MMP-9-cleaved osteopontin isoform mediates tumor immune escape by inducing expansion of myeloid-derived suppressor cells.

As an extracellular matrix protein, osteopontin (OPN) has been shown to play an important role in regulation of the immune response to tumors. Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid progenitors, are major components of the immune suppressive tumor microenvironment and contribute to tumor evasion of the immune response. However, the specific regulating mechanisms underlying MDSCs expansion remain unclear. Here, we found that MDSCs accumulated in the spleen and tumors of 3LL tumor-bearing mice. Supernatant collected from 3LL cells was able to induce the expansion of MDSCs in peripheral blood mononuclear cell (PBMC) in vitro. Results of enzyme linked immunosorbent assay showed high levels of OPN and matrix metalloproteinase-9 (MMP-9) in this supernatant. Silencing OPN can effectively reduce MDSCs frequency in vivo and in vitro. Furthermore, a specific fragment of OPN, OPN-32 kDa cleaved by MMP-9 was detected in the supernatant from 3LL cells. Overexpression of OPN-32 kDa in 3LL cells induced MDSCs expansion. Inhibition of MMP-9 by monoclonal antibody and inhibitor (TIMP-1) reduced MDSCs expansion in vitro and in vivo. These findings suggest that the MMP-9-cleaved OPN fragment, OPN-32kDa, was responsible for MDSCs expansion, which may contribute to tumor's evasion of the immune response.

Myeloid-derived suppressor cells regulate immune response in patients with chronic hepatitis B virus infection through PD-1-induced IL-10.

Although myeloid-derived suppressor cells (MDSCs) are well known for their immunosuppressive function in several pathological conditions, the role of MDSCs in hepatitis B virus infection remains obscure. In this study, we investigated the frequency and function of MDSCs in the peripheral blood and liver of 91 chronic hepatitis B (CHB) patients. A higher percentage of MDSCs, defined as CD14(+)HLA-DR(-/low), was detected in peripheral blood of CHB patients than that of the healthy controls. Moreover, high expression of programmed death 1 (PD-1) and secretion of IL-10 in this population were determined. The frequency of MDSCs was positively correlated with serum viral load, but it was negatively correlated with liver inflammatory injury. These cells were also abundant in liver tissue of CHB patients and were related to necroinflammatory activity. Furthermore, we found that these cells could suppress hepatitis B virus-specific CD8(+) T cell response, including reduced proliferation and IFN-γ production, and inhibit degranulation of CD8(+) T cells, including reduced production of granzyme B and perforin. Importantly, PD-1-induced IL-10 production by MDSCs was responsible for the suppressive activity. To our knowledge, for the first time our study proved that CD14(+)HLA-DR(-/low)PD-1(+) MDSCs in CHB patients contribute to an inadequate immune response against the virus and lead to chronic infection, which represents a potential target for therapeutic intervention.

Thymosin α1 promotes the activation of myeloid-derived suppressor cells in a Lewis lung cancer model by upregulating Arginase 1.

Thymosin α1 (Tα1) has been tested for cancer therapy for several years, in most cases, the anti-tumor effect of Tα1 was limited, especially when Tα1 was used as a single agent. The role of Tα1 in cancer treatment and the regulatory mechanisms by which Ta1 takes effects are not yet completely understood. Using a Lewis lung caner model, here we report that Tα1 used alone elevated CD8(+) T cells, but failed to inhibit tumor growth. Furthermore, immunosuppressive myeloid-derived suppressor cells (MDSCs) showed heightened Arginase 1 production in response to Tα1 treatment, which led to stronger suppression of anti-tumor immunity. In addition, the upregulation of ARG1 was dependent on TLRs/MyD88 signaling, blocking MyD88 signaling abrogated the enhanced ARG1 expression and restored the anti-tumor efficacy of Tα1. This study provides the first demonstration that Tα1 treatment activates but not expands MDSCs via MyD88 signaling, which indicates better immunotherapeutic strategy of Tα1 against cancer.

Elevated plasma interleukin-35 levels predict poor prognosis in patients with non-small cell lung cancer.

Interleukin-35 (IL-35) has recently been implicated in tumor immunity. The aim of this study was to explore the clinical role of plasma IL-35 in patients with non-small cell lung cancer (NSCLC). Plasma collected from 106 patients with NSCLC cases and 78 healthy controls (HC) were subjected to IL-35 enzyme-linked immunosorbent assay (ELISA) and relationships between plasma IL-35 levels and clinical characteristics were evaluated. The correlation of IL-35 and overall survival was analyzed using Kaplan-Meier method. The prognostic value of IL-35 was tested using univariate and multivariate analysis. Circulating IL-35 levels were significantly higher in the NSCLC group in comparison with the HC group (21.37 ± 11.55 pg/ml vs. 10.09 ± 5.32 pg/ml, p < 0.001). Correlation analysis by subgroup indicated that plasma IL-35 correlated positively with tumor TNM stage (p < 0.001) and lymph node metastases (p < 0.0001). Using a cutoff level of 20.26 pg/ml (median value), IL-35 showed an inverse correlation with overall survival. Univariate and multivariate analysis indicated that plasma IL-35 was an independent prognostic factor for NSCLC patients. Circulating IL-35 in NSCLC patients significantly increased. IL-35 is a promising potential biomarker in prognostication of clinical outcome of NSCLC patients and the regulation of IL-35 expression may provide a new target for the treatment of NSCLC patients.

Anti-osteopontin monoclonal antibody prevents ovariectomy-induced osteoporosis in mice by promotion of osteoclast apoptosis.

Osteopontin (OPN) is abundant in mineralized tissues and has long been implicated in bone remodeling. However, the therapeutic effect of targeting OPN in bone loss diseases and the underlying molecular mechanism remain largely unknown. Here, we reported that anti-OPN mAb (23C3) could protect against ovariectomy-induced osteoporosis in mice, demonstrated by microcomputed tomography analysis and histopathology evaluation. In vitro assay showed that 23C3 mAb reduced osteoclasts (OCs)-mediated bone resorption through promotion of mature OC apoptosis. Thus, the study has important implications for understanding the role of OPN in OC bone resorption and survival, and OPN antagonists may have therapeutic potential for osteoporosis and other osteopenic diseases.

Increased CD14(+)HLA-DR (-/low) myeloid-derived suppressor cells correlate with extrathoracic metastasis and poor response to chemotherapy in non-small cell lung cancer patients.

Accumulating evidence has demonstrated that myeloid-derived suppressor cells (MDSCs), a heterogeneous population of cells, play an important role in the subversion, inhibition, and downregulation of the immune response to cancer. However, the characteristics of these cells, particularly clinical relevance, in malignant tumors remain unclear due to a lack of specific markers. In this study, we characterized peripheral CD14(+)HLA-DR(-/low) cells, a new human MDSC subpopulation, in 89 patients with non-small cell lung cancer (NSCLC). As expected, both frequency and absolute number of CD14(+)HLA-DR(-/low) cells were significantly increased in the peripheral blood of NSCLC patients compared with that of the healthy controls and indicated an association with metastasis, response to chemotherapy, and progression-free survival. These cells showed decreased expression of CD16 and CD86 compared with HLA-DR(+) monocytes. Unlike classical monocytes, these populations showed significantly decreased allostimulatory activity and showed the ability to inhibit autologous T cell proliferation and IFN-γ production in a cell-contact-dependent manner. Furthermore, we demonstrated that CD14(+)HLA-DR(-/low) cells expressed the NADPH oxidase component gp91(phox) and generated high level of reactive oxygen species (ROS). Moreover, inactivation of ROS reversed their immunosuppressive capacity on T cell response. These results prove, for the first time, the existence of ROS-producing CD14(+)HLA-DR(-/low) myeloid-derived suppressor cells in NSCLC patients, which mediate tumor immunosuppression and might thus represent a potential target for therapeutic intervention.

Identification of a novel functional domain of ricin responsible for its potent toxicity.

Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate the universally conserved α-sarcin loop of large rRNAs. They have received attention in biological and biomedical research because of their unique biological activities toward animals and human cells as cell-killing agents. A better understanding of the depurination mechanism of RIPs could allow us to develop potent neutralizing antibodies and to design efficient immunotoxins for clinical use. Among these RIPs, ricin exhibited remarkable efficacy in depurination activity and highly conserved tertiary structure with other RIPs. It can be considered as a prototype to investigate the depurination mechanism of RIPs. In the present study, we successfully identified a novel functional domain responsible for controlling the depurination activity of ricin, which is located far from the enzymatic active site reported previously. Our study indicated that ricin A-chain mAbs binding to this domain (an α-helix comprising the residues 99-106) exhibited an unusual potent neutralizing ability against ricin in vivo. To further investigate the potential role of the α-helix in regulating the catalytic activity of ricin, ricin A-chain variants with different flexibility of the α-helix were rationally designed. Our data clearly demonstrated that the flexibility of the α-helix is responsible for controlling the depurination activity of ricin and determining the extent of protein synthesis inhibition, suggesting that the conserved α-helix might be considered as a potential target for the prevention and treatment of RIP poisoning.

A functional motif QLYxxYP is essential for osteopontin induced T lymphocyte activation and migration.

Osteopontin (OPN) plays an important role in regulating lymphocyte adhesion and cytokine production associated with inflammatory processes and autoimmune diseases. Here we developed and characterized a monoclonal antibody F8E11 specific for human OPN (hOPN). F8E11 could inhibit OPN-induced lymphocyte activation and migration. Epitope mapping showed that F8E11 could specifically recognize the peptide QLYxxYP. In addition, a synthesized mimetic peptide F8P (EEKQLYNKYPDA) could block the binding of F8E11 to hOPN and significantly inhibit the hOPN-induced lymphocyte migration. Moreover, mutations on the QLYxxYP motif of hOPN also markedly diminished its activity for lymphocyte activation and migration. The functioning assay indicated that this novel epitope is critically involved in the lymphocyte migration through activating MAPK/ERK/AP-1 pathway, which can be inhibited by the motif QLYxxYP blocking antibody, F8E11. These results suggest that this novel epitope of OPN may provide a potential therapeutic target for the treatment of T cell mediated-immune diseases.

Leukemia-related protein 16 (LRP16) promotes tumor growth and metastasis in pancreatic cancer.

BACKGROUND: Leukemia related protein 16 (LRP16), one of the genes belonging to the macro domain family, has been found up-regulated in various tumors including testicles, ovaries and mucosa of colon and is associated with poor clinical outcomes. PURPOSE: The objective of this study was to investigate expression pattern and biological roles of LRP16 in pancreatic cancer. PATIENTS AND METHODS: Western blot and immunohistochemistry were used to investigate the expression of LRP16 in pancreatic cancer cell lines and tissues. qRT-PCR was utilized to examine LRP16 mRNA expression. Lentivirus based overexpression and knockdown of LRP16 was carried out in four pancreatic cancer cell lines (Panc1, CFPAC1, SW1990 and AsPC1). Cell proliferation, migration and invasion were determined by MTS and transwell assay, respectively. Flow cytometry was performed to investigate cell apoptosis. In vivo, the tumorigenic ability of LRP16 was determined in a NOD/SCID mouse model. RESULTS: In the present study, we found that LRP16 expression was increased in pancreatic tumor samples, compared with normal tissues. Moreover, the LRP16 expression was positive in 60.9% of 156 specimens and correlated with tumor size, clinical stage, distant metastasis and tumor differentiation. Multivariate Cox regression analysis revealed that the level of LRP16 expression was an independent prognostic factor for overall survival in pancreatic cancer patients. Furthermore, silencing of LRP16 significantly accelerated apoptosis, decrease proliferation, migration and invasion of pancreatic cancer cell lines in vitro. In contrast, overexpression of LRP16 attenuated apoptosis, promoted proliferation, migration and invasion. In addition, in vivo study revealed that down regulation of LRP16 could attenuate tumor growth and prolong the survival. On the contrary, up-regulation of LRP16 could promote tumor growth and shorten their survival. CONCLUSION: These findings suggest that LRP16 played an oncogenic role in pancreatic carcinoma.

LRP16 prevents hepatocellular carcinoma progression through regulation of Wnt/ß-catenin signaling.

Elevated LRP16 expression is associated with poor clinical outcomes in multiple malignancies. We detected LRP16 expression in hepatocellular carcinoma (HCC) and found that it was downregulated in tumor samples and HCC cell lines. In a cohort of 80 HCC patients, high level of LRP16 expression in HCC tumors was associated with well differentiation, less lymph node metastasis, and good overall survival (OS). Overexpression of LRP16 in the HepG2 and MHCC-97L cell lines increased cell apoptosis, attenuated cell proliferation, migration, and invasion ability in vitro, and drastically diminished tumor growth and metastasis in vivo. Silencing LRP16 in HCC-LM3 and SMMC-7721 cell lines showed opposite results. Microarray evaluation of tumor cells overexpressing LRP16 revealed the effects on decreased activity in the Wnt signaling pathway. These results were confirmed by qRT-PCR and Western blots. Furthermore, inhibition of Wnt signaling decreased proliferation, migration, and invasion of HCC cell lines. Mechanism conducted showed that LRP16 overexpression could prevent ß-catenin from entering the nucleus. Our study demonstrated that LRP16 suppresses tumor growth in HCC by modulating Wnt/ß-catenin signaling. KEY MESSAGES: LRP16 was low expression in HCC tissue and cell lines. Low expression of LRP16 in HCC was associated with poor prognosis. LRP16 inhibits activation of the Wnt/ß-catenin pathway in HCC. LRP16 prevents ß-catenin from entering the nucleus.

Blockade of LIGHT/HVEM and B7/CD28 signaling facilitates long-term islet graft survival with development of allospecific tolerance.

BACKGROUND: Previous studies have shown that blockade of LIGHT, a T-cell costimulatory molecule belonging to the tumor necrosis factor (TNF) superfamily, by soluble lymphotoxin beta receptor-Ig (LTbetaR-Ig) inhibited the development of graft-versus-host disease. The cardiac allografts were significantly prolonged in LIGHT deficient mice. No data are yet available regarding the role of the LIGHT/HVEM pathway in more stringent fully allogeneic models such as skin and islet transplantation models. METHODS: Streptozotocin-induced chemical diabetic BALB/C mice underwent transplantation with allogeneic C57BL/6 islets and were treated with LTbetaR-Ig, CTLA4-Ig or a combination of both in the early peritransplant period. RESULTS: Administration of CTLA4-Ig or LTbeta R-Ig alone only increased graft survival to 55 days and 27 days respectively, whereas simultaneous blockade of both pathways significantly prolonged the islet allograft survival for more than 100 days. Long-term survivors were retransplanted with donor-specific (C57BL/6) islets and the grafted islets remained functional for more than 100 days. All of islet allografts were protected against rejection when the mixtures of 1x10(6) CD4+ T cells from tolerant mice and islet allografts were cotransplanted under the renal capsule of the naïve BALB/c recipients. CONCLUSIONS: These data indicate that: 1) a synergistic effect for prolonged graft survival can be obtained by simultaneously blocking LIGHT and CD28 signaling in the stringent model of islet allotransplantation; 2) development of donor-specific immunological tolerance is associated with the presence of regulatory T-cell activity; and 3) local cotransplantation of the allografts with the regulatory T cells can effectively prevent allograft rejection and induce donor-specific tolerance in lymphocytes-sufficient recipients.

DRB genotyping in cynomolgus monkeys from China using polymerase chain reaction -sequence-specific primers.

Cynomolgus macaques are relevant models for human diseases and transplantation. In each case, a complete understanding of these models requires knowledge of the macaque major histocompatibility complex (MHC). Because of high polymorphism and multiple genes per haplotype, it has been difficult to develop a rapid typing method for cynomolgus monkey MHC class II. We developed a simple and rapid polymerase chain reaction-sequence specific primer (PCR-SSP) strategy for Chinese cynomolgus monkey DRB locus typing. Forty Chinese cynomolgus monkeys originating from the Guangxi Province in China were included in the study. Twenty nine cynomolgus monkey allele-specific primer pairs were designed based on published Macaca fascicularis (Mafa)-DRB locus gene sequences. Allele-specific PCR products ranged in size from 143 to 253 bp; 5' and 3' Mafa-DRB locus allele specific primers were located in the second exon. Specific PCR product gel purification was followed by direct sequencing in both directions. Our data showed prominent variability in the number of Mafa-DRB sequences ranging from 2 to 7 per animal. This analysis demonstrated that most of the amplicons were identical to Mafa-DRB sequences that our PCR primers were to amplify. However, 98 to 99% similarity was noticed in the case of Mafa-DRB4*0101, Mafa-DRB*W2101, and Mafa-DRB*W4901 sequences. The observed mismatches were located in nonpolymorphic regions. Thus, haplotype analysis confirmed the existence of allelic associations published earlier. In addition, we propose a new DRB sequence. The established medium-resolution PCR-SSP technique appears to be a highly reproducible and discriminatory typing method for detecting polymorphisms of DRB genes in Chinese cynomolgus monkeys.

Functional and stable expression of recombinant human FOXP3 in bacterial cells and development of antigen-specific monoclonal antibodies.

FOXP3 is a member of the forkhead/winged-helix family of transcriptional regulators which plays a key role in CD4+CD25+ regulatory T-cell (Tregs) function and represents a specific marker for these cells. In order to understand the functional role of FOXP3 and identify Tregs both in normal development and relevant diseases, protein-DNA and protein-protein interaction studies involving this factor are essentially required. Such investigations would be facilitated by the availability of significant amounts of purified FOXP3 protein and specific McAb against FOXP3. Here, we report the purification of human FOXP3 (HFOXP3) expressed in Escherichia coli as a soluble and functional glutathione-S-transferase (GST) fusion protein. Through a single purification procedure, 4 mg of GST-HFOXP3 recombinant protein was obtained per litre of bacterial culture. The biological activity of the recombinant protein was verified by EMSA assay. The yield of folded FOXP3 in the purified GST-FOXP3 was determined by reverse phase HPLC. Besides, we generated and obtained two specific monoclonal antibodies by immunizing BALB/c mouse with the purified GST-HFOXP3 protein. The resulting HFOXP3 protein and anti-HFOXP3 McAbs might provide a useful tool in studying Tregs development and immune regulations.

Successful in vivo hyperthermal therapy toward breast cancer by Chinese medicine shikonin-loaded thermosensitive micelle.

The Chinese traditional medicine Shikonin is an ideal drug due to its multiple targets to tumor cells. But in clinics, improving its aqueous solubility and tumor accumulation is still a challenge. Herein, a copolymer with tunable poly(N-isopropylacrymaide) and polylactic acid block lengths is designed, synthesized, and characterized in nuclear magnetic resonance. The corresponding thermosensitive nanomicelle (TN) with well-defined core-shell structure is then assembled in an aqueous solution. For promoting the therapeutic index, the physical-chemistry properties of TNs including narrow size, low critical micellar concentration, high serum stability, tunable volume phase transition temperature (VPTT), high drug-loading capacity, and temperature-controlled drug release are systematically investigated and regulated through the fine self-assembly. The shikonin is then entrapped in a degradable inner core resulting in a shikonin-loaded thermosensitive nanomicelle (STN) with a VPTT of ~40°C. Compared with small-molecular shikonin, the in vitro cellular internalization and cytotoxicity of STN against breast cancer cells (Michigan Cancer Foundation-7) are obviously enhanced. In addition, the therapeutic effect is further enhanced by the programmed cell death (PCD) specifically evoked by shikonin. Interestingly, both the proliferation inhibition and PCD are synergistically promoted as T > VPTT, namely the temperature-regulated passive targeting. Consequently, as intravenous injection is administered to the BALB/c nude mice bearing breast cancer, the intratumor accumulation of STNs is significantly increased as T > VPTT, which is regulated by the in-house developed heating device. The in vivo antitumor assays against breast cancer further confirm the synergistically enhanced therapeutic efficiency. The findings of this study indicate that STN is a potential effective nanoformulation in clinical cancer therapy.

Umbilical cord-derived mesenchymal stem cells alleviate liver fibrosis in rats.

AIM: To evaluate the efficacy of umbilical cord-derived mesenchymal stem cells (UC-MSCs) transplantation in the treatment of liver fibrosis. METHODS: Cultured human UC-MSCs were isolated and transfused into rats with liver fibrosis induced by dimethylnitrosamine (DMN). The effects of UC-MSCs transfusion on liver fibrosis were then evaluated by histopathology; serum interleukin (IL)-4 and IL-10 levels were also measured. Furthermore, Kupffer cells (KCs) in fibrotic livers were isolated and cultured to analyze their phenotype. Moreover, UC-MSCs were co-cultured with KCs in vitro to assess the effects of UC-MSCs on KCs' phenotype, and IL-4 and IL-10 levels were measured in cell culture supernatants. Finally, UC-MSCs and KCs were cultured in the presence of IL-4 antibodies to block the effects of this cytokine, followed by phenotypical analysis of KCs. RESULTS: UC-MSCs transfused into rats were recruited by the injured liver and alleviated liver fibrosis, increasing serum IL-4 and IL-10 levels. Interestingly, UC-MSCs promoted mobilization of KCs not only in fibrotic livers, but also in vitro. Co-culture of UC-MSCs with KCs resulted in increased production of IL-4 and IL-10. The addition of IL-4 antibodies into the co-culture system resulted in decreased KC mobilization. CONCLUSION: UC-MSCs could increase IL-4 and promote mobilization of KCs both in vitro and in vivo, subsequently alleviating the liver fibrosis induced by DMN.

Prognostic significance of osteopontin in patients with non-small cell lung cancer: results from a meta-analysis.

BACKGROUNDS: Non-small cell lung cancer (NSCLC) is one of the most common malignancies with a high mortality level. Recently, a variety of studies explored the role of osteopontin (OPN) expression in the prognosis of NSCLC, but the results were controversial. METHODS: We performed a meta-analysis of eligible studies to evaluate the prognostic significance of OPN expression in NSCLC patients. In order to assess the association between OPN and OS and DFS/PFS, hazard ratio (HR) with 95% confidence interval (CI) was calculated. RESULTS: A total of ten studies comprising 1420 patients were included in the meta-analysis. The summary results indicated that high OPN expression was a poor predictor for OS (HR = 2.19, 95% CI: 1.6-2.98), and DFS/PFS (HR = 2, 95% CI: 1.66-2.41). Subgroup analysis revealed that high OPN expression was a negative prognostic marker for OS and DFS/PFS regardless of ethnicity background, treatment and OPN detection method. CONCLUSION: Our results showed that increased OPN expression significantly correlated with poor OS and DPS/PFS in NSCLC patients.

Increased circulating CD14(+)HLA-DR-/low myeloid-derived suppressor cells are associated with poor prognosis in patients with small-cell lung cancer.

BACKGROUND: High expression of CD14(+)HLA-DR-/low myeloid-derived suppressor cells (MDSCs) is correlated with immunosuppressive activity in various cancers, however, no studies have shown a correlation of these immunosuppressive cells with clinical outcomes in small-cell lung cancer (SCLC) patients. OBJECTIVE: The purpose of the study was to investigate the number, frequency and clinical significance of CD14(+)HLA-DR-/low MDSCs in SCLC patients. METHODS: The peripheral blood of 42 patients with SCLC and 37 healthy controls was analyzed by using flow cytometry. The relationships between the number and frequency of MDSCs, clinicopathological features and overall survival(OS) were analyzed. The prognostic value of MDSCs was tested by using univariate and multivariate analysis. RESULTS: Our results demonstrated that number and frequency of peripheral CD14(+)HLA-DR-/low MDSCs were significantly increased in SCLC patients than in controls (p< 0.0001 and p< 0.0001, respectively). The frequency of MDSCs correlated with tumor stage (p= 0.02), serum LDH levels (p= 0.001) and with the poorer OS (log-rank test, p= 0.017). Univariate and multivariate analyses suggested that frequency of CD14(+)HLA-DR-/low MDSCs as an independent biomarker for poor prognosis in SCLC patients during follow-up. Our study provides the first evidence that the frequency of CD14(+)HLA-DR-/low MDSCs negatively correlates with clinical outcomes in SCLC patients. CONCLUSIONS: The frequency of CD14(+)HLA-DR-/low MDSCs could be considered as a poor prognostic predictor in SCLC and the elimination of MDSCs during medical interventions may improve the prognosis of SCLC patients.

Prognostic value of serum IL-17 and VEGF levels in small cell lung cancer.

OBJECTIVE: To explore the prognostic value of serum interleukin-17 (IL-17) and vascular endothelial growth factor (VEGF) levels in patients with small cell lung cancer (SCLC). MATERIALS AND METHODS: IL-17 and VEGF levels were determined by a commercially available ELISA method. RESULTS: Serum IL-17 and VEGF levels were higher in the SCLC group than the control group (p<0.001 and p<0.0001, respectively). In addition, there was a significant correlation between IL-17 and VEGF. The level of IL-17 showed significant correlations with tumor stage and tumor metastasis, while serum VEGF levels were significantly associated only with tumor metastasis. Univariate and multivariate analysis indicated that an elevated IL-17 level was an independent prognostic factor for shorter overall survival in SCLC. CONCLUSIONS: IL-17 may play an important role in tumor dissemination in SCLC patients and measurement of IL-17 could have useful prognostic value for worse overall survival in patients with SCLC.

Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression.

Although HER2-targeting antibody trastuzumab confers a substantial benefit for patients with HER2-overexpressing breast and gastric cancer, overcoming trastuzumab resistance remains a large unmet need. In this study, we revealed a STAT3-centered positive feedback loop that mediates the resistance of trastuzumab. Mechanistically, chronic exposure of trastuzumab causes the upregulation of fibronection (FN), EGF and IL-6 in parental trastuzumab-sensitive breast and gastric cells and convergently leads to STAT3 hyperactivation. Activated STAT3 enhances the expression of FN, EGF and IL-6, thus constituting a positive feedback loop which amplifies and maintains the STAT3 signal; furthermore, hyperactivated STAT3 signal promotes the expression of MUC1 and MUC4, consequently mediating trastuzumab resistance via maintenance of persistent HER2 activation and masking of trastuzumab binding to HER2 respectively. Genetic or pharmacological inhibition of STAT3 disrupted STAT3-dependent positive feedback loop and recovered the trastuzumab sensitivity partially due to increased apoptosis induction. Combined trastuzumab with STAT3 inhibition synergistically suppressed the growth of the trastuzumab-resistant tumor xenografts in vivo. Taken together, our results suggest that feedback activation of STAT3 constitutes a key node mediating trastuzumab resistance. Combinatorial targeting on both HER2 and STAT3 may enhance the efficacy of trastuzumab or other HER2-targeting agents in HER2-positive breast and gastric cancer.

Human umbilical cord blood-derived mesenchymal stem cells producing IL15 eradicate established pancreatic tumor in syngeneic mice.

Mesenchymal stem cells (MSC) represent a new tool for delivery of therapeutic agents to cancer sites because of their strong tropism toward tumors. IL15 has demonstrated a potent antitumor activity in various animal models as well as clinical trials. However, because of its short half-life, effective therapeutic effects usually require a high dose, which often results in undesired side effects; thus, new strategies for overcoming this disadvantage are needed. In this study, human MSCs were isolated from umbilical cord blood as delivery vehicles and transduced with lentivirus vector expressing murine IL15 (MSC-IL15). In vitro assays of lymphocyte activation and proliferation demonstrated that IL15 produced by MSCs was biofunctional. In syngeneic mice bearing Pan02 pancreatic tumors, systemic administration of MSC-IL15 significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice, which were associated with tumor cell apoptosis, and natural killer (NK)- and T-cell accumulation. Furthermore, we confirmed that MSC-IL15 could migrate toward tumor and secreted IL15 in tumor-specific sites. Depletion of NK and CD8(+) T cells abolished the antitumor activity of MSC-IL15, suggesting that NK and CD8(+) T cells play a key role for MSC-IL15-mediated effect. Interestingly, cured mice after MSC-IL15 treatment were resistant to Pan02 pancreatic tumor rechallenge, and adoptive transfer of lymphocytes from cured mice also could cause rejection of Pan02 tumor inoculation in naïve mice, indicating that MSC-IL15 induced tumor-specific T-cell immune memory response. Overall, these data support that MSCs producing IL15 might represent an innovative strategy for therapy of pancreatic tumor.