Reconnaissance of tumor immune microenvironment spatial heterogeneity in metastatic renal cell carcinoma and correlation with immunotherapy response.

A clearer understanding of the tumor immune microenvironment (TIME) in metastatic clear cell renal cell carcinoma (ccRCC) may help to inform precision treatment strategies. We sought to identify clinically meaningful TIME signatures in ccRCC. We studied tumors from 39 patients with metastatic ccRCC using quantitative multiplexed immunofluorescence and relevant immune marker panels. Cell densities were analyzed in three regions of interest (ROIs): tumor core, tumor-stroma interface and stroma. Patients were stratified into low- and high-marker density groups using median values as thresholds. Log-rank and Cox regression analyses while controlling for clinical variables were used to compare survival outcomes to patterns of immune cell distributions. There were significant associations with increased macrophage (CD68+ CD163+ CD206+ ) density and poor outcomes across multiple ROIs in primary and metastatic tumors. In primary tumors, T-bet+ T helper type 1 (Th1) cell density was highest at the tumor-stromal interface (P = 0·0021), and increased co-expression of CD3 and T-bet was associated with improved overall survival (P = 0·015) and survival after immunotherapy (P = 0·014). In metastatic tumor samples, decreased forkhead box protein 3 (FoxP3)+ T regulatory cell density correlated with improved survival after immunotherapy (P = 0·016). Increased macrophage markers and decreased Th1 T cell markers within the TIME correlated with poor overall survival and treatment outcomes. Immune markers such as FoxP3 showed consistent levels across the TIME, whereas others, such as T-bet, demonstrated significant variance across the distinct ROIs. These findings suggest that TIME profiling outside the tumor core may identify clinically relevant associations for patients with metastatic ccRCC.

Fibronectin induction abrogates the BRAF inhibitor response of BRAF V600E/PTEN-null melanoma cells.

The mechanisms by which some melanoma cells adapt to Serine/threonine-protein kinase B-Raf (BRAF) inhibitor therapy are incompletely understood. In the present study, we used mass spectrometry-based phosphoproteomics to determine how BRAF inhibition remodeled the signaling network of melanoma cell lines that were BRAF mutant and PTEN null. Short-term BRAF inhibition was associated with marked changes in fibronectin-based adhesion signaling that were PTEN dependent. These effects were recapitulated through BRAF siRNA knockdown and following treatment with chemotherapeutic drugs. Increased fibronectin expression was also observed in mouse xenograft models as well as specimens from melanoma patients undergoing BRAF inhibitor treatment. Analysis of a melanoma tissue microarray showed loss of PTEN expression to predict for a lower overall survival, with a trend for even lower survival being seen when loss of fibronectin was included in the analysis. Mechanistically, the induction of fibronectin limited the responses of these PTEN-null melanoma cell lines to vemurafenib, with enhanced cytotoxicity observed following the knockdown of either fibronectin or its receptor α5ß1 integrin. This in turn abrogated the cytotoxic response to BRAF inhibition via increased AKT signaling, which prevented the induction of cell death by maintaining the expression of the pro-survival protein Mcl-1. The protection conveyed by the induction of FN expression could be overcome through combined treatment with a BRAF and PI3K inhibitor.

Targeting PYK2 mediates microenvironment-specific cell death in multiple myeloma.

Multiple myeloma (MM) remains an incurable malignancy due, in part, to the influence of the bone marrow microenvironment on survival and drug response. Identification of microenvironment-specific survival signaling determinants is critical for the rational design of therapy and elimination of MM. Previously, we have shown that collaborative signaling between ß1 integrin-mediated adhesion to fibronectin and interleukin-6 confers a more malignant phenotype via amplification of signal transducer and activator of transcription 3 (STAT3) activation. Further characterization of the events modulated under these conditions with quantitative phosphotyrosine profiling identified 193 differentially phosphorylated peptides. Seventy-seven phosphorylations were upregulated upon adhesion, including PYK2/FAK2, Paxillin, CASL and p130CAS consistent with focal adhesion (FA) formation. We hypothesized that the collaborative signaling between ß1 integrin and gp130 (IL-6 beta receptor, IL-6 signal transducer) was mediated by FA formation and proline-rich tyrosine kinase 2 (PYK2) activity. Both pharmacological and molecular targeting of PYK2 attenuated the amplification of STAT3 phosphorylation under co-stimulatory conditions. Co-culture of MM cells with patient bone marrow stromal cells (BMSC) showed similar ß1 integrin-specific enhancement of PYK2 and STAT3 signaling. Molecular and pharmacological targeting of PYK2 specifically induced cell death and reduced clonogenic growth in BMSC-adherent myeloma cell lines, aldehyde dehydrogenase-positive MM cancer stem cells and patient specimens. Finally, PYK2 inhibition similarly attenuated MM progression in vivo. These data identify a novel PYK2-mediated survival pathway in MM cells and MM cancer stem cells within the context of microenvironmental cues, providing preclinical support for the use of the clinical stage FAK/PYK2 inhibitors for treatment of MM, especially in a minimal residual disease setting.

Aurora-A is a determinant of tamoxifen sensitivity through phosphorylation of ERα in breast cancer.

Despite the clinical success of tamoxifen, its resistance remains a major challenge in breast cancer. Here we show that Aurora-A determines tamoxifen sensitivity by regulation of oestrogen receptor (ER)α. Ectopic expression of Aurora-A decreases and depletion of Aurora-A enhances tamoxifen sensitivity in ERα-positive breast cancer. Elevated Aurora-A was significantly associated with the recurrence of ERα-positive tumours. Notably, Aurora-A inhibitor MLN8237, which is currently in clinical trial, synergizes with tamoxifen and overcomes tamoxifen resistance. Furthermore, Aurora-A interacts with and phosphorylates ERα on serine-167 and -305, leading to increase in ERα DNA-binding and transcriptional activity. Elevated levels of Aurora-A are significantly associated with disease-free survival in ERα-positive but not ERα-negative breast cancers. These data suggest that Aurora-A has a pivotal role in tamoxifen resistance and ERα is a bona fide substrate of Aurora-A. Thus, Aurora-A represents a prognostic marker in ERα-positive tumour and a critical therapeutic target in tamoxifen-resistant breast cancer, and Aurora-A inhibitor could be used as either an independent or concurrent agent in tamoxifen-resistant tumour.

Ultraviolet/matrix-assisted laser desorption/ionization mass spectrometric characterization of 2,5-dihydroxybenzoic acid-induced reductive hydrogenation of oligonucleotides on cytosine residues.

The changes in the ion signals in the isotope cluster, mass resolution, signal-to-noise ratio and mass accuracy for matrix-assisted laser desorption/ionization (MALDI) of DNA oligonucleotides (dGGATC, dCAGCt, and dAACCGTT) and their fragment ions were evaluated, and these data were compared with those obtained using 3-hydroxypicolinic acid. Mass spectra obtained by using 2,5-dihydroxybenzoic acid (2,5-DHB) appear to have differences from the theoretical isotopic clusters, which arise by reductive hydrogenation producing a second peak at the M + 2 isotope of the native oligonucleotide. Based on the patterns of the isotopic envelope observed in the in-source decay fragments, we propose that cytosine is the site of reduction. We do not find evidence of reduction of oligonucleotides, viz. dTGGGGTT, that do not contain cytosine; however, 2'-deoxycytidine and 2'-deoxycytidine-5'-monophosphate undergo reductive hydrogenation. Several experiments were carried out in an effort to determine whether the reductive hydrogenation occurs during sample preparation or as a result of laser irradiation. The results of these experiments suggest that it occurs during sample preparation. The relative intensities of ion signals corresponding to the reduced base can be altered by using different matrix additives (aminonaphthalenes) or a different substrate (copper). Also, the oxidized form of 2,5-DHB is trapped by reaction with the side chain of cysteine in glutathione, providing evidence that the reaction occurs in solution as the matrix crystallizes.

Improvement of resolution, mass accuracy, and reproducibility in reflected mode DE-MALDI-TOF analysis of DNA using fast evaporation--overlayer sample preparations.

DNA analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is hindered by two processes: alkali metal adduction and fragmentation of the intact ionized molecule. The adverse effects of both processes can be reduced by adding ammonium ion salts or compounds such as fructose to the sample preparations. Matrix additives improve sensitivity and resolution of DNA analysis by MALDI. In addition, spot-to-spot reproducibility, resolution, and mass accuracy for DNA oligonucleotides (< or = 12 mer) can be improved by the use of overlayer sample preparations with matrixes that have low aqueous solubilities, such as alpha-cyano-4-hydroxycinnamic acid, ferulic acid, and 2,4,6-trihydroxyacetophenone. For example, resolution for 5-12-mer oligonucleotides is greater than 7000 using overlayer matrix preparations and mass accuracy values are well below 20 ppm. In addition to these methods, a new method for analyzing DNA in positive ion mode is reported using acidified 3-hydroxypicolinic acid. This method does not lose sensitivity for higher mass oligonucleotides as quickly as overlayer methods, and spectra retain > 6000 resolution and mass accuracies of approximately 20 ppm between different overlayer depositions.

The structure of an insect chymotrypsin.

The South American imported fire ant (Solenopsis invicta), without natural enemies in the United States, widely infests the southern United States, causing more than a half billion dollars in health and agriculture-related damage annually in Texas alone. Fire ants are resistant to most insecticides, so control will require a more fundamental understanding of their biochemistry and metabolism leading to the design of selective, ecologically safe insecticides. The 4th instar larvae play a crucial role in the nutrition of the colony by secreting proteinases (especially chymotrypsin) which digest food products for the entire colony. The first structure of an ant proteolytic enzyme, fire ant chymotrypsin, was determined to atomic resolution (1.7 A). A structural comparison of the ant and mammalian structures confirms the "universality" of the serine proteinase motif and reveals a difference at residues 147-148, which are proteolytically removed in the bovine enzyme but are firmly intact in the ant chymotrypsin, suggesting a different activation mechanism for the latter. Likewise, the absence of the covalently attached propeptide domain (1-15) further suggests an uncharacteristic activation mechanism. The presence of Gly189 in the S1 site is an atypical feature of this chymotrypsin and is comparable only to human leukocyte elastase, hornet chymotrypsin and fiddler crab collagenase. Binding studies confirm the chymotrypsin nature of this novel enzyme.

Mapping of surrogate markers of cellular components and structures using laser desorption/ionization mass spectrometry.

Laser desorption/ionization mass spectrometry (LDI-MS) has been used to assess the potential of using surrogate markers, bound to cellular structures containing nucleic acids, to image or map the position of these structures within biological samples. In this study, organic dyes were used as markers because of their established use in the histochemical marking of nucleic acids, and also because they are amenable to LDI-MS. Eight cationic dyes were tested and all could be desorbed from nucleic acid samples without additional matrix after specifically binding to these molecules. Methylene Blue was the best of these based on its sensitivity to detection by LDI-MS and the fact that it can be washed from the tissue in areas where it was not specifically bound to provide low-intensity background signals. Experiments are reported which characterize the M(+) ion signal obtained from Methylene Blue with regard to sensitivity, reproducibility and possible use for quantitation. This dye was used to map (with a lateral resolution of 25 microm) several nucleic acid-containing samples spotted on prepared surfaces, and to image the location of nucleic acids in two model tissues, retinal vertical sections and thyroid whole mount sections.

Calcium repletion-induced arrhythmias after short periods of calcium-free perfusion in the isolated rat heart.

The occurrence of arrhythmias was studied in the "calcium-paradox" model in the isolated rat heart. Clear relationships were found between the duration of calcium-free perfusion and (a) the occurrence of calcium-free-induced electrophysiological changes, (b) the incidence and duration of subsequently induced calcium-repletion arrhythmias and (c) mechanical recovery at the end of the repletion period. The first signs of electrophysiological changes (i.e. decreased heart rate, T-wave amplitude and increased PQ-interval) and irreversible loss of myocardial recovery occurred during or after 60-90 s of calcium-free perfusion. The occurrence of calcium-repletion induced ventricular tachycardia parallelled this onset of irreversible cardiac injury. These results suggest that the process of calcium washout and subsequent sudden calcium overloading may play a role as a trigger in the pathogenesis of ventricular arrhythmias.

The role of lipid peroxidation in acute doxorubicin-induced cardiotoxicity as studied in rat isolated heart.

Doxorubicin induces an acute cardiotoxicity that becomes manifest in isolated hearts as a deterioration in mechanical function. The oxidative component in this myocardial damage has been investigated. The effects of doxorubicin on the activity of superoxide dismutase and the capacity of the glutathione system, factors of the cellular protective mechanism against free radicals, were examined in rat isolated heart. Doxorubicin was found to reduce the capacity of the protective mechanisms. Whether oxidative membrane damage due to excessive free radical formation plays a role in the pathogenesis of the acute cardiotoxic action of doxorubicin was also examined. Its acute effect on myocardial contraction amplitude, frequency of beating, coronary flow and on the above mentioned biochemical parameters was compared in rat hearts sufficient or deficient in vitamin E. Peroxidation of lipids was measured as the formation of malondialdehyde, one of the final products of this process. Vitamin E deficiency neither aggravated the decrease in the capacity of the cellular protective factors nor worsened the reduction in myocardial function. Nor did induction of lipid peroxidation by doxorubicin occur in vitamin E-deficient hearts. It was concluded that lipid peroxidative damage most probably is not decisive in the development of the acute cardiomyopathy in rats.

Decreased defence against free radicals in rat heart during normal reperfusion after hypoxic, ischemic and calcium-free perfusion.

Excessive formation of free radicals possibly plays an important role in the origin of irreversible damage of the heart after hypoxic, ischemic or Ca2+-free treatment. The effect of these treatments on the activity of superoxide dismutase and the glutathione system was studied on isolated rat heart. These activities reflect the protective capacity of the heart against reactive substances. In addition the peroxidation of lipids is determined in the treated hearts using malondialdehyde formation as an indicator. All experiments were performed using a Langendorff-apparatus with recirculating perfusion. The observed changes in the components of the glutathione system and superoxide dismutase activity both after hypoxic, ischemic and Ca2+-free perfusion, as measured upon reperfusion, indicate a decrease in cellular defense mechanisms in the heart against free radicals. The effect was most pronounced upon Ca2+-repletion after a period of Ca2+-free perfusion. No malondialdehyde could however be detected either in the tissue of the treated hearts or in the perfusate. Our data give reason to expect beneficial effects of an adequate pharmacological treatment, which replenishes the cellular defence systems.

Evidence for lipid peroxidation during the calcium paradox in vitamin E-deficient rat heart.

Vitamin E is known to play an important role in the protective capacity of tissues as a free radical scavenger. Rats were made deficient in vitamin E, in order to demonstrate more clearly the formation of free radicals after exposing the rat heart to sudden changes in calcium homeostasis. The formation of malondialdehyde was taken as measure for lipid peroxidation. Malondialdehyde was detected in appreciable amounts both in heart tissue and coronary perfusate of vitamin E-deficient rat hearts after exposing them to the sudden changes in calcium concentration as seen during the calcium paradox. These findings emphasize a the hearts of normally fed rats no malondialdehyde could be detected in tissue or coronary perfusate after the calcium paradox. Therefore an essential role for vitamin E against oxidative stress in heart tissue is also indicated.

Biphasic positive inotropic actions of ouabain on rat, guinea-pig and cat heart: a mathematical description.

The inotropic action of ouabain on isolated perfused hearts of rat, guinea-pig, and cat was studied over a wide concentration range (10(-12)-5 X 10(-3) M). In all three species used, the positive inotropic effect (PIE) of ouabain appeared to be biphasic in character. However, there was a remarkable difference in the course of the logdose-response curves of ouabain on guinea-pig and cat heart as compared with than on rat heart. The first two species showed, at very low concentrations of ouabain (guinea-pig heart: 10(-9) M and cat heart: 10(-10) M), a typical bell-shaped increase in cardiac contractile activity, while at higher concentrations (10(-8)-10(-6) M and 10(-9)-10(-7) M, respectively) the normally observed S-shaped increase in contractile activity occurred. On the contrary, rat hearts showed a flat S-curve between 10(-8) and 10(-6) M and a steep one between 10(-6) and 10(-4) M of ouabain. In order to explain the biphasic action of ouabain a hypothetical model for the mechanism(s) of action of ouabain is discussed. Mathematical description of this model is based on the existence of two different receptor-types for ouabain. It is suggested that sarcolemma-bound calcium may play an important role in both mechanisms of inotropic action of ouabain.

Ca2+-free perfusion of isolated rat heart: early irreversible changes and discrepancy between functional impairments and release of cellular constituents.

The effects of repeated (10 X), brief Ca2+-free perfusions (20 to 120 s) on myocardial contractility, coronary flow and release of cellular constituents of isolated rat heart were investigated. Ten successive Ca2+-deprivations of 20 or 40 s had no irreversible influences on cardiac performance. Ca2+-deprivations, however, of 60 s or longer, resulted in a substantial depression of contractile activity during recovery, which effect was parallelled by the development of myocardial contracture. Both effects appeared to be additive in character (and thus irreversible) and were dependent upon the duration and number of Ca2+-deprivations. The effect on coronary flow was biphasic: after an initial, rapid increase coronary flow steadily declined and was almost normal at the end of the experiments. The release of cellular constituents into the coronary effluent only occurred during reperfusion with Ca2+ after Ca2+-free periods of 60 s or longer. This loss of cellular material from the heart was observed mainly after the first Ca2+-deprivation and was only small or even negligible during the subsequent Ca2+-deprivations. The overall release of cellular material was clearly dependent on the duration of the Ca2+-free period, but was badly related to the overall loss in contractile activity. It was concluded that a relatively brief Ca2+-free perfusion period may induce a small but irreversible damage to the heart. This damage becomes visible, and more deleterious, when the brief Ca2+-washout is repeated several times. In addition, it was concluded that Ca2+-free-induced functional impairments and the release of cellular constituents of the heart are badly correlated and may be caused by two separate, but probably interrelated, mechanisms.

Myocardial recovery from global ischemia and reperfusion: effects of pre- and/or post-ischemic perfusion with low-Ca2+.

The effects of brief (5 min) pre- and/or post-ischemic treatment with low-Ca2+ (10(-4)M) on cardiac mechanical performance during and after increasing periods of total global ischemia were investigated on isolated perfused rat heart. Mechanical parameters studied were changes in diastolic resting length (delta DRL), ventricular contraction amplitude, its first derivative dL/dtmax, and cardiac responsiveness to variations in extracellular Ca2+. The results demonstrate that pre-ischemic low-Ca2+ may completely prevent: (a) the development of myocardial contracture during 45 min ischemia, (b) the occurrence of post-ischemic contracture and (c) a loss in myocardial contractile activity after 30 min of total ischemia. Post-ischemia low-Ca2+ was clearly less effective in protecting the heart against ischemia-induced loss in contractile function and offered no additional benefit when combined with pre-ischemic low-Ca2+. Post-ischemic mechanical recovery appeared to correlate letter with (end-)reperfusion-contracture than with (end-)ischemic contracture. Cardiac responsiveness to perfusate-Ca2+ decreased with increasing duration of the ischemic period (greater than 30 min), but was not significantly altered by the low-Ca2+-treatments. It was concluded from the results that the protective effect of a pre-ischemic low-Ca2+ treatment is superior to that of a similar post-ischemic treatment. The mechanism of protection and the role of Ca2+ in the ischemic process are discussed in terms of changes in the availability of intracellular free-Ca2+.

Magnesium and the calcium paradox: the occurrence of "spasmodic contractions" during Ca2+-Mg2+ -free perfusion of isolated rat heart.

The effects of omission of Mg2+ during Ca2+-free perfusion (3 min) of either spontaneously beating and electrically stimulated rat hearts were studied. Ca2+-free perfusion per se induced cardiac arrest and coronary vasodilation, and increased intrinsic pulse rate of the heart. Upon reperfusion with Ca2+, cardiac function was lost and parallelled by a sudden and massive release of cellular constituents ("calcium paradox"). Mg2+-free perfusion evoked effects opposite from Ca2+-free, with exception of heart rate which was increased. During Ca2+-Mg2+-free perfusion the electrocardiogram became irregular within 30 s, and this effect was followed by transient "spasmodic contractions". The effects of normal reperfusion were indistinguishable from those observed after Ca2+-free perfusion in the presence of Mg2+. Addition of Mn2+ or La3+ to the Ca2+-Mg2+-free perfusion medium completely inhibited the induction of electrical irregularities and spasmodic contractions. The typical effects of Ca2+-Mg2+-free perfusion are discussed in terms of Mg-Ca and Mg-K interactions at the sarcolemmal surface. It was concluded that our results may contribute indirectly to an explanation of the protective effect of high Mg2+-concentrations during Ca2+-free as well as ischemic perfusion of mammalian hearts.

A concentration-dependent biphasic positive inotropic action of ouabain on isolated heart of rat and guinea-pig.

Log dose-response curves of ouabain on rat and guinea-pig heart revealed the existence of a concentration-dependent, biphasic inotropic effect of ouabain. With rat as with guinea pig heart the "first" effect observed at the lowest concentrations of ouabain, was relatively small (rat: 115% of control, at 10(-6) M ouabain; guinea-pig: 110% of control, at 6.25.10(-10) M ouabain) but statistically highly significant (p less than or equal to 0.001). The shape of the concentration-effect relationship for this first action differed between species. With rat heart it was sigmoid in character and had a flat slope, with guinea-pig on the contrary it was almost bell-shaped (i.e. the effect disappeared with increasing ouabain concentration). The shape of the concentration-effect relationship for the "second" action was sigmoid in shape for both species used (rat: between 10(-6) - 10(-4) M; guinea-pig: between 10(-8) - 10(-6) M ouabain). From the results is was concluded that the dual positive inotropic effect of ouabain supports the existence of at least two different mechanisms of action of this drug. Furthermore, it was suggested that a species-dependent binding of calcium to the cardiac sarcolemma may be responsible for the different shapes of the "first" positive inotropic effect of ouabain.