No abstract

Using genetic interventions, we previously determined that C-C motif chemokine ligand 2 (CCL2) promotes malignant pleural effusion (MPE) formation in mice. Here we conducted preclinical studies aimed at assessing the specific therapeutic potential of antibody-mediated CCL2 blockade against MPE. For this, murine MPEs or skin tumors were generated in C57BL/6 mice by intrapleural or subcutaneous delivery of lung (LLC) or colon (MC38) adenocarcinoma cells. Human lung adenocarcinoma cells (A549) were used to induce MPEs in severe combined immunodeficient mice. Intraperitoneal antibodies neutralizing mouse CCL2 and/or CCL12, a murine CCL2 ortholog, were administered at 10 or 50 mg/kg every three days. We found that high doses of CCL2/12 neutralizing antibody treatment (50 mg/kg) were required to limit MPE formation by LLC cells. CCL2 and CCL12 blockade were equally potent inhibitors of MPE development by LLC cells. Combined CCL2 and CCL12 neutralization was also effective against MC38-induced MPE and prolonged the survival of mice in both syngeneic models. Mouse-specific CCL2-blockade limited A549-caused xenogeneic MPE, indicating that host-derived CCL2 also contributes to MPE precipitation in mice. The impact of CCL2/12 antagonism was associated with inhibition of immune and vascular MPE-related phenomena, such as inflammation, new blood vessel assembly and plasma extravasation into the pleural space. We conclude that CCL2 and CCL12 blockade are effective against experimental MPE induced by murine and human adenocarcinoma in mice. These results suggest that CCL2-targeted therapies may hold promise for future use against human MPE.

The lungs are ubiquitous receptacles of metastases originating from various bodily tumors. Although osteopontin (SPP1) has been associated with tumor dissemination, the role of its isoforms in lung-directed metastasis is incompletely understood. We employed syngeneic mouse models of spontaneous and induced lung-targeted metastasis in C57BL/6 mice competent and deficient in both Spp1 alleles. Tumorderived osteopontin expression was modulated using either stable anti-Spp1 RNA interference, or forced overexpression of intracellular and secreted Spp1 isoforms. Identified osteopontin's downstream partners were validated using lung adenocarcinoma cells conditionally lacking the Trp53 gene and Ccr2-deficient mice. We determined that host-derived osteopontin was dispensable for pulmonary colonization by different tumor types. Oppositely, tumor-originated intracellular osteopontin promoted tumor cell survival by preventing tumor-related protein 53-mediated apoptosis, while the secretory osteopontin functioned in a paracrine mode to accelerate lung metastasis by enhancing tumor-derived C-C-motif chemokine ligand 2 signaling to cognate host receptors. As new ways to target osteopontin signaling are becoming available, the cytokine may constitute an important therapeutic target against pulmonary involvement by cancers of other organs. KEYWORDSC-C-motif chemokine ligand 2; inflammation and cancer; secreted phosphoprotein 1; spontaneous lung metastasis; tumor-related protein 53

Nuclear factor (NF)-κB signalling is required for lung adenocarcinoma development in mice, and both of its subunits RelA and RelB were independently reported to be highly expressed in human non-small cell lung cancer (NSCLC). To comprehensively examine NF-κB expression in NSCLC, we analyzed serial sections of primary tumor samples from 77 well-documented patients (36 adenocarcinomas, 40 squamous cell carcinomas and 3 large cell carcinomas) for immunoreactivity of RelA, RelB, P50, and P52/P100. Tumor and intratumoral stroma areas were discriminated based on proliferating cell nuclear antigen immunoreactivity and inflammatory infiltration was assessed in intratumoral stroma areas. NF-κB immunoreactivity was quantified by intensity, extent, and nuclear localization and was cross-examined with tumor cell proliferation, inflammatory infiltration, and clinical-pathologic data. We found that the expression of the different NF-κB subunits was not concordant, warranting our integral approach. Overall, RelA, RelB, and P50 were expressed at higher levels compared with P52/P100. However, RelA and P50 were predominantly expressed in intratumoral stroma, but RelB in tumor cells. Importantly, tumor area RelA expression was correlated with the intensity of inflammatory infiltration, whereas RelB expression was identified in proliferating tumor cells. Using multiple logistic regression, we identified that tumor RelB expression was an independent predictor of lymph node metastasis, and tumor P50 was an independent predictor of TNM6 stage IIB or higher, whereas tumor RelA was an independent predictor of inflammatory infiltration. We conclude that pathologic studies of NF-κB expression in cancer should include multiple pathway components. Utilizing such an approach, we identified intriguing associations between distinct NF-κB subunits and clinical and pathologic features of NSCLC.

BackgroundThe hippocampus is a functionally heterogeneous brain structure and specializations of the intrinsic neuronal network may crucially support the functional segregation along the longitudinal axis of the hippocampus. Short-term synaptic plasticity plays fundamental roles in information processing and may be importantly involved in diversifying the properties of local neuronal network along the hippocampus long axis. Therefore, we aimed to examine the properties of the cornu ammonis 1 (CA1) synapses along the entire dorsoventral axis of the rat hippocampus using field excitatory postsynaptic potentials from transverse rat hippocampal slices and a frequency stimulation paradigm.ResultsApplying a ten-pulse stimulus train at frequencies from 0.1 to 100 Hz to the Schaffer collaterals we found a gradually diversified pattern of frequency-dependent synaptic effects along the dorsoventral hippocampus axis. The first conditioned response was facilitated along the whole hippocampus for stimulus frequencies 10–40 Hz. However, steady-state responses or averaged responses generally ranged from maximum synaptic facilitation in the most dorsal segment of the hippocampus to maximum synaptic depression in the most ventral segment of the hippocampus. In particular, dorsal synapses facilitated for stimulus frequency up to 50 Hz while they depressed at higher frequencies (75–100 Hz). Facilitation at dorsal synapses was maximal at stimulus frequency of 20 Hz. On the contrary, the most ventral synapses showed depression regardless of the stimulus frequency, only displaying a transient facilitation at the beginning of 10–50 Hz stimulation. Importantly, the synapses in the medial hippocampus displayed a transitory behavior. Finally, as a whole the hippocampal synapses maximally facilitated at 20 Hz and increasingly depressed at 50–100 Hz.ConclusionThe short-term synaptic dynamics change gradually along the hippocampal long axis in a frequency-dependent fashion conveying distinct properties of information processing to successive segments of the structure, thereby crucially supporting functional segregation along the dorsoventral axis of the hippocampus.

Hippocampus is importantly involved in dopamine-dependent behaviors and dopamine is a significant modulator of synaptic plasticity in the hippocampus. Moreover, the dopaminergic innervation appears to be disproportionally segregated along the hippocampal longitudinal (dorsoventral) axis with unknown consequences for synaptic plasticity. In this study we examined the actions of endogenously released dopamine and the effects of exogenous D1/D5 dopamine receptor agonists on theta-burst stimulation-induced long-term potentiation (LTP) of field excitatory synaptic potential (fEPSP) at Schaffer collateral-CA1 synapses in slices from dorsal (DH) and ventral hippocampus (VH). Furthermore, we quantified D1 receptor mRNA and protein expression levels in DH and VH. We found that blockade of D1/D5 receptors by SCH 23390 (20 μM) significantly reduced the magnitude of LTP in both DH and VH similarly suggesting that dopamine endogenously released during TBS, presumably mimicking low activity of DA neurons, exerts a homogeneous modulation of LTP along the hippocampal long axis. Moderate to high concentrations of the selective partial D1/D5 receptor agonist SKF 38393 (50-150 μM) did not significantly change LTP in either hippocampal segment. However, the full D1 receptor selective agonist SKF 82958 (10 μM) significantly enhanced LTP in VH but not DH. Furthermore, the expression of D1 receptor mRNA and protein was considerably higher in VH compared with DH. These results suggest that the dynamic range of D1/D5 receptor-mediated dopamine effects on LTP may be higher in VH than DH and that VH may be specialized to acquire information about behaviorally relevant strong stimuli signaled by the dopamine system.

No abstract

Mast cells (MC) have been identified in human lung adenocarcinoma (LADC) tissues, but their functional role has not been investigated in vivo. For this, we applied three mouse models of KRAS-mutant LADC to two different MC-deficient mouse strains (cKit Wsh and Cpa3.Cre). Moreover, we derived MC gene signatures from murine bone marrow-derived MC and used them to interrogate five human cohorts of LADC patients. Tumor-free cKit Wsh and Cpa3.Cre mice were deficient in alveolar and skin KITdependent (KIT+) MC, but cKit Wsh mice retained normal KIT-independent (KIT-) MC in the airways. Both KIT+ and KIT-MC infiltrated murine LADC to varying degrees, but KIT+ MC were more abundant and promoted LADC initiation and progression through interleukin-1β secretion. KIT+ MC and their transcriptional signature were significantly enriched in human LADC compared to adjacent normal tissue, especially in the subset of patients with KRAS mutations. Importantly, MC density increased with tumor stage and high overall expression of the KIT+ MC signature portended poor survival. Collectively, our results indicate that KIT+ MC foster LADC development and represent marked therapeutic targets.