Distinct tumor-infiltrating lymphocyte landscapes are associated with clinical outcomes in localized non-small-cell lung cancer.

BACKGROUND: Despite the importance of tumor-infiltrating T lymphocytes (TILs) in cancer biology, the relationship between TIL phenotypes and their prognostic relevance for localized non-small-cell lung cancer (NSCLC) has not been well established. PATIENTS AND METHODS: Fresh tumor and normal adjacent tissue was prospectively collected from 150 patients with localized NSCLC. Tissue was comprehensively characterized by high-dimensional flow cytometry of TILs integrated with immunogenomic data from multiplex immunofluorescence, T-cell receptor sequencing, exome sequencing, RNA sequencing, targeted proteomics, and clinicopathologic features. RESULTS: While neither the magnitude of TIL infiltration nor specific TIL subsets were significantly prognostic alone, the integration of high-dimensional flow cytometry data identified two major immunotypes (IM1 and IM2) that were predictive of recurrence-free survival independent of clinical characteristics. IM2 was associated with poor prognosis and characterized by the presence of proliferating TILs expressing cluster of differentiation 103, programmed cell death protein 1, T-cell immunoglobulin and mucin-domain containing protein 3, and inducible T-cell costimulator. Conversely, IM1 was associated with good prognosis and differentiated by an abundance of CD8+ T cells expressing cytolytic enzymes, CD4+ T cells lacking the expression of inhibitory receptors, and increased levels of B-cell infiltrates and tertiary lymphoid structures. While increased B-cell infiltration was associated with good prognosis, the best prognosis was observed in patients with tumors exhibiting high levels of both B cells and T cells. These findings were validated in patient tumors from The Cancer Genome Atlas. CONCLUSIONS: Our study suggests that although the number of infiltrating T cells is not associated with patient survival, the nature of the infiltrating T cells, resolved in distinct TIL immunotypes, is prognostically relevant in NSCLC and may inform therapeutic approaches to clinical care.

The Wnt pathway regulator DKK1 is preferentially expressed in hormone-resistant breast tumours and in some common cancer types.

In addition to new tumour antigens, new prognostic and diagnostic markers are needed for common cancers. In this study, we report the expression of Dickkopf-1 (DKK1) in multiple common cancers. This constitutes a comprehensive analysis of the DKK1 expression profile. Dickkopf-1 expression was evaluated by classical and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbant assay for protein determination, in cancer lines and clinical specimens of several cancer origins. For breast cancer, expression was correlated with clinicopathological parameters. Dickkopf-1 expression was confirmed in several cancer cell lines derived from breast and other common cancers. Dickkopf-1 protein secretion was documented in breast, prostate and lung cancer lines, but was negligible in melanoma. Analysis of DKK1 expression in human cancer specimens revealed DKK1 expression in breast (21 out of 73), lung (11 out of 23) and kidney cancers (six out of 20). Interestingly, DKK1 was preferentially expressed in oestrogen and progesterone receptor-negative tumours (ER(-)/PR(-); P=0.005) and in tumours from women with a family history of breast cancer (P=0.024). Importantly, DKK1 protein production was confirmed in multiple breast cancer specimens that were positive by RT-PCR. This work establishes DKK1 as a potential prognostic and diagnostic marker for cohorts of breast cancer patients with poor prognosis. Dickkopf-1 may also become a relevant candidate target for immunotherapy of different cancers.

IL-8 inhibits isoproterenol-stimulated ciliary beat frequency in bovine bronchial epithelial cells.

Mucociliary clearance is a critical host defense that protects the lung. The mechanisms by which mucociliary function is altered by inflammation are poorly defined. Chronic exposure to cigarette smoke decreases ciliary beating and interferes with proper airway clearance. Bronchoalveolar lavage (BAL) fluid from smokers and ex-smokers has increased amounts of IL-8, which has played a critical role in airway inflammation. We hypothesized that IL-8 might interfere with stimulated ciliary beating in airway epithelium. To test this hypothesis, we stimulated bovine ciliated bronchial epithelial cells (BBECs) with a known activator of ciliary beat frequency (CBF), isoproterenol (ISO; 100 microM), in the presence or absence of IL-8 (100 pg/mL). We measured CBF digitally using the Sisson-Ammons Video Analysis (SAVA) system. CBF increased in untreated cells exposed to ISO (approximately 3 Hz) over baseline. In contrast, cells pre-incubated with IL-8 failed to respond to ISO. Pretreatment with IL-8 also blocked ISO-stimulated cAMP-dependent kinase (PKA) activation, which is known to control ISO-stimulated CBF. In addition, IL-8 pretreated cells revealed a marked decrease in PKA activity when cells were stimulated with forskolin (FSK; 10 microM). Cells were assayed specifically for cAMP-phosphodiesterase (PDE) activity. ISO-stimulated cells demonstrated an increase in PDE activity as compared to control. Pretreatment with IL-8 had no effect on ISO-stimulated PDE activity. Collectively, these data suggest that IL-8 appears to mediate its effect at the level of adenylyl cyclase. It is also possible that IL-8 may not only act as a chemotactic agent, but also as a potential autocrine/paracrine inhibitor of PKA-mediated stimulation of ciliary motility. In conclusion, IL-8 inhibits beta-agonist dependent ciliostimulation and such inhibition of stimulated ciliary activity may contribute to the impaired mucociliary clearance seen in airway diseases. Furthermore, since IL-8 levels are increased in the airway of cigarette smokers, it is likely they may be more resistant to the cilio and muco-ciliostimulating effects of beta-agonists.

Relaxin stimulates bronchial epithelial cell PKA activation, migration, and ciliary beating.

Relaxin is an insulin-like serum protein secreted during pregnancy and found in many tissues, including the lung. Relaxin is reported to stimulate epithelial cell proliferation, but the effects of relaxin on airway epithelium are unknown. We tested the hypothesis that relaxin would stimulate the increased migration of bronchial epithelial cells (BEC) in response to wounding. Using monolayers of BEC in a wound-healing model, relaxin augmented wound closure with maximal closure occurring at 12 hr (1 micro M). Unlike cytokines, relaxin did not stimulate increased BEC interleukin-8 (IL-8) release. Relaxin caused a significant stimulation of ciliary beat frequency (CBF) in BEC. Because protein kinase (PKA) activation increases CBF and relaxin can elevate intracellular cAMP levels, we measured PKA activity in BEC treated with relaxin. Relaxin increased PKA activity 3-4 fold by approximately 4 hr, with a return to baseline levels by 8-10 hr. Relaxin-stimulated PKA activity differs temporally from the rapid (1 hr) beta-adrenergic activation of PKA in BEC. These data suggest that relaxin augments epithelial repair by increasing airway cell migration and CBF via PKA-dependent mechanisms.

The differential effect of VIP and PACAP on guinea pig gallbladder in vitro.

OBJECTIVES: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a recently identified member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family. Like VIP, PACAP is largely inhibitory in the gastrointestinal tract. The aim of our work was to characterize the effects of PACAP on both contraction and relaxation of guinea pig gallbladder (GPGB) muscle. METHODS: Gallbladder muscle strips were obtained from male Dunkin-Hartley guinea pigs (250-350 g). Isometric tension was measured in strips suspended in gassed (95% O2, 5% CO2) Krebs' solution at 37 degrees C and equilibrated for 60 min. Cumulative additions of VIP or PACAP (10(-9)-10(-6) mol/l) were performed with strips at basal tone or with strips pre-contracted with cholecystokinin-octapeptide (CCK-8). RESULTS: VIP had no effect on basal tone, in contrast with PACAP which produced concentration-dependent contraction to a maximum of 57.9 +/- 24.3% of control (CCK 3 x 10(-7) mol/l). The highest concentration (10(-6) mol/l) of VIP produced a 32 +/- 6% relaxation of 3 x 10(-9) mol/l CCK-8-contracted GPGB. With 3 x 10(-8) mol/l CCK-contracted GPGB strips, VIP produced a 26.7 +/- 6.6% relaxation. PACAP produced a further concentration-dependent contraction of 3 x 10(-9) mol/l CCK-contracted strips which reached 17.5 +/- 9.9% at the maximal concentration used (10(-6) mol/l). PACAP produced a concentration-dependent relaxation of 3 x 10(-8) mol/l CCK-contracted strips which reached a maximum relaxation of 19 +/- 5% of the control. CONCLUSIONS: PACAP has a dual effect on guinea pig gallbladder motility in vitro, producing contraction in the basal state, and both contraction and relaxation in the CCK-contracted state. This is in contrast to the effects of VIP, a closely related peptide.

Physiological roles of matrix metalloproteinases: implications for tumor growth and metastasis.

Physiological processes involving remodelling of the extracellular matrix, such as wound healing, embryogenesis, angiogenesis, and the female reproductive cycle, require the activity of matrix metalloproteinases (MMPs). This group of proteases degrades basal membranes and connective tissues and plays an essential role in the homeostasis of the extracellular matrix. An imbalance in the expression or activity of MMPs can have important consequences in diseases such as multiple sclerosis, Alzheimer's disease, or the development of cancers. Because of the pathophysiological importance of MMPs, their activity is highly controlled in order to confine them to specific areas. An activation cascade, initiated by the proteolysis of plasminogen, cleaves proMMPs, and every step is controlled by specific activators or inhibitors. MMPs destabilize the organization of the extracellular matrix and influence the development of cancer by contributing to cell migration, tumor cell proliferation, and angiogenesis. Accordingly, these proteases possess an important role in cell-matrix interactions by affecting fundamental processes such as cell differentiation and proliferation. Therefore, the characterization of MMPs involved in specific types and stages of tumors will significantly improve the diagnosis and treatment of these cancers in humans.

Biological and molecular analyses of structurally reduced analogues of endothelin-1.

Structurally reduced analogues of endothelin-1 (ET-1) were synthesized through linking with an aliphatic spacer [aminocaproic acid (Aca)], segment 3-11 of ET-1 to carboxyl-terminal fragments of various lengths (16-21, 17-21,...,21). The peptides were prepared in their linear or cyclic form, and a formyl group was or was not introduced on the Trp21 side chain. Pharmacological studies were carried out with the guinea pig lung parenchyma paradigm and the rat thoracic aorta bioassay. In the rat aorta, an ET(A) receptor preparation, all of the analogues were inactive. However, in the lung parenchyma, we observed that among the linear formylated derivatives, [Cys(Acm)3,11,Trp(For)21]-(3-11)-Aca-(17-21)ET was a partial agonist. In this series, the presence of His16, as in [Cys(Acm)3,11,Trp(For)21]-(3-11)-Aca-(16-21)ET, caused a decrease in contractile activity, suggesting that the imidazole group disfavors the proper interaction of the linear molecule with the ETB receptors of the lung parenchyma. The loss of biological activity of the deformylated linear analogues strongly suggested that the formyl group played a stabilizing role in the structure of the linear molecules. Interestingly, molecular modeling studies indicated the adoption of different conformations by the formylated and the nonformylated analogues. In contrast, the stabilizing effect of the formyl group was not observed with the cyclic compounds. Furthermore, the presence of His16 favored the contractile activity of the cyclic peptides. Finally, the results demonstrated that the carboxyl-terminal residues 18-21 are required for the activity in the guinea pig lung parenchyma ETB receptors.

Potential roles for endothelins in systemic inflammatory response syndrome with a particular relationship to cytokines.

Endothelins (ETs) are multifunctional isopeptides and their role in several pathophysiologies is slowly emerging. They are possible therapeutic targets in sepsis and other systemic inflammatory response syndromes (SIRS). In such conditions, elevated concentrations of ETs have been reported, together with other proinflammatory markers such as several cytokines. Some of the cytokines even modulate the expression, production, and release of ETs from cells and also the biological responsiveness of ETs into the circulation. Here, we systematically review the literature and discuss the role of these peptides in affecting vascular and inflammatory responses in SIRS. This review also intends to provide a better understanding of the mechanisms of ETs and their interrelationships to other mediators, mainly cytokines, in SIRS. There is no doubt that ETs are useful markers of vascular injury in SIRS. From experimental evidence in animals, endothelins, as potent vasoconstrictors, play a beneficial central compensatory role against the loss of vascular tone associated with SIRS. Conversely, endothelins compromise the circulation in several vascular beds and exacerbate conditions in which inadequate perfusion already exists during the early stages of SIRS. Since no single magic solution has been found for complex diseases related to SIRS, we should look toward a group of mediators, such as cytokines and endothelins, acting as team players.

Structure-activity studies of the C-terminal segment of structurally reduced analogues of ET-1.

Structurally reduced analogues of endothelin-1 (ET-1) were synthesized by linking via aminocaproic acid (Aca) the segment 3-11 of ET-1 to C-terminal fragments of various lengths [16-21, 17-21,...,21]. Analogues were studied in their linear or cyclic form in the absence or presence of a formyl group on the Trp21 side-chain, and their biologic activities were tested using guinea pig lung parenchymal strips. The absence of the first disulfide bridge and the presence of the Aca spacer caused a slight decrease in activity. The presence of His16 is essential for the contractile activity of the monocyclic peptides. Formylation of these monocyclic analogues did not modify this behavior. Similarly, linear analogues carrying S-acetamidomethyl (Acm) functions and an Aca linker were still active. However, most formylated linear derivatives were partial agonists, whereas the addition of His16 caused a decrease in contractile activity. In these latter analogues, molecular modeling studies suggested that formylation produces different conformations.