CCL21 Programs Immune Activity in Tumor Microenvironment.

CCL21 promotes immune activity in the tumor microenvironment (TME) by colocalizing dendritic cells (DC) and T cells programing ectopic lymph node architectural structures that correlate with cancer prognosis. Innovative strategies to deliver CCL21 in cancer patients will reactivate the downregulated immune activity in the TME. Immune escape mechanisms are upregulated in the TME that promote tumor immune evasion. CCL21 combined with inhibition of dominant pathways of immune evasion will aid in the development of effective immunotherapy for cancer.

MicroRNA profile in HBV-induced infection and hepatocellular carcinoma.

BACKGROUND: MicroRNAs (miRNAs) exhibit essential regulatory functions related to cell growth, apoptosis, development and differentiation. Dysregulated expression of miRNAs is associated with a wide variety of human diseases. As such miRNA signatures are valuable as biomarkers for disease and for making treatment decisions. Hepatitis B virus (HBV) is a major risk factor for hepatocellular carcinoma (HCC). Here we screened for miRNAs in chronic HBV associated HCC. METHODS: To determine the miRNAs in HCC occurrence associated with HBV infection, we analyzed global miRNA expression profiles in 12 pairs of HCC and adjacent matched non-HCC tissues from HBV-positive and HBV-negative patients using microarray analyses. The microarray result was validated by real-time PCR in 32 HBV-positive and 24 HBV-negative patient HCC samples. The potential candidate target genes of the miRNAs were predicted by miRWalk software. Genes simultaneously predicted as targets by two or more miRNAs were subjected to GO and KEGG pathway analysis. The miRNA regulatory network analysis was performed using the Ingenuity Pathway Analysis (IPA) software. RESULTS: Eight miRNAs (miR-223, miR-98, miR-15b, miR-199a-5p, miR-19b, miR-22, miR-451, and miR-101) were involved in HBV-unrelated HCC, 5 miRNAs (miR-98, miR-375, miR-335, miR-199a-5p, and miR-22) were involved in HBV infection, and 7 miRNAs (miR-150, miR-342-3p, miR-663, miR-20b, miR-92a-3p, miR-376c-3p and miR-92b) were specifically altered in HBV-related HCC. Gene Ontology and KEGG analyses predict that these HBV-related HCC miRNAs are involved in the regulation of: transcription, RNA polymerase II promoter, phosphorylation of proteins through MAPK signaling pathway, focal adhesion, and actin cytoskeleton. IPA analysis also suggest that these miRNAs act on AGO2, TP53, CCND1, and 11 other genes that significantly influence HCC occurrence and HBV infection. CONCLUSION: Our data indicates that the unique 7 miRNAs expression signature could be involved in the development HBV- related HCC.

Immunotherapy for malignant pleural mesothelioma. Current status and future prospects.

Malignant pleural mesothelioma (MPM) is a rare malignancy of the pleura that is frequently resistant to conventional therapies. Immunotherapy is a promising investigational approach for MPM that has shown some evidence of clinical benefit in select patients. However, tumor-induced immunosuppression is likely a major impediment to achieving optimal clinical responses to immunotherapeutic intervention. MPM contains a variable degree of infiltrating T-regulatory cells and M2 macrophages, which are believed to facilitate tumor evasion from the host immune system. Additional immunosuppressive factors identified in other human tumor types, such as tumor-associated programmed death ligand-1 expression, may be relevant for investigation in MPM. Conventional cytoreductive therapies, such as radiation, chemotherapy, and surgery, may play a critical role in successful immunotherapeutic strategies by ablating intratumoral and/or systemic immunosuppressive factors, thus creating a host environment more amenable to immunotherapy. This article reviews the immunotherapeutic approaches being evaluated in patients with MPM and discusses how immunotherapy might be rationally combined with conventional tumor cytoreductive therapies for this disease.

Role of PAX8 in the regulation of MET and RON receptor tyrosine kinases in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancers (NSCLC) are highly heterogeneous at the molecular level and comprise 75% of all lung tumors. We have previously shown that the receptor tyrosine kinase (RTK) MET frequently suffers gain-of-function mutations that significantly promote lung tumorigenesis. Subsequent studies from our lab also revealed that PAX5 transcription factor is preferentially expressed in small cell lung cancer (SCLC) and promotes MET transcription. PAX8, however, is also expressed in NSCLC cell lines. We therefore investigated the role of PAX8 in NSCLC. METHODS: Using IHC analysis, PAX8 protein expression was determined in archival NSCLC tumor tissues (n = 254). In order to study the effects of PAX8 knockdown on NSCLC cellular functions such as apoptosis and motility, siRNA against PAX8 was used. Confocal fluorescence microscopy was used to monitor the localization of MET, RON and PAX8. The combinatorial effect of PAX8 knockdown and MET inhibition using SU11274 was investigated in NSCLC cell viability assay. RESULTS: Relative levels of PAX8 protein were elevated (≥ + 2 on a scale of 0-3) in adenocarcinoma (58/94), large cell carcinoma (50/85), squamous cell carcinoma (28/47), and metastatic NSCLC (17/28; lymph node). Utilizing early progenitors isolated from NSCLC cell lines and fresh tumor tissues, we observed robust overexpression of PAX8, MET, and RON. PAX8 knockdown A549 cells revealed abrogated PAX8 expression with a concomitant loss in MET and the related RON kinase expression. A dramatic colocalization between the active form of MET (also RON) and PAX8 upon challenging A549 cells with HGF was visualized. A similar colocalization of MET and EGL5 (PAX8 ortholog) proteins was found in embryos of C. elegans. Most importantly, knockdown of PAX8 in A549 cells resulted in enhanced apoptosis (~6 fold) and decreased cell motility (~45%), thereby making PAX8 a potential therapeutic target. However, the combinatorial approach of PAX8 knockdown and treatment with MET inhibitor, SU11274, had marginal additive effect on loss of NSCLC cell viability. CONCLUSION: PAX8 provides signals for growth and motility of NSCLC cells and is necessary for MET and RON expression. Further investigations are necessary to investigate the therapeutic potential of PA8 in NSCLC.

Bmi-1 confers adaptive radioresistance to KYSE-150R esophageal carcinoma cells.

Radiotherapy (RT) is a major modality of cancer treatment. However, tumors often acquire radioresistance, which causes RT to fail. The exact mechanisms by which tumor cells subjected to fractionated irradiation (FIR) develop an adaptive radioresistance are largely unknown. Using the radioresistant KYSE-150R esophageal squamous cell carcinoma (ESCC) model, which was derived from KYSE-150 parental cells using FIR, the role of Bmi-1 in mediating the radioadaptive response of ESCC cells to RT was investigated. The results showed that the level of Bmi-1 expression was significantly higher in KYSE-150R cells than in the KYSE-150 parental cells. Bmi-1 depletion sensitized the KYSE-150R cells to RT mainly through the induction of apoptosis, partly through the induction of senescence. A clonogenic cell survival assay showed that Bmi-1 depletion significantly decreased the radiation survival fraction in KYSE-150R cells. Furthermore, Bmi-1 depletion increased the generation of reactive oxygen species (ROS) and the expression of oxidase genes (Lpo, Noxo1 and Alox15) in KYSE-150R cells exposed to irradiation. DNA repair capacities assessed by γ-H2AX foci formation were also impaired in the Bmi-1 down-regulated KYSE-150R cells. These results suggest that Bmi-1 plays an important role in tumor radioadaptive resistance under FIR and may be a potent molecular target for enhancing the efficacy of fractionated RT.

Early intervention with a small molecule inhibitor for tumor necrosis factor-α prevents cognitive deficits in a triple transgenic mouse model of Alzheimer's disease.

BACKGROUND: Chronic neuroinflammation is an important component of Alzheimer's disease and could contribute to neuronal dysfunction, injury and loss that lead to disease progression. Multiple clinical studies implicate tumor necrosis factor-α as an inflammatory mediator of neurodegeneration in patients with Alzheimer's because of elevated levels of this cytokine in the cerebrospinal fluid, hippocampus and cortex. Current Alzheimer's disease interventions are symptomatic treatments with limited efficacy that do not address etiology. Thus, a critical need exists for novel treatments directed towards modifying the pathophysiology and progression. METHODS: To investigate the effect of early immune modulation on neuroinflammation and cognitive outcome, we treated triple transgenic Alzheimer's disease mice (harboring PS1(M146V), APP(Swe), and tau(P301L) transgenes) with the small molecule tumor necrosis factor-α inhibitors, 3,6'-dithiothalidomide and thalidomide, beginning at four months of age. At this young age, mice do not exhibit plaque or tau pathology but do show mild intraneuronal amyloid beta protein staining and a robust increase in tumor necrosis factor-α. After 10 weeks of treatment, cognitive performance was assessed using radial arm maze and neuroinflammation was assessed using biochemical, stereological and flow cytometric endpoints. RESULTS: 3,6'-dithiothalidomide reduced tumor necrosis factor-α mRNA and protein levels in the brain and improved working memory performance and the ratio of resting to reactive microglia in the hippocampus of triple transgenic mice. In comparison to non-transgenic controls, triple transgenic Alzheimer's disease mice had increased total numbers of infiltrating peripheral monomyelocytic/granulocytic leukocytes with enhanced intracytoplasmic tumor necrosis factor-α, which was reduced after treatment with 3,6'-dithiothalidomide. CONCLUSIONS: These results suggest that modulation of tumor necrosis factor-α with small molecule inhibitors is safe and effective with potential for the long-term prevention and treatment of Alzheimer's disease.

IL-7 promotes CXCR3 ligand-dependent T cell antitumor reactivity in lung cancer.

We are evaluating the immune enhancing activities of cytokines for their optimum utility in augmenting cellular immune responses against lung cancer. In this study, we evaluated the mechanism of antitumor responses following IL-7 administration to mice bearing established Lewis lung cancer. IL-7 decreased tumor burden with concomitant increases in the frequency of CD4 and CD8 T lymphocyte subsets, T cell activation markers CXCR3, CD69, and CD127(low), effector memory T cells, and T cell cytolytic activity against parental tumor cells. Accompanying the antitumor responses were increases in IFN-gamma, CXCL9, CXCL10, and IL-12. Individual neutralization of CD4, CD8 T lymphocytes, or the CXCR3 ligands CXCL9 and CXCL10 reversed the antitumor benefit of IL-7, indicating their importance for optimal responses in vivo. Furthermore, IL-7 decreased the tumor-induced apoptosis of T cells with subsequent decrease of the proapoptotic marker Bim. We assessed the impact of IL-7 treatment on regulatory T cells that negatively impact antitumor immune responses. IL-7 decreased regulatory T Foxp3 as well as cell suppressive activity with a reciprocal increase in SMAD7. These results indicate that IL-7 induces CXCR3 ligand-dependent T cell antitumor reactivity in lung cancer.

Targeting BMI-1-mediated epithelial-mesenchymal transition to inhibit colorectal cancer liver metastasis.

Liver is the most common metastatic site for colorectal cancer (CRC), there is no satisfied approach to treat CRC liver metastasis (CRCLM). Here, we investigated the role of a polycomb protein BMI-1 in CRCLM. Immunohistochemical analysis showed that BMI-1 expression in liver metastases was upregulated and associated with T4 stage, invasion depth and right-sided primary tumor. Knockdown BMI-1 in high metastatic HCT116 and LOVO cells repressed the migratory/invasive phenotype and reversed epithelial-mesenchymal transition (EMT), while BMI-1 overexpression in low metastatic Ls174T and DLD1 cells enhanced invasiveness and EMT. The effects of BMI-1 in CRC cells were related to upregulating snail via AKT/GSK-3ß pathway. Furthermore, knockdown BMI-1 in HCT116 and LOVO cells reduced CRCLM using experimental liver metastasis mice model. Meanwhile, BMI-1 overexpression in Ls174T and DLD1 significantly increased CRCLM. Moreover, sodium butyrate, a histone deacetylase and BMI-1 inhibitor, reduced HCT116 and LOVO liver metastasis in immunodeficient mice. Our results suggest that BMI-1 is a major regulator of CRCLM and provide a potent molecular target for CRCLM treatment.

Technical Feasibility and Safety of Repeated Computed Tomography-Guided Transthoracic Intratumoral Injection of Gene-Modified Cellular Immunotherapy in Metastatic NSCLC.

INTRODUCTION: To assess the technical feasibility and safety of repeated percutaneous computed tomography (CT)-guided transthoracic biopsies and intratumoral injections of gene-modified dendritic cells in metastatic NSCLC. METHODS: A total of 15 patients with 15 NSCLC lesions measuring greater than 1.0 cm underwent two cycles of intratumoral biopsies and CCL21 dendritic cell injections separated by 7 days. All needle placements and injections were done under CT guidance. Clinical and imaging follow-up was done approximately 4 weeks after the first procedure. Safety and feasibility were determined as: (1) safety and feasibility similar to that of single-needle biopsy, and (2) an absence of serious adverse events defined as grade greater than or equal to three according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0. RESULTS: A total of 30 percutaneous, transthoracic intratumoral biopsies and injections into the lung cancer were performed, two cycles (at d 0 and 7) received by each patient (311 biopsies and 96 intratumoral injections). All percutaneous cases achieved technical success with respect to needle placement for both biopsy and injection of CCL21 dendritic cells. Only minor complications were observed (grade <3), including pneumothorax (n = 10, 33%) and small postbiopsy hemorrhage (n = 2, 7%). Pneumothorax was moderate (n = 1) or trace (n = 9), with resolution of the moderate pneumothorax after manual aspiration without chest tube placement. No patient required chest tube placement. No other complications or serious adverse effects related to the biopsy or dendritic cell injection were noted. All patients were in stable condition after up to 4 hours in the recovery unit and were discharged home on the same day. No procedure-related complications were observed on imaging or clinical follow-up at 4 weeks. CONCLUSIONS: Repeated percutaneous, transthoracic CT-guided biopsies and intratumoral gene-modified cell-based immunotherapy injections into lung cancers are technically feasible, safe, and reproducible. There were no procedure-related serious (defined as grade ≥3) adverse events.

PD-1 Immune Checkpoint Blockade Promotes Therapeutic Cancer Vaccine to Eradicate Lung Cancer.

Background: Targeting inhibitory immune checkpoint molecules has highlighted the need to find approaches enabling the activation of immune responses against cancer. Therapeutic vaccination, which induces specific immune responses against tumor antigens (Ags), is an attractive option. Methods: Utilizing a K-RasG12Dp53null murine lung cancer model we determined tumor burden, tumor-infiltrating T cell (TIL) cytolysis, immunohistochemistry, flow cytometry, and CD4 and CD8 depletion to evaluate the efficacy of PD-1 blockade combined with CCL21-DC tumor lysate vaccine. Results: Anti-PD-1 plus CCL21-DC tumor lysate vaccine administered to mice bearing established tumors (150 mm3) increased expression of perforin and granzyme B in the tumor microenvironment (TME), increased tumor-infiltrating T cell (TIL) activity, and caused 80% tumor eradication. Mice with treatment-induced tumor eradication developed immunological memory, enabling tumor rejection upon challenge and cancer-recurrence-free survival. The depletion of CD4 or CD8 abrogated the antitumor activity of combined therapy. PD-1 blockade or CCL21-DC tumor lysate vaccine monotherapy reduced tumor burden without tumor eradication. Conclusion: Immune checkpoint blockade promotes the activity of the therapeutic cancer vaccine. PD-1 blockade plus CCL21-DC tumor lysate vaccine therapy could benefit lung cancer patients.