumorcells in suspension cultures by amobarbital is coupled to an increased glycolytic activity as shown by lactic acid pro duction and glucose consumption; higher concentrations of amobarbital than 1 m M enhance the A TP/A D P ratio of the total cell. The actual activity of pyruvate dehydrogenase of intact cells is completely inhibited in the presence of 2 m M amobarbital as was shown by the 14C 0 2 evolution from [1-14C] pyruvate or the incorporation of 14C 0 2 into the total lipid fraction of the cells from [U-14C] pyruvate or from [U-14C] lactate. The pyruvate dehydrogenase complex from Ehrlich ascites tumorcells is completely inhibited by 1 m M amobarbital in vitro . The activity of a-oxoglutarate dehydrogenase is inhibited by amobarbital, too, as was shown by measuring the 14|C 0 2 evolution from [1-14C] glutamate with intact cells.
The direct effects of ionizing radiation exposure on different immune cells and their stem cells and especially their radiation sensitivity were recently summarized in three reviews ( 20 – 22 ), therefore, the readers are referred to these reviews and other reviews for a detailed description of the immune cells, an overview of their function and the direct radiation effects. Shortly, granu- locytes (eosinophils, basophils, neutrophils), natural killer (NK) cells and mast cells are the major players in the innate immune sys- tem. T-lymphocytes with their subtypes [cytotoxic T-cells (CTLs), helper T-cells (Ths) with the subpopulations Th1, Th2, Th17, regulatory T-cells (Tregs), memory T-cells] and B-lymphocytes ( 23 ) [plasma cells, and memory B cells] represent the adaptive arm of the immune system. T-lymphocytes are the key players in the cell-mediated immune response, while B-lymphocytes mediate the humoral reactions. The circulating peripheral blood lymphocytes represent only <2% of the lymphocytes in lym- phoid tissues ( 24 ). At the interface of the innate and the adaptive immune system, macrophages derived from monocytes and dendritic cells (DCs) act as antigen-presenting cells (APCs). NKT cells show features of NK cells and T-lymphocytes. The direct effects encompass reduced survival, proliferation, cell cycle alterations, diminished function, gene expression changes ( 25 – 27 ), chromosomal aberrations, mutations, and possible transformation ( 28 ). In vivo, mitotic catastrophe is usually fol- lowed by necrosis resulting in an inflammatory reaction ( 29 , 30 ). Mitotic catastrophe contributes strongly to the death of tumorcells induced by ionizing radiation ( 29 ), and is now assumed to be the major cell death pathway in solid tumors following radiotherapy ( 31 ). In tumor radiotherapy, this might result in enhanced tumor cell killing by cytotoxic immune cells and also in damage to the normal tissue ( 32 ).
Cell proliferation, viability, DNA-, RNA-, protein synthesis, amino acid transport, respiration and lactate/glucose quotient of Ehrlich ascites tumorcells grown in suspension culture in serum free medium supplemented with albumin charges of different origin were studied. Optimal cell growth was obtained in nutrient medium supplemented with 1 % bovine serum albumin (Cohn- fraction V, Serva). Cell proliferation under these culture conditions was delayed to 50% as compar ed to controls in normal medium; the rate of synthesis of macromolecules was reduced; energy metabolism was not significantly impaired. The trend of the cells in albumin medium to attach to glass was independent from the pH of the cultures between 7.2 and 8.0; it was enhanced by fatty acid deprivation of the albumin.
In order to survive, tumorcells use adaptation processes well-established by previous reports. The altered energy metabolism of cancer cells was first reported in 1924 by Otto Warburg, the Nobel laureate honored for discovering cytochrome c oxidase. In the presence of oxygen normal cells primarily convert glucose into pyruvate within the cytosol through the process of glycolysis and then oxidize the pyruvate in the mitochondrial tricarboxylic acid (TCA) cycle. During this reaction the reduced form of nicotinamide adenine dinucleotide (NADH) is produced which is essential for the oxidative phosphorylation with up to 36 ATPs per molecule glucose (Vander Heiden et al., 2009). However, under anaerobic conditions normal cells are limited to using only glycolysis and then converting pyruvate into lactate. This pathway generates only 2 ATPs per molecule glucose. In contrast to normal cells, many types of cancer cells mostly rely on glycolysis producing large amounts of lactate regardless of the availability of oxygen. This ‘aerobic glycolysis’ of cancer cells has been termed the Warburg effect (Warburg, 1956). Many tumors show an elevated expression of hypoxia-inducible lactate dehydrogenase-A (LDH-A), which is involved in interconversion between pyruvate and lactate (Firth et al., 1995). Compensation of the reduced energy results in increased glucose consumption, which is assumed to be an adaptation to intermittent hypoxia in tumors (Gatenby and Gillies, 2004). It was also shown that the increase of glucose uptake correlates with tumor aggressiveness and poor prognosis (Younes et al., 1996). As a result, increased lactate production leads to acidosis which is a hallmark of hypoxia in tissues (Seagroves et al., 2001). Furthermore, the increased glucose uptake in hypoxia is associated with increased expression of the glucose transporters GLUT-1 and GLUT-3, which are involved in glycolysis (Ebert et al., 1995; O'Rourke et al., 1996). Hypoxia also regulates cancer epigenetics and induces alteration of histone modifications associated with general transcriptional repression (Johnson et al., 2008).
The substrate specificity and the effects of nucleotides and SH-blocking agents on the p-nitro- phenylphosphatase activity of intact Ehrlich ascites tumorcells (EAT) cells were studied. DL-ß- Glycerophosphate, o-phosphoethanolamine, cholinephosphate, glucose-6-phosphate, o-carboxyphenyl- phosphate,, phosphoenolpyruvate and AMP were not attacked by intact cells. ATP > GTP > UTP > PPj > pNPP were cleaved with decreasing velocity. A stimulation of the cleavage of p-NPP by the following nucleotides was observed with decreasing effectivity: ATP > ADP > GTP > UTP; AMP was ineffective. The phosphatase activity was not affected by malate, tartrate and glutathion disulfide. The SH blocking agents diamide and thimerosal were more effective in hibitors of the pNPPase than of the ATPase activity, whereas the hydrolysis of ATP is more affected by the ATP analog adenylylimidodiphosphate. The present data are best compatible with a double headed enzyme: Both active sites interact with ATP, only one is active against p-NPP and sensitive against SH-blocking agents.
2.2.3 Tumor-associated macrophages in breast cancer
MΦs represent the bulk of the immune cell infiltrate in breast tumors. In contrast to resident MΦs in healthy tissues, they mainly originate from circulating blood monocytes (MOs) that differentiate into MΦs upon extravasation into tissue. Such tumor-associated macrophages (TAMs) can exert several protumorigenic functions and are typically associated with tumor progression and poor prognosis . They exhibit a M2-like or mixed phenotype, however, a minor subpopulation showing a M1- like phenotype can often be detected in tumors. It is important to acknowledge that the above described M1/M2 activation states represent two extremes along a continuum of observed MΦ phenotypes. For TAMs, this simplified terminology is used in order to roughly distinguish phenotypes associated with tumoricidal (M1-like) or tumor supporting (M2-like) functions without the claim to adequately reflect the existing phenotypical plasticity . The recruitment of MΦs to the tumor site and their functional properties are the result of different factors deriving from tumor as well as from stromal cells. In breast cancer, most of the chemoattractive potential for MΦs can be attributed to C-C chemokine ligand 2 (CCL2) and CCL5, secreted mostly by tumorcells, but also by stromal cells. In response to CCL2 and CCL5, recruited MΦs promote migration, angiogenesis, and further recruitment of leukocytes by secreting MMP9, IL8, VEGF, and additional CCL2 and CCL5. TAMs prevalently accumulate in hypoxic regions. Hypoxia further induces protumorigenic functions like VEGF upregulation leading to increased angiogenesis. Since VEGF was shown to mediate MΦ chemoattraction, this might result in a positive feedback which amplifies not only their recruitment but also a protumorigenic shaping of the whole tumor microenvironment. TAMs can further support such an immunosuppressive tumor microenvironment by suppressing cytotoxic T cell responses via secretion of IL10 and TGFβ as well as by recruiting myeloid-derived suppressor cells and regulatory T- cells.
Without question the search for molecular markers that predict the presence of cancer and the development of reliable assays that detect them is one of the most promising fields of oncology. The “Identification of predictive biomarkers in circulating tumorcells has the potential to become a breakthrough in cancer diagnostics and drug devel- opment.” (Parkinson et al. 2012). Moreover, pediatric brain tumors have distinct patho- genesis and biology, compared with their adult counterparts. Some of the molecular features are so specific to a particular tumor type, such as SMARCB1 mutations in AT/RT that they could serve as a diagnostic marker on their own (Ichimura et al. 2012). The advantage of circulating tumor markers is their easy obtainment by peripheral blood or alternatively cerebrospinal fluid sampling. The analysis of circulating tumorcells is also termed “liquid biopsy”, which can be repeated on a regular basis, allowing real- time monitoring of metastatic progression (Bednarz-Knoll et al. 2011). Molecular markers could be used on various different levels ranging from diagnosis, detection of metastatic tumor tissue to the monitoring of cancer patients. They could also be prog- nostic for survival or predictive of response to therapy. Furthermore, molecular markers could be used as indicators in the selection of therapy, in terms of personalized health care and individualized treatment. Molecular profiling of circulating tumorcells could also accelerate drug development and promote targeted therapies against signaling pro- teins (Parkinson et al. 2012).
balanced microenvironment within the niche, CSCs are not only pampered in a tumor-promoting way, but are in consequence also protected from treatments like radiation therapy or chemotherapy. As some tumors are very resistant to therapies and others have a high relapse rate, the CSC-niche is thought to contribute to the extent of response to therapy. Despite pathophysiological characterization, the morphological distinction of cancer cells and CSCs is quite complex [ 108 ]: CSCs can be distinguished from other cells using surface markers, but there is not an individual specific CSC marker. However, CD133 expression has often associated to CSC, and in fact, CD133-expression was shown to be a sufficient condition for brain tumorcells to initiate new tumors upon transplantation into NOD-SCID immune-deficient mouse brains [ 102 ]. A more comprehensive characterization of CSC surface markers has been performed in breast cancer stem cells, with suggested markers being Bcrp 1, ALDH, CD133, CD176, CD56, CD16, CD44 and CD24 [ 111 ]. 6. Connexins in Cancer Stem Cells
A critical issue concerning CAP application is the selectivity of the treatment towards tumorcells. First insights into a possible cell selectivity by CAP was gained by treatment of non- tumorous primary astrocytes and an ex vivo murine brain slice model containing implanted glioma cells. The significant cell cycle arrest in G2/ M phase achieved in tumorcells by 60 seconds and longer was not detectable in primary murine astrocytes by CAP treatment times of up to 180 seconds (figure 23). However, an increase of cells residing in the S phase of the cell cycle after 180 seconds of CAP was observed. These results indicate that a “therapeutic window” - where tumorcells are affected but non-tumorous, healthy cells persist unharmed - might be found. It remains speculative why primary astrocytes are less prone to CAP treatment, however one hypothesis is that brain cells proliferate less than tumorcells and thus have a reduced uptake of reactive species produced by CAP. Furthermore, non-tumorous cells have lower ROS levels compared to tumorcells which exhibit a high energy metabolism. ROS amounts in tumorcells will reach cytotoxic levels earlier than the ones in non-tumorous cells, an outcome that is enforced by an insufficient anti-oxidant system in tumorcells.
PARP inhibitors (PARPis) have been used to induce synthetic lethality in BRCA-deficient tumors in clin- ical trials with limited success. We hypothesized that RAD52-mediated DNA repair remains active in PARPi-treated BRCA-deficient tumorcells and that targeting RAD52 should enhance the synthetic lethal effect of PARPi. We show that RAD52 inhibitors (RAD52is) attenuated single-strand annealing (SSA) and residual homologous recombination (HR) in BRCA-deficient cells. Simultaneous targeting of PARP1 and RAD52 with inhibitors or dominant-nega- tive mutants caused synergistic accumulation of DSBs and eradication of BRCA-deficient but not BRCA-proficient tumorcells. Remarkably, Parp1 / ; Rad52 / mice are normal and display prolonged latency of BRCA1-deficient leukemia compared with Parp1 / and Rad52 / counterparts. Finally, PARPi+RAD52i exerted synergistic activity against BRCA1-deficient tumors in immunodeficient mice with minimal toxicity to normal cells and tissues. In conclusion, our data indicate that addition of RAD52i will improve therapeutic outcome of BRCA- deficient malignancies treated with PARPi.
cell types, such as the brain, heart, lungs, liver, kidney and pancreas (Chakrabarti et al., 2015; Lee et al., 2006). HDAC8 can deacetylate all core histones in vitro, although it predominantly deacetylates histones H3 and H4 (Lee et al., 2006). However, it is still controversial whether histones can be considered as primary HDAC8 substrates in vivo. Thus, there exist also many non-histone proteins that are deacetylated by HDAC8, like structural maintenance of chromosomes 3 (SMC3), cohesin, estrogen receptor α (ERRα), cortical actin-binding protein (cortactin) or p53. So HDAC8 is involved in the control of a variety of mechanisms, such as sister chromatid separation, energy homeostasis, muscle contraction regulation, cell cycle and tumorigenesis (Chakrabarti et al., 2015). HDAC8 seems to have a major impact on telomerase activity by preventing the hEST1B (human ever-shorter telomeres 1B) from undergoing ubiquitin-mediated degradation (Lee et al., 2006; Chakrabarti et al., 2015). This process is controlled by protein kinase A-dependent phosphorylation of HDAC8. In normal cells, the telomeres shorten with each cell division because the telomerase enzyme is repressed. By contrast, tumorcells avoid the associated senescence since telomerase activity is often highly upregulated. A loss of HDAC8 leads to a significant reduction in telomerase activity (Lee et al., 2006). The expression of HDAC8 has been detected in various types of tumors, such as colon, lung, pancreatic and cervical cancer, as well as in pediatric neuroblastoma. But also in viral and parasitic diseases, such as schistosomiasis, and in X-linked intellectual disability, HDAC8 is considered to play a significant role (Chakrabarti et al., 2015).
Statistical analysis was performed with SPSS 20 (SPSS, Inc., Chicago IL, USA). The association of PD1 expression and clinicopathologic parameters was analyzed using the Chi square test, t test, or Mann–Whitney U test. For the calculation of OS, the time between primary surgery and the patient’s death was analyzed. Disease-free survival (DFS) was defined as the time from primary surgery until the first evidence of disease progression. For the calcula- tion of both OS and DFS, patients without complete resection (n = 23) were excluded from the analyses. The influence of PD1 expression on tumorcells and lympho- cytes as well as the influence of other clinicopathologic findings on OS and DFS was evaluated with the Kaplan– Meier method, log-rank tests, and the Cox proportional hazard model. All tests were performed in a two-sided manner, and p values lower than 0.05 were considered to be statistically significant.
When blood samples from SCCHN patients with locally advanced, inoperable disease were analyzed by both flow cytometry methods described in this study, CTCs were successfully detected at similar frequencies and low numbers. High numbers of CTCs have been reported mainly in cancer patients with distant metastatic disease (6,7,11,33,34) and the general occurrence of a low number of CTCs in the blood of locally advanced SCCHN patients is therefore expected, since very few cases present with clinically detectable distant metastases at the time of diagnosis (35). Moreover, the same low numbers of CTCs observed in our study have been recently detected in advanced SCCHN by using the CellSearch system, which is the only F.D.A. approved platform for CTC monitoring (36). Thus, after detecting CTCs in negatively enriched peripheral blood samples of 42 patients with advanced SCCHN by flow cytometry, it was asked whether their presence correlates with clinical parameters such as tumor size or nodal spread, since CTCs may either result from tumor development or represent the reservoir for metastasis formation. The observed lack of correlation between CTCs and the T stage or tumor volume and their significant correlation with the number of affected lymph nodes implies that the spread of the detected tumorcells in the blood of head and neck cancer patients is not a result of their passive detachment from growing primary tumors, but of an active process of tumor cell transformation and dissemination to the lymph nodes and distant organs via the circulation system, which has been suggested as the main route for metastasis of head and neck cancer (37-39). Indeed, the number of lymph nodes involved is an established prognostic factor for the development of distant metastasis in SCCHN (40-44). Therefore, the presence of CTCs in patient blood might serve as an additional marker predicting the metastasis-free survival of SCCHN patients. Additionally, in this study CTCs have been also detected, though rarely, in patients without clinically detectable lymph node metastasis, which suggests that CTCs may also represent a novel diagnostic tool for the detection of early metastasis.
reasonable that in EAT cells norm ally meeting most of their demand by uptake o f exogenous cholesterol from the m edium [ 8 , 9] cholesterol synthesis is stimulated, but the present result is difficult to explain. Although anom alies in the control o f cho lesterol biosynthesis currently discussed for tum or cells [ 10 , 11 ] could explain an exceeding synthesis under modified culture conditions, it seems more probable that the ratio of phospholipids to choles terol has to be corrected due to an enhanced synthesis of phospholipids as revealed by the in corporation rates o f isotopic choline and ethanol- amine. Changes in the physico-chemical state o f cell membranes seem to be possible as a response to the
Pancreatic neuroendocrine tumors (pNETs) are a heterogeneous group of malignancies originating from cells of the diffuse endocrine system of the pancreas [ 1 ]. Only 1–5% of patients with pancreatic tumors are diagnosed with pNETs, resulting in an overall incidence lower than 1/100,000 cases per year [ 2 , 3 ]. Functional pNETs display distinct symptoms due to hypersecretion of specific hormones, including insulin (insulinoma), gastrin (gastrinoma), glucagon (glucagonoma), and vasoactive intestinal polypeptide (VIPoma), whereas non-functional pNETs display unspecific symptoms or stay asymptomatic [ 1 , 4 , 5 ]. Compared to other pancreatic tumors, pNETs are associated with a better prognosis, but still lack specific treatment options [ 6 , 7 ]. Multimodal therapy with surgical removal of the primary tumor and regional lymph nodes combined with chemotherapeutic drugs represents the currently established treatment [ 5 , 6 ]. Thus far, many underlying mechanisms of pNET tumorigenesis and tumor progression as well as risk factors are still unknown. Recently, alterations of epigenetic mechanisms have been found in several malignancies [ 8 – 10 ]. Histone deacetylases (HDACs) play a key role in epigenetics by altering post-translational histone modifications through removal of the acetyl group of N-terminal lysine histone residues [ 11 ], thus repressing expression through tighter DNA-packaging around histones. Dysfunction and or expression changes of HDACs have been observed in a wide range of tumors, including breast cancer, melanoma, and prostate cancer [ 10 , 12 – 16 ]. This led to the development of the first pharmaceutical HDAC inhibitor (HDACi), which received FDA approval in 2006, and several more being recently approved or being investigated in trials [ 17 – 19 ]. A recent report describes a phase II study on the use of the pan-HDAC inhibitor, panobinostat, in 15 low-grade NET patients indicating a low response rate, but encouraging stable disease rate and
Alterations in pathways of cell communication, the endocrine system and circadian rhythm are new aspects involved in describing the action of Viscum album extracts on cancer cells. Cell communication did not depend on lectin, which could not overregulate any of the pathways involved, but was influenced mainly by Iscador A in breast cancer cell lines and by Iscador A and M in lymphomas. In contrast, the endocrine system was lectin-dependent and was induced by lymphoma and breast cancer cells. Finally, 22-40% of the genes in the circadian rhythm pathway were changed by isolated mistletoe lectin in HCC-1937, MCF-7 and KPL-1, so that this aspect has to be pursued in future studies in vitro as well in vivo. Human Diseases
HUVEC random migration was assessed by single cell tracking, a well established in vitro assay for measuring cell motility. HUVEC were seeded in 12-well plates at a concentration of 20,000 cells/ml, first cultured in complete endoprime medium (C/E), then starved in media consisting of 5% C/E and 95% D/F for 16 hours. After the addition of different concentrations and types of SNs, 2 positions in each well were selected under the microscope with a magnification of 4×10-fold. Pictures were taken every 30 minutes during 24 hours. Then ImageJ software was used to track at least 30 cells in each movie by clicking on the position of the nuclear of a selected cell at each time point resulting in a list of tracks for each cell. Finally, using the Chemotaxis and Migration Tool from Ibidi the average velocity (µm/h), total distance, and other parameters, were calculated. Data was presented as mean ± SE of the average cell velocity of at least 3 independent experiments.
Immunocytochemistry was performed on routine slides previously fixed according to Delaunay and stained according to Papanicolaou. On these slides, cells of interest were marked by felt-tip pen. Coverslips were then removed in xylene at room temperature (RT). The coverslips fell off within 24 hours. If there were not enough slides from a patient to apply different antibodies, the slides were divided into two or three regions using a DakoPen (Dako, Glostrup, Denmark, Nr: S2002). Thus more than one antibody could be applied simultaneously on the same slide. The avidin-biotin complex method (ABC) was applied for the visualization of immunologic reactions. The incubations were carried out in a horizontal position of the slides in a humidified chamber. All other steps were carried out in an upright position of slides in cuvettes. Endogenous peroxidase activity was stopped by incubation with 1 ml H 2 O 2 (30% Perhydrol in 100