Anti-CCL2 antibody combined with etoposide prolongs survival in a minimal residual disease mouse model of neuroblastoma.

C-C motif chemokine ligand 2 (CCL2) is a monocyte chemoattractant that promotes metastatic disease and portends a poor prognosis in many cancers. To determine the potential of anti-CCL2 inhibition as a therapy for recurrent metastatic disease in neuroblastoma, a mouse model of minimal residual disease was utilized in which residual disease was treated with anti-CCL2 monoclonal antibody with etoposide. The effect of anti-CCL2 antibody on neuroblastoma cells was determined in vitro with cell proliferation, transwell migration, and 2-dimensional chemotaxis migration assays.

Anti-disialoganglioside antibody internalization by neuroblastoma cells as a mechanism of immunotherapy resistance.

Neuroblastoma (NBL) accounts for a disproportionate number of deaths among childhood malignancies despite intensive multimodal therapy that includes antibody targeting disialoganglioside GD2, a NBL antigen. Unfortunately, resistance to anti-GD2 immunotherapy is frequent and we aimed to investigate mechanisms of resistance in NBL. GD2 expression was quantified by flow cytometry and anti-GD2 antibody internalization was measured using real-time microscopy in 20 human NBL cell lines.

Combined immune checkpoint blockade increases CD8+CD28+PD-1+ effector T cells and provides a therapeutic strategy for patients with neuroblastoma.

Immune checkpoint therapy has resulted in minimal clinical response in many pediatric cancers. We sought to understand the influence of immune checkpoint inhibition using anti-PD-1 and anti-CTLA-4 antibodies individually, in combination, and after chemotherapy on immune responses in minimal and established murine neuroblastoma models. We also sought to understand the role of the tumor microenvironment (TME) and PD-L1 expression and their alteration post-chemotherapy in our models and human tissues.

Initiation of immunotherapy with activated natural killer cells and anti-GD2 antibody dinutuximab prior to resection of primary neuroblastoma prolongs survival in mice.

Background: Immunotherapy with anti-disialoganglioside dinutuximab has improved survival for children with high-risk neuroblastoma (NB) when given after induction chemotherapy and surgery. However, disease recurrence and resistance persist. Dinutuximab efficacy has not been evaluated when initiated before primary tumor removal.

Circulating T cell subsets are associated with clinical outcome of anti-VEGF-based 1st-line treatment of metastatic colorectal cancer patients: a prospective study with focus on primary tumor sidedness.

Background: In a prospective study with long-term follow-up, we analyzed circulating T cell subsets in patients with metastatic colorectal cancer (mCRC) in the context of primary tumor sidedness, KRAS status, and clinical outcome. Our primary goal was to investigate whether baseline levels of circulating T cell subsets serve as a potential biomarker of clinical outcome of mCRC patients treated with an anti-VEGF-based regimen.

Methods: The study group consisted of 36 patients with colorectal adenocarcinoma who started first-line chemotherapy with bevacizumab for metastatic disease.

Anti-CD105 Antibody Eliminates Tumor Microenvironment Cells and Enhances Anti-GD2 Antibody Immunotherapy of Neuroblastoma with Activated Natural Killer Cells.

Purpose: We determined whether elimination of CD105 cells in the tumor microenvironment (TME) with anti-CD105 antibodies enhanced anti-disialoganglioside (GD2) antibody dinutuximab therapy of neuroblastoma when combined with activated natural killer (aNK) cells.

Experimental Design: The effect of MSCs and monocytes on antibody-dependent cellular cytotoxicity (ADCC) mediated by dinutuximab with aNK cells against neuroblastoma cells was determined . ADCC with anti-CD105 mAb TRC105 and aNK cells against MSCs, monocytes, and endothelial cells, which express CD105, was evaluated.

Activated Natural Killer Cells in Combination with Anti-GD2 Antibody Dinutuximab Improve Survival of Mice after Surgical Resection of Primary Neuroblastoma.

Purpose: Immunotherapy of neuroblastoma that remains after myeloablative chemotherapy with anti-GD2 antibody dinutuximab has increased the two-year event-free and overall survival of high-risk neuroblastoma patients; however, 40% of patients develop recurrent disease during or after this treatment. To determine the potential of such antibody-based immunotherapy earlier in treatment, a mouse model was developed in which surgical resection of the primary tumor was followed by therapy of residual disease with dinutuximab combined with -activated human natural killer (aNK) cells.

Experimental Design: The effect of combining dinutuximab with human aNK cells was determined with cellular cytotoxicity and Matrigel invasion assays.

Colony stimulating factor 1 receptor blockade improves the efficacy of chemotherapy against human neuroblastoma in the absence of T lymphocytes.

Tumor-associated macrophages can promote growth of cancers. In neuroblastoma, tumor-associated macrophages have greater frequency in metastatic versus loco-regional tumors, and higher expression of genes associated with macrophages helps to predict poor prognosis in the 60% of high-risk patients who have MYCN-non-amplified disease. The contribution of cytotoxic T-lymphocytes to anti-neuroblastoma immune responses may be limited by low MHC class I expression and low exonic mutation frequency.

Phase I trial of dasatinib, lenalidomide, and temozolomide in children with relapsed or refractory central nervous system tumors.

Single agent studies targeting the tumor microenvironment in central nervous system (CNS) tumors have largely been disappointing. Combination therapies targeting various pathways and cell types may be a more effective strategy. In this phase I study, we evaluated the combination of dasatinib, lenalidomide, and temozolomide in children with relapsed or refractory primary CNS tumors.

Tumor-associated macrophages promote neuroblastoma via STAT3 phosphorylation and up-regulation of c-MYC.

Tumor-associated macrophages (TAMs) are strongly associated with poor survival in neuroblastomas that lack amplification. To study TAM action in neuroblastomas, we used a novel murine model of spontaneous neuroblastoma lacking amplification, and observed recruitment and polarization of TAMs, which in turn enhanced neuroblastoma proliferation and growth. In both murine and human neuroblastoma cells, we found that TAMs increased STAT3 activation in neuroblastoma cells and transcriptionally up-regulated the oncogene.

Cancer-Associated Fibroblasts Share Characteristics and Protumorigenic Activity with Mesenchymal Stromal Cells.

Cancer-associated fibroblasts (CAF) have been suggested to originate from mesenchymal stromal cells (MSC), but their relationship with MSCs is not clear. Here, we have isolated from primary human neuroblastoma tumors a population of αFAP- and FSP-1-expressing CAFs that share phenotypic and functional characteristics with bone marrow-derived MSCs (BM-MSC). Analysis of human neuroblastoma tumors also confirmed the presence of αFAP- and FSP-1-positive cells in the tumor stroma, and their presence correlated with that of M2 tumor-associated macrophages.

TGFβR1 Blockade with Galunisertib (LY2157299) Enhances Anti-Neuroblastoma Activity of the Anti-GD2 Antibody Dinutuximab (ch14.18) with Natural Killer Cells.

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.

Preservation of high glycolytic phenotype by establishing new acute lymphoblastic leukemia cell lines at physiologic oxygen concentration.

Cancer cells typically exhibit increased glycolysis and decreased mitochondrial oxidative phosphorylation, and they continue to exhibit some elevation in glycolysis even under aerobic conditions. However, it is unclear whether cancer cell lines employ a high level of glycolysis comparable to that of the original cancers from which they were derived, even if their culture conditions are changed to physiologically relevant oxygen concentrations. From three childhood acute lymphoblastic leukemia (ALL) patients we established three new pairs of cell lines in both atmospheric (20%) and physiologic (bone marrow level, 5%) oxygen concentrations.

Lenalidomide overcomes suppression of human natural killer cell anti-tumor functions by neuroblastoma microenvironment-associated IL-6 and TGFβ1.

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.

Membrane-bound TRAIL supplements natural killer cell cytotoxicity against neuroblastoma cells.

Neuroblastoma cells have been reported to be resistant to death induced by soluble, recombinant forms of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (CD253/TNFSF10) because of low or absent expression of caspase-8 and/or TRAIL-receptor 2 (TRAIL-R2/DR5/CD262/TNFRSF10b). However, their sensitivity to membrane-bound TRAIL on natural killer (NK) cells is not known. Comparing microarray gene expression and response to NK cell-mediated cytotoxicity, we observed a correlation between TRAIL-R2 expression and the sensitivity of 14 neuroblastoma cell lines to the cytotoxicity of NK cells activated with interleukin (IL)-2 plus IL-15.

Critical role of STAT3 in IL-6-mediated drug resistance in human neuroblastoma.

Drug resistance is a major cause of treatment failure in cancer. Here, we have evaluated the role of STAT3 in environment-mediated drug resistance (EMDR) in human neuroblastoma. We determined that STAT3 was not constitutively active in most neuroblastoma cell lines but was rapidly activated upon treatment with interleukin (IL)-6 alone and in combination with the soluble IL-6 receptor (sIL-6R).

Growth and activation of natural killer cells ex vivo from children with neuroblastoma for adoptive cell therapy.

Purpose: Adoptive transfer of natural killer (NK) cells combined with tumor-specific monoclonal antibodies (mAb) has therapeutic potential for malignancies. We determined if large numbers of activated NK (aNK) cells can be grown ex vivo from peripheral blood mononuclear cells (PBMC) of children with high-risk neuroblastoma using artificial antigen-presenting cells (aAPC).

Experimental Design: Irradiated K562-derived Clone 9.

Meningitic Escherichia coli K1 penetration and neutrophil transmigration across the blood-brain barrier are modulated by alpha7 nicotinic receptor.

Alpha7 nicotinic acetylcholine receptor (nAChR), an essential regulator of inflammation, is abundantly expressed in hippocampal neurons, which are vulnerable to bacterial meningitis. However, it is unknown whether α7 nAChR contributes to the regulation of these events. In this report, an aggravating role of α7 nAChR in host defense against meningitic E.

Combination of vorinostat and flavopiridol is selectively cytotoxic to multidrug-resistant neuroblastoma cell lines with mutant TP53.

As p53 loss of function (LOF) confers high-level drug resistance in neuroblastoma, p53-independent therapies might have superior activity in recurrent neuroblastoma. We tested the activity of vorinostat, a histone deacetylase inhibitor, and flavopiridol, a pan-Cdk inhibitor, in a panel of multidrug-resistant neuroblastoma cell lines that included lines with wild-type (wt) and transcriptionally active TP53 (n = 3), mutated (mt), and LOF TP53 (n = 4) or p14(ARF) deletion (n = 1). The combination of vorinostat and flavopiridol was synergistic and significantly more cytotoxic (P < 0.

Activity of vincristine, L-ASP, and dexamethasone against acute lymphoblastic leukemia is enhanced by the BH3-mimetic ABT-737 in vitro and in vivo.

Defects in apoptosis signaling contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL), and overexpression of antiapoptotic Bcl-2 (Bcl-2 and Bcl-X(L)) family proteins has been observed in ALL. ABT-737 is a small-molecule BH3-mimetic that inhibits the antiapoptotic Bcl-2 family proteins. We evaluated the cytotoxicity of ABT-737 in combination with vincristine, dexamethasone, and L-asparaginase (VXL) in 7 ALL cell lines.

HER2 signaling downregulation by trastuzumab and suppression of the PI3K/Akt pathway: an unexpected effect on TRAIL-induced apoptosis.

We investigated whether HER2 downregulation by trastuzumab modulates the responsiveness of breast cancer cells to TNF-related apoptosis-inducing ligand (TRAIL). Interestingly, in contrast to increased response to TRAIL in SKBr3 cells, trastuzumab decreased the susceptibility of BT474 cells to TRAIL. This decrease was also observed after exogenous inhibition of PI3-K/Akt kinase, but not MAPK/ERK kinase (MEK)/mitogen-activated protein kinase (MAPK).

Dimethyl sulfoxide potentiates death receptor-mediated apoptosis in the human myeloid leukemia U937 cell line through enhancement of mitochondrial membrane depolarization.

Dimethyl sulfoxide (DMSO) is a widely used prototypical chemical inducer of cell differentiation. In the present study, the effects of DMSO on susceptibility of human myeloid leukemia U937 cells towards ligation of distinct death receptors (DRs) were investigated. DMSO sensitized cells towards induction of apoptosis by anti-Fas antibody, tumour necrosis factor-alpha or Apo2 ligand/TNF-related apoptosis-inducing ligand (TRAIL).

Developmental status of CD4-CD8+ and CD4+CD8- thymocytes with medium expression of CD3.

In normal mice, more than 10% of thymocytes in the CD4+CD8- and CD4-CD8+ single-positive (SP) subsets express a medium level of CD3 on the cell surface. However, the fate of CD3medium cells is unclear. The CD3medium SP subpopulations might contain (i) cells in an immature stage of the pathways leading to CD3high cells, (ii) cells in developmental pathways that do not lead to CD3high cells, or (iii) cells that have been negatively selected.

Role of p53 in regulating constitutive and X-radiation-inducible CD95 expression and function in carcinoma cells.

The p53 tumor suppressor protein is known to regulate the expression of the CD95 (Fas/APO-1) death receptor in a small subset of normal cell types as well as in many cancer cell types. However, whether p53-dependent regulation of CD95 expression is consistently associated with increased susceptibility to CD95-mediated cell death is poorly understood. To address this issue, we examined constitutive and induced CD95 surface expression and function in wild-type p53-expressing carcinoma cells relative to their isogenic p53-inactivated counterparts.

Release of cytokeratin-18 and -19 fragments (TPS and CYFRA 21-1) into the extracellular space during apoptosis.

Serum fragments of cytokeratins-18 and -19 (measured as TPS and CYFRA 21-1, respectively) have traditionally been considered as markers of tumor proliferation, although the evidence is scarce for a causative relationship between proliferation and levels of TPS and CYFRA 21-1. We examined whether apoptosis might produce TPS and CYFRA 21-1 fragments. MCF-7 breast cancer cells were treated with mitomycin C or agonistic anti-CD95 antibody, and levels of TPS and CYFRA 21-1 in tissue culture supernatants were compared with the frequency of cells exhibiting the following markers of cell death: intracellular cytokeratin-18 cleavage, surface staining with annexin-V, propidium iodide uptake, DNA fragmentation.