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PurposeThe NB5 assay was performed in bone marrow (BM) and peripheral blood (PB) to detect neuroblastomas (NBs) with micrometastases. The sensitivity and factors influencing the NB5 assay were preliminarily evaluated.MethodsThe NB5 assay uses RT-PCR to detect the co-expression of five mRNAs from the neuroblastoma-associated genes, CHGA, DCX, DDC, PHOX2B, and TH. We enrolled 180 cases of neuroblastoma and 65 cases of non-neuroblastoma. Bone marrow and peripheral blood were collected from every patient. The gold standard for the diagnosis of NB was pathological evaluation of solid tumor specimens or bone marrow biopsies (BMBs) from hematological tumors. STATA version 15 and SPSS version 17 software were used for analysis.ResultsWe found that 17 patients were BMB (+), and they were diagnosed as the International Neuroblastoma Staging System (INSS) stage IV and the high-risk group. All 17 patients were BM (+), while 15 patients were PB (+) (15/17, 88.2%). Among the 163 children who were BMB (−), 56 were BM (+), 40 were PB (+), and 36 were BM (+) and PB (+). The sensitivity of the NB5 assay in BM (40.5%) and PB (30.5%) was significantly higher than the sensitivity of BMB (9.4%, P = 0.000). In the non-NB group, four cases were BM (+) and one case was PB (+). The specificity of the NB5 assay in BM and PB was 93.8% and 98.5%, respectively. The sensitivity of the NB5 assay in both BM and PB in INSS stage IV patients was significantly higher than that in INSS stage I–II patients (P ConclusionsThe NB5 assay had significantly higher sensitivity than the pathological analysis of BMB in detecting NB with micrometastases. The NB5 assay had higher sensitivity in INSS stage IV or the high-risk group. Liver metastases and bone metastases were the primary factors that affected the sensitivity of the NB5 assay.

Cancer immunotherapy has been firmly established as a new milestone for cancer therapy, with the development of multiple immune cells as therapeutic tools. Natural killer (NK) cells are innate immune cells endowed with potent cytolytic activity against tumors, and meanwhile act as regulatory cells for the immune system. The efficacy of NK cell-mediated immunotherapy can be enhanced by immune stimulants such as cytokines and antibodies, and adoptive transfer of activated NK cells expanded ex vivo. In addition, NK cells can arm themselves with chimeric antigen receptors (CARs), which may greatly enhance their anti-tumor activity. Most recently, extracellular vesicles (EVs) derived from NK cells show promising anti-tumor effects in preclinical studies. Herein, we carefully review the current progress in these NK cell-based immunotherapeutic strategies (NK cells combined with stimulants, adoptive transfer of NK cells, CAR-NK cells, and NK EVs) for the treatment of cancers, and discussed the challenges and opportunities for opening a new horizon for cancer immunotherapy.

Background: miRNA, a small non-coding RNA molecule containing about 22 nucleotides, functions in RNA silencing and post-transcriptional regulation of gene expression. With these qualities, miRNAs modulate multiple signaling pathways involved in cancer development, such as cellular proliferation, apoptosis, and migration. Methods: The goal of this review is to discuss possible miRNAs-based therapeutic strategies, through defining performance of miRNAs in carcinogenesis, in particular in lung cancer. Results: miRNA, a non-coding RNA, is divided into two main types: tumor suppressor miRNAs and oncogenic miRNAs. In addition, special processed miRNAs can be an assistant therapeutic tool. Conclusion: In order to develope antitumor therapy, miRNAs-based therapeutic strategies are worth of deeper studies. In this process, the stability, effectiveness, and side effects should be considered.

Purpose: Immunotherapy of high-risk neuroblastoma using the anti-GD2 antibody dinutuximab induces antibody-dependent cell-mediated cytotoxicity (ADCC). Galunisertib, an inhibitor of TGFβR1, was examined for its ability to enhance the efficacy of dinutuximab in combination with human ex vivo activated NK (aNK) cells against neuroblastoma. Experimental Design: TGFB1 and TGFBR1 mRNA expression was determined for 249 primary neuroblastoma tumors by microarray analysis. The ability of galunisertib to inhibit SMAD activity induced by neuroblastoma patient blood and bone marrow plasmas in neuroblastoma cells was tested. The impact of galunisertib on TGFβ1-induced inhibition of aNK cytotoxicity and ADCC in vitro and on anti-neuroblastoma activity in NOD-scid gamma (NSG) mice was determined. Results: Neuroblastomas express TGFB1 and TGFBR1 mRNA. Galunisertib suppressed SMAD activation in neuroblastoma cells induced by exogenous TGFβ1 or by patient blood and bone marrow plasma, and suppressed SMAD2 phosphorylation in human neuroblastoma cells growing in NSG mice. In NK cells treated in vitro with exogenous TGFβ1, galunisertib suppressed SMAD2 phosphorylation and restored the expression of DNAM-1, NKp30, and NKG2D cytotoxicity receptors and the TRAIL death ligand, the release of perforin and granzyme A, and the direct cytotoxicity and ADCC of aNK cells against neuroblastoma cells. Addition of galunisertib to adoptive cell therapy with aNK cells plus dinutuximab reduced tumor growth and increased survival of mice injected with two neuroblastoma cell lines or a patient-derived xenograft. Conclusions: Galunisertib suppresses activation of SMAD2 in neuroblastomas and aNK cells, restores NK cytotoxic mechanisms, and increases the efficacy of dinutuximab with aNK cells against neuroblastoma tumors. Clin Cancer Res; 23(3); 804–13. ©2016 AACR. See related commentary by Zenarruzabeitia et al., p. 615

Neuroblastoma is the most common extracranial solid tumor in the pediatric population. Sorafenib (Nexavar), a multikinase inhibitor, blocks cell proliferation and induces apoptosis in certain types of cancers. Here, we tested antitumor effects of sorafenib (≤ 10 µM) on four human neuroblastoma cell lines, CHLA255, CHLA171, CHLA90 and SK-N-AS. Sorafenib inhibited cell proliferation and induced apoptosis of neuroblastoma tumor cells in a dose-dependent manner. Sorafenib inhibited phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) proteins at Tyr705 in these cells, associated with inhibition of phosphorylated JAK2, an upstream kinase that mediates STAT3 phosphorylation. Expression of a constitutively-activated STAT3 mutant (pSTAT3-C) partially blocked the antitumor effects of sorafenib on neuroblastoma cells. Sorafenib also inhibited the phosphorylation of STAT3 induced by IL-6 and sphingosine-1-phosphate (S1P), a recently identified regulator for STAT3, in these tumor cells. Moreover, sorafenib downregulated phosphorylation of MAPK (p44/42) in neuroblastoma cells, consistent with inhibition of their upstream regulators MEK1/2. Sorafenib inhibited expression of cyclin E, cyclin D1/D2/D3, key regulators for cell cycle, and the antiapoptotic proteins Mcl-1 and survivin. Finally, sorafenib suppressed the growth of human neuroblastoma cells in a mouse xenograft model. Taken together, these findings suggest the potential use of sorafenib for the treatment of pediatric neuroblastomas.

Natural killer (NK) cells-based immunotherapy is one of the most promising treatments for incurable malignant tumors. NK cells in combination with monoclonal antibodies to surface antigens of the tumor cell have been proved to be effective in a number of clinical trials. A limiting step in the development of successful cellular immunotherapy lies in developing an efficient and economic method to expand appropriate amount of NK cells and CD8(+) T cells. In this study, we constructed a humanized IL-15Ralpha-IgG1-Fc, which mimicked IL-15 trans-presentation. The feasibility of expanding populations of the human NK and CD8(+) T cells by IL-15Ralpha-IgG1-Fc complexes was tested. We then measured the cytotoxicity of expanded NK and CD8(+) T cells against tumor cell lines and primary tumor cells. When tested ex vivo, IL-2/IL-15Ralpha-IgG1-Fc complexes significantly enhanced NK and CD8(+) T cells expansion, isolated or non-isolated from PBMCs. The effect of IL-15Ralpha-IgG1-Fc was dependent on the presence of IL-2 or IL-15. IL-15Ralpha-IgG1-Fc complexes increased NK, CD8(+) T and NKT cells ratio in PBMC and BMMC after 14 days incubation. High level of granzyme B expression was observed in the supernatant of the complexes-treated NK cells. Expanded NK and CD8(+) T cell populations had cytotoxic function against the PC3, LNCaP, K562 and chronic lymphocytic leukemia (CLL) patient primary B cell lymphoma. We concluded that IL-2/IL-15Ralpha-IgG1-Fc significantly enhanced NK, CD8(+) T and NKT cells expansion, which possess strong anti-tumor activity. These data support clinical testing IL-2/IL-15Ralpha-IgG1-Fc expanded NK cells in patients with prostate cancer and CLL.

IMiDs immunomodulatory drugs, including lenalidomide and pomalidomide represent a novel class of small molecule anticancer and anti-inflammatory drugs with broad biologic activities. However, the molecular mechanism through which these drugs exert their effects is largely undefined. Using pomalidomide and primary human monocytes, we report that pomalidomide rapidly and selectively activated RhoA and Rac1, but not Cdc42 or Ras, in the absence of any costimulation. Consistent with the activation of Rho GTPases, we found that pomalidomide enhanced F-actin formation, stabilized microtubules, and increased cell migration, all of which were blocked by selective inhibitors of ROCK1 and Rac1. Further, we showed that in Swiss 3T3 cells, pomalidomide only activated RhoA, not Rac1 or Cdc42, and potently induced stress fiber formation. The pomalidomide effect on actin cytoskeleton was blocked by the ROCK1 inhibitor, but not Rac1 inhibitor. Finally, we demonstrated that pomalidomide was able to regulate the activity of Rho GTPases and the formation of F-actin in primary human T cells as it did in monocytes and showed that the activation of RhoA was essential for pomalidomide-induced interleukin-2 expression in T cells. These novel activities provide what we believe a critical mechanism by which IMiDs drugs function as therapeutic immunomodulatory agents.

The anti-inflammatory properties of aqueous extracts from Lonicera japonica (LJ) flower, an anti-inflammatory treatment in traditional Chinese medicine, were tested by radioimmunoassay of cyclooxygenase isoenzyme-generated prostaglandin E2 (PGE2) synthesis as well as by Western and Northern blot analysis of COX-2 protein and mRNA expression, respectively. Boiled LJ aqueous extracts directly inhibited both COX-1 and COX-2 activity, while non-boiled extracts stimulated COX-1. Boiled LJ extracts also inhibited expression of IL-1beta-induced COX-2 protein expression and suppressed its mRNA induction by IL-1beta in A549 cells. Suppression of COX-2 mRNA induction required a significantly higher dose of aqueous extract than did suppression of protein expression, indicating that compounds in the extract act translationally or post-translationally at lower doses and transcriptionally or post-transcriptionally at higher doses. Direct inhibition of COX isoenzymes as well as down-regulation of COX-2 mRNA and protein may represent the mechanism by which this ancient herbal treatment decreases inflammation.

Background Treatment for children with high-risk neuroblastoma with anti-disialoganglioside mAb ch14.18, IL-2, and GM-CSF plus 13-cis-retinoic acid after myeloablative chemotherapy improves survival, but 40 % of patients still relapse during or after this therapy. The microenvironment of high-risk neuroblastoma tumors includes macrophages, IL-6, and TGFβ1. We hypothesized that this microenvironment suppresses anti-tumor functions of natural killer (NK) cells and that lenalidomide, an immune-modulating drug, could overcome suppression. Methods Purified NK cells were cultured with IL-2, neuroblastoma/monocyte-conditioned culture medium (CM), IL-6, TGFβ1, and lenalidomide in various combinations and then characterized using cytotoxicity (direct and antibody-dependent cell-mediated cytotoxicity), cytokine, flow cytometry, and Western blotting assays. Anti-tumor activity of NK cells with lenalidomide, ch14.18, or both was evaluated with a xenograft model of neuroblastoma. Results CM from neuroblastoma/monocyte co-cultures contains IL-6 and TGFβ1 that suppress IL-2 activation of NK cell cytotoxicity and IFNγ secretion. IL-6 and TGFβ1 activate the STAT3 and SMAD2/3 pathways in NK cells and suppress IL-2 induction of cytotoxicity, granzymes A and B release, perforin expression, and IFNγ secretion. Lenalidomide blocks IL-6 and TGFβ1 activation of these signaling pathways and inhibits their suppression of NK cells. Neuroblastoma cells in NOD/SCID mice exhibit activated STAT3 and SMAD2/3 pathways. Their growth is most effectively inhibited by co-injected peripheral blood mononuclear cells (PBMC) containing NK cells when mice are treated with both ch14.18 and lenalidomide. Conclusion Immunotherapy with anti-tumor cell antibodies may be improved by lenalidomide, which enhances activation of NK cells and inhibits their suppression by IL-6 and TGFβ1. Electronic supplementary material The online version of this article (doi:10.1007/s00262-013-1466-y) contains supplementary material, which is available to authorized users.