Tumor infiltration with Vα24-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.

These data suggest that interactions between tumor and inflammatory cells may contribute to the clinical metastatic neuroblastoma phenotype, improve prognostication, and reveal novel therapeutic targets.

Cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU), and has been investigated extensively as a potential tool for selective cellular eradication. In this paper, genetic constructs were designed to express the CD enzyme fused to the transmembrane and extracellular domains of the human nerve growth factor receptor (NGFR), thus allowing for positive identification of transduced cells by flow cytometry and positive selection by magnetic bead technology. Constructs were designed to encode a [Gly(4)Ser](2) flexible linker between the nucleic acid coding sequences for the NGFR and CD genes. Retroviral vectors constructed with wild-type CD and NG/CD fusion genes were used to transduce 3T3 fibroblasts and the human T cell line CEM. The function of CD fusion genes was comparable to that of wild-type genes as determined in cytotoxicity assays. By flow cytometry, the NGFR antigen was detectable after expression of the fusion gene derived from either Escherichia coli (NG/CDe) or Saccharomyces cerevisiae (NG/CDs), but the greatest antigen density was observed in cells transduced with the NG/CDs vector. Similarly, superior 5-FC sensitivity was observed with NG/CDs fusion gene in both murine fibroblasts and human T cells. In addition, CEM cells expressing NG/CDs were more efficiently eliminated in vivo. Engineering of cells utilizing the chimeric NG/CD genes provides a new modality in gene therapy allowing positive and negative selection using a single protein-coding sequence.

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-associated inflammation may play a critical role in tumor progression but its mechanism is yet poorly understood. We have detected elevated levels of IL-6 (2.9 – 88.9 pg/ml) in serum from 10 patients with recurrent stage 4 neuroblastoma. Although none of 5 tested neuroblastoma cell lines produced either IL-6 or IL-10, their conditioned medium selectively induced both cytokines in short-term culture of normal PBMC in a time- and dose-dependent manner as determined by 9-plex Luminex® assay. Treatment with rhIL-6 enhanced neuroblastoma cell proliferation as measured by BrdU incorporation and was sufficient for their survival in a serum-free medium. IL-6 induced rapid STAT3 phosphorylation in neuroblastoma cells and production of yet unidentified soluble factors that inhibited dendritic cell maturation. Gene expression analysis with TaqMan® Low Density Arrays of primary metastatic tumors from 40 patients revealed that elevated expression of IL-6 or IL-10 was associated with a poor 5-year event-free survival: 35% +/−11% vs. 75 +/−10% when IL-6 or IL-10 expression was above and below median levels, respectively (P < 0.02). Thus, although neuroblastoma cells do not express IL-6 and IL-10, they can induce expression of these cytokines by inflammatory cells. These cytokines can then have a paracrine effect on tumor cells and other elements of tumor microenvironment that may promote tumor progression.