Purpose: Although existence of humoral immunity has been previously shown in malignant pleural effusions, only a limited number of immunogenic tumor-associated antigens (TAA) have been identified and associated with lung cancer. In this study, we intended to identify moreTAAs in pleural effusion^derived tumor cells. Experimental Design: Using morphologically normal lung tissues as a control lysate inWestern blotting analyses, 54 tumor samples were screened with autologous effusion antibodies. Biochemical purification and mass spectrometric identification of TAAs were done using established effusion tumor cell lines as antigen sources. We identified a p48 antigen as a-enolase (ENO1). Semiquantitative immunohistochemistry was used to evaluate expression status of ENO1 in the tissue samples of 80 patients with non^small cell lung cancer (NSCLC) and then correlated with clinical variables. Results: Using ENO1-specifc antiserum, up-regulation of ENO1expression in effusion tumor cells from 11of 17 patients was clearly observed compared with human normal lung primary epithelial and non-cancer-associated effusion cells. Immunohistochemical studies consistently showed high level of ENO1 expression in all the tumors we have examined thus far. Log-rank and Cox's analyses of ENO1 expression status revealed that its expression level in primary tumors was a key factor contributing to overall-and progression-free survivals of patients (P < 0.05). The same result was also obtained in the early stage of NSCLC patients, showing that tumors expressing relatively higher ENO1level were tightly correlated with poorer survival outcomes. Conclusions: Our data strongly support a prognostic role of ENO1in determining tumor malignancy of patients with NSCLC.

Chronic inflammation, coupled with alcohol, betel quid, and cigarette consumption, is associated with oral squamous cell carcinoma (OSCC). Interleukin-1 beta (IL-1b) is a critical mediator of chronic inflammation and implicated in many cancers. In this study, we showed that increased pro-IL-1b expression was associated with the severity of oral malignant transformation in a mouse OSCC model induced by 4-Nitroquinolin-1-oxide (4-NQO) and arecoline, two carcinogens related to tobacco and betel quid, respectively. Using microarray and quantitative PCR assay, we showed that pro-IL-1b was upregulated in human OSCC tumors associated with tobacco and betel quid consumption. In a human OSCC cell line TW2.6, we demonstrated nicotine-derived nitrosamine ketone (NNK) and arecoline stimulated IL-1b secretion in an inflammasome-dependent manner. IL-1b treatment significantly increased the proliferation and dysregulated the Akt signaling pathways of dysplastic oral keratinocytes (DOKs). Using cytokine antibodies and inflammation cytometric bead arrays, we found that DOK and OSCC cells secreted high levels of IL-6, IL-8, and growth-regulated oncogene-a following IL-1b stimulation. The conditioned medium of IL-1b-treated OSCC cells exerted significant proangiogenic effects. Crucially, IL-1b increased the invasiveness of OSCC cells through the epithelial-mesenchymal transition (EMT), characterized by downregulation of E-cadherin, upregulation of Snail, Slug, and Vimentin, and alterations in morphology. These findings provide novel insights into the mechanism underlying OSCC tumorigenesis. Our study suggested that IL-1b can be induced by tobacco and betel quid-related carcinogens, and participates in the early and late stages of oral carcinogenesis by increasing the proliferation of dysplasia oral cells, stimulating oncogenic cytokines, and promoting aggressiveness of OSCC.

In previous research, we found α-enolase to be inversely correlated with progression-free and overall survival in lung cancer patients and detected α-enolase on the surface of lung cancer cells. Based on these findings, we hypothesized that surface α-enolase has a significant role in cancer metastasis and tested this hypothesis in the current study. We found that α-enolase was co-immunoprecipitated with urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen in lung cancer cells and interacted with these proteins in a cell-free dot blotting assay, which can be interrupted by α-enolase-specific antibody. α-Enolase in lung cancer cells co-localized with these proteins and was present at the site of pericellular degradation of extracellular matrix components. Treatment with antibody against α-enolase in vitro suppressed cell-associated plasminogen and matrix metalloproteinase activation, collagen and gelatin degradation, and cell invasion. Examination of the effect of treatment with shRNA plasmids revealed that down regulation of α-enolase decreases extracellular matrix degradation by and the invasion capacity of lung cancer cells. Adoptive transfer of α-enolase-specific antibody to mice resulted in accumulation of antibody in subcutaneous tumor and inhibited the formation of tumor metastasis in lung and bone. This study demonstrated that surface α-enolase promotes extracellular matrix degradation and invasion of cancer cells and that targeting surface α-enolase is a promising approach to suppress tumor metastasis.

SummaryMesenchymal stromal cells (MSCs) are multilineage progenitors with immunomodulatory properties, including expansion of immunomodulatory leukocytes such as regulatory T lymphocytes (Tregs) and tolerogenic dendritic cells. We report that human MSCs can expand CD14−CD11b+CD33+ human myeloid-derived suppressor cells (MDSCs). MSC-expanded MDSCs suppress allogeneic lymphocyte proliferation, express arginase-1 and inducible nitric oxide synthase, and increase the number of Tregs. This expansion occurs through the secretion of hepatocyte growth factor (HGF), with effects replicated by adding HGF singly and abrogated by HGF knockdown in MSCs. In wild-type mice, the liver, which secretes high levels of HGF, contains high numbers of Gr-1+CD11b+ MDSCs, and injection of HGF into mice significantly increases the number of MDSCs. Expansion of MDSCs by MSC-secreted HGF involves c-Met (its receptor) and downstream phosphorylation of STAT3, a key factor in MDSC expansion. Our data further support the strong immunomodulatory nature of MSCs and demonstrate the role of HGF, a mitogenic molecule, in the expansion of MDSCs.

Monocytes are a population of leukocytes that terminally differentiate into macrophages and DCs. Whereas these differentiated progeny have inflammatory and resident--which are more immunomodulatory--phenotypes, less has been reported on the plasticity of monocytes themselves. We found that MSCs, a population of somatic stem cells, can rapidly induce human and murine monocytes through secretion of HGF to acquire an immunomodulatory phenotype to suppress T cell effector function. MSCs are multilineage postnatal progenitor cells with strong immunomodulatory effects toward T lymphocytes, NK lymphocytes, and DCs, but less is known regarding their interactions with monocytes. We found that CD14(+) human monocytes express c-Met, the receptor for HGF, and both depletion of HGF-treated CD14(+) monocytes and knockdown of HGF secretion in MSCs abrogate the suppression of anti-CD3/28-activated T cell proliferation. HGF-treated monocytes remain undifferentiated and can alter activated T cell cytokine expression from a Th1 toward Th2 profile. Moreover, monocytes cocultured with MSCs or treated with HGF alone can produce high levels of IL-10, a potent immunomodulatory cytokine. Injection of HGF to WT mice also results in an increase in IL-10(+)-expressing monocytes from the spleen, a known reservoir for circulating monocytes. Mechanistically, HGFs modulate IL-10 production in monocytes through the ERK1/2 pathway. Our data demonstrate further the pleomorphic nature of MSC immunomodulation, as well as highlight the important role of immunomodulatory monocytes in altering T cell effector function.

The broad immunomodulatory properties of human mesenchymal stem cells (MSCs) have allowed for wide application in regenerative medicine as well as immune/ inflammatory diseases, including unmatched allogeneic use. The novel coronavirus disease COVID-19 has unleashed a pandemic in record time accompanied by an alarming mortality rate mainly due to pulmonary injury and acute respiratory distress syndrome. Because there are no effective preventive or curative therapies currently, MSC therapy (MSCT) has emerged as a possible candidate despite the lack of preclinical data of MSCs for COVID-19. Interestingly, MSCT preclinical data specifically on immune/inflammatory disorders of the lungs were among the earliest to be reported in 2003, with the first clinical use of MSCT for graft-vs-host disease reported in 2004. Since these first reports, preclinical data showing beneficial effects of MSC immunomodulation have accumulated substantially, and as a consequence, over a third of MSCT clinical trials now target immune/inflammatory diseases. There is much preclinical evidence for MSCT in noninfectious-including chronic obstructive pulmonary disease, asthma, and idiopathic pulmonary fibrosis-as well as infectious bacterial immune/inflammatory lung disorders, with data generally demonstrating therapeutic effects; however, for infectious viral pulmonary conditions, the preclinical evidence is more scarce with some inconsistent outcomes. In this article, we review the mechanistic evidence for clinical use of MSCs in pulmonary immune/inflammatory disorders, and survey the ongoing clinical trials-including for COVID-19-of MSCT for these diseases, with some perspectives and comment on MSCT for COVID-19.

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DNA vaccine has been suggested to use in cancer therapy, but the efficacy remains to be improved. The immunostimulatory effect of a fungal immunomodulatory protein Ling Zhi-8 (LZ-8) isolated from Ganoderma lucidum has been reported. In this study, we tested the adjuvanticity of LZ-8 for HER-2/neu DNA vaccine against p185(neu) expressing tumor MBT-2 in mice. We found that recombinant LZ-8 stimulated mouse bone marrow-derived dendritic cells (DCs) via TLR4 and its stimulatory effect was not due to any microbe contaminant. In addition, LZ-8 enhanced the ability of DCs to induce antigen-specific T cell activation in vitro and in a subunit vaccine model in vivo. Surprisingly, LZ-8 cotreatment strongly improved the therapeutic effect of DNA vaccine against MBT-2 tumor in mice. This increase in antitumor activity was attributed to the enhancement of vaccine-induced Th1 and CTL responses. Consistent with the results from DCs, the promoting effect of LZ-8 on DNA vaccine was diminished when the MBT-2 tumor cells were grown in TLR4 mutant mice. Thus, we concluded that LZ-8 may be a promising adjuvant to enhance the efficacy of DNA vaccine by activating DCs via TLR4.