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. Cell lines established in 20% oxygen exhibited lower proliferation, survival, expression of glycolysis genes, glucose consumption, and lactate production. Interestingly, the effects of oxygen concentration used during cell line initiation were only partially reversible when established cell cultures were switched from one oxygen concentration to another for eight weeks. These observations indicate that ALL cell lines established at atmospheric oxygen concentration can exhibit relatively low levels of glycolysis and these levels are semi-permanent, suggesting that physiologic oxygen concentrations may be needed from the time of cell line initiation to preserve the high level of glycolysis commonly exhibited by leukemias in vivo.
•Establishing new ALL cell lines in 5% oxygen resulted in higher glycolytic expression and function.•Establishing new ALL cell lines in 5% oxygen resulted in higher proliferation and lower cell death.•The divergent metabolic phenotypes selected in 5% and 20% oxygen are semi-permanent.
Consolidation using high-dose chemotherapy with autologous stem cell transplantation (ASCT) is an important component of frontline therapy for children with high-risk neuroblastoma. The optimal ...preparative regimen is uncertain, although recent data support a role for busulfan/melphalan (BuMel). The Children's Oncology Group (COG) conducted a trial (ANBL12P1) to assess the tolerability and feasibility of BuMel ASCT following a COG induction. Patients with newly diagnosed high-risk neuroblastoma who did not progress during induction therapy and met organ function requirements received i.v. busulfan (every 24 hours for 4 doses based on age and weight) and melphalan (140 mg/m
for 1 dose), followed by ASCT. Busulfan doses were adjusted to achieve to an average daily area under the curve (AUC) <5500 µM × minute. The primary endpoint was the occurrence of severe sinusoidal obstruction syndrome (SOS) or grade ≥4 pulmonary complications within the first 28 days after completion of consolidation therapy. A total of 146 eligible patients were enrolled, of whom 101 underwent BuMel ASCT. The overall incidence of protocol-defined unacceptable toxicity during consolidation was 6.9% (7 of 101). Six patients (5.9%) developed SOS, with 4 (4%) meeting the criteria for severe SOS. An additional 3 patients (3%) experienced grade ≥4 pulmonary complications during consolidation. The median busulfan AUC was 4558 µM × min (range, 3462 to 5189 µM × minute) for patients with SOS and 3512 µM × min (2360 to 5455 µM × minute) (P = .0142). No patients died during consolidation. From the time of study enrollment, the mean 3-year event-free survival for all 146 eligible patients was 55.6 ± 4.2%, and the mean 3-year overall survival was 74.5 ± 3.7%. The BuMel myeloablative regimen following COG induction was well tolerated, with acceptable pulmonary and hepatic toxicity.
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 TGFbeta1. We hypothesized that this microenvironment suppresses anti-tumor functions of natural killer (NK) cells and that lenalidomide, an immune-modulating drug, could overcome suppression. Purified NK cells were cultured with IL-2, neuroblastoma/monocyte-conditioned culture medium (CM), IL-6, TGFbeta1, 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. CM from neuroblastoma/monocyte co-cultures contains IL-6 and TGFbeta1 that suppress IL-2 activation of NK cell cytotoxicity and IFNγ secretion. IL-6 and TGFbeta1 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 TGFbeta1 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. 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 TGFbeta1.PUBLICATION ABSTRACT
Medulloblastoma, the most common malignant brain tumor in children, occurs with increased frequency in individuals with Fanconi anemia who have biallelic germline mutations in BRCA2. We describe an ...8-year-old child who had disseminated anaplastic medulloblastoma and a deleterious heterozygous BRCA2 6174delT germline mutation. Molecular profiling was consistent with Group 4 medulloblastoma. The posterior fossa mass was resected and the patient received intensive chemotherapy and craniospinal irradiation. Despite this, the patient succumbed to a second recurrence of his medulloblastoma, which presented 8 months after diagnosis as malignant pleural and peritoneal effusions. Continuous medulloblastoma cell lines were isolated from the original tumor (CHLA-01-MED) and the malignant pleural effusion (CHLA-01R-MED). Here, we provide their analyses, including in vitro and in vivo growth, drug sensitivity, comparative genomic hybridization, and next generation sequencing analysis. In addition to the BRCA2 6174delT, the medulloblastoma cells had amplification of MYC, deletion at Xp11.2, and isochromosome 17, but no structural variations or overexpression of GFI1 or GFI1B. To our knowledge, this is the first pair of diagnosis/recurrence medulloblastoma cell lines, the only medulloblastoma cell lines with BRCA2 6174delT described to date, and the first reported case of a child with medulloblastoma associated with a germline BRCA2 6174delT who did not also have Fanconi anemia.
Abstract
Tumor infiltration with CD1d-reactive Vα24-invariant NKT cells associates with favorable outcome in neuroblastoma and other cancers. However, the function of NKTs in tumor immunity remains ...largely unknown. We demonstrate that CD68+ tumor-associated monocytes/macrophages (TAMs) represent the majority of CD1d-expressing cells in primary neuroblastomas. TAMs in vitro and in vivo stimulated neuroblastoma growth via IL-6 production and were the source of IL-6 in primary tumors and metastatic bone marrows in patients. Gene expression analysis with TaqMan® low-density arrays of 129 primary MYCN non-amplified neuroblastomas revealed that high level expression of TAM genes (CD14, CD16, IL-6, IL-6R, and TGFβ) was associated with poor 5-year event-free survival. While NKTs were not cytotoxic against neuroblastoma cells, they effectively killed monocytes pulsed with tumor cell lysate. The killing was CD1d-restricted since it was inhibited by anti-CD1d mAb. Co-transfer of human monocytes and NKTs to tumor-bearing NOD/SCID mice decreased monocyte number at the tumor site and delayed tumor growth compared to mice transferred with monocytes alone. Thus, killing of TAMs reveals a novel mechanism of NKT cell anti-tumor activity that relates to the disease outcome and should be considered for NKT-based therapeutic strategies. Supported by NIH grants: RO1 CA116548 (LSM) and PO1 CA81403 (RCS).
Tumor infiltration with Valpha24-invariant NKT cells (NKTs) associates with favorable outcome in neuroblastoma and other cancers. Although NKTs can be directly cytotoxic against CD1d+ cells, the ...majority of human tumors are CD1d-. Therefore, the role of NKTs in cancer remains largely unknown. Here, we demonstrate that CD68+ tumor-associated monocytes/macrophages (TAMs) represented the majority of CD1d-expressing cells in primary human neuroblastomas. TAMs stimulated neuroblastoma growth in human cell lines and their xenografts in NOD/SCID mice via IL-6 production. Indeed, TAMs produced IL-6 in primary tumors and in the BM of patients with metastatic neuroblastoma. Gene expression analysis using TaqMan low-density arrays of 129 primary human neuroblastomas without MYCN amplification revealed that high-level expression of TAM-specific genes (CD14, CD16, IL6, IL6R, and TGFB1) was associated with poor 5-year event-free survival. While NKTs were not cytotoxic against neuroblastoma cells, they effectively killed monocytes pulsed with tumor cell lysate. The killing of monocytes was CD1d restricted because it was inhibited by a CD1d-specific mAb. Cotransfer of human monocytes and NKTs to tumor-bearing NOD/SCID mice decreased monocyte number at the tumor site and suppressed tumor growth compared with mice transferred with monocytes alone. Thus, killing of TAMs reveals what we believe to be a novel mechanism of NKT antitumor activity that relates to the disease outcome.
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