Osteosarcomagenesis: Biology, Development, Metastasis, and Mechanisms of Pain

Osteosarcoma is the most common primary cancer of the bone and third most common cancer in children and adolescents with approximately 900 new cases annually in the United States. A major facet of osteosarcoma is its high level of genomic instability, in particular chromosomal instability, which is the result of increased or decreased chromosome number in a cell. Furthermore, pain is the most common symptomatic feature of osteosarcoma that lacks effective therapy. Pain in osteosarcoma is relatively more complicated than many other painful conditions requiring a more thorough understanding of its etiology, pathobiology, and neurobiology to allow the development of better therapies for reducing pain in osteosarcoma patients. Studies are underway to define the diverse modalities of presentation, growth, development, metastases, and nociception in osteosarcoma. New data from human studies in combination with data from studies incorporating transgenic mouse models of osteosarcoma are providing valuable insights into the mechanisms underlying the development of both the tumor and the tumor-induced pain. These new data will undoubtedly lead to improved prognoses, as well as the development of novel therapeutics that will significantly decrease bone cancer pain

Relative importance of βcyto- and γcyto-actin in primary mouse embryonic fibroblasts

The highly homologous β (βcyto) and γ (γcyto) cytoplasmic actins are hypothesized to carry out both redundant and unique essential functions, but studies using targeted gene knockout and siRNA-mediated transcript knockdown to examine βcyto- and γcyto-isoform--specific functions in various cell types have yielded conflicting data. Here we quantitatively characterized actin transcript and protein levels, as well as cellular phenotypes, in both gene- and transcript-targeted primary mouse embryonic fibroblasts. We found that the smooth muscle αsm-actin isoform was the dominantly expressed actin isoform in WT primary fibroblasts and was also the most dramatically up-regulated in primary βcyto- or β/γcyto-actin double-knockout fibroblasts. Gene targeting of βcyto-actin, but not γcyto-actin, led to greatly decreased cell proliferation, decreased levels of cellular ATP, and increased serum response factor signaling in primary fibroblasts, whereas immortalization induced by SV40 large T antigen supported fibroblast proliferation in the absence of βcyto-actin. Consistent with in vivo gene-targeting studies in mice, both gene- and transcript-targeting approaches demonstrate that the loss of βcyto-actin protein is more disruptive to primary fibroblast function than is the loss of γcyto-actin

Engineering a hyperactive TcBuster transposase for efficient gene delivery for cell therapy applications

Please click Additional Files below to see the full abstrac

Engineering Genetic Predisposition in Human Neuroepithelial Stem Cells Recapitulates Medulloblastoma Tumorigenesis.

Human neural stem cell cultures provide progenitor cells that are potential cells of origin for brain cancers. However, the extent to which genetic predisposition to tumor formation can be faithfully captured in stem cell lines is uncertain. Here, we evaluated neuroepithelial stem (NES) cells, representative of cerebellar progenitors. We transduced NES cells with MYCN, observing medulloblastoma upon orthotopic implantation in mice. Significantly, transcriptomes and patterns of DNA methylation from xenograft tumors were globally more representative of human medulloblastoma compared to a MYCN-driven genetically engineered mouse model. Orthotopic transplantation of NES cells generated from Gorlin syndrome patients, who are predisposed to medulloblastoma due to germline-mutated PTCH1, also generated medulloblastoma. We engineered candidate cooperating mutations in Gorlin NES cells, with mutation of DDX3X or loss of GSE1 both accelerating tumorigenesis. These findings demonstrate that human NES cells provide a potent experimental resource for dissecting genetic causation in medulloblastoma

Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity.

Chimeric antigen receptors (CARs) significantly enhance the anti-tumor activity of immune effector cells. Although most studies have evaluated CAR expression in T cells, here we evaluate different CAR constructs that improve natural killer (NK) cell-mediated killing. We identified a CAR containing the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain to mediate strong antigen-specific NK cell signaling. NK cells derived from human iPSCs that express this CAR (NK-CAR-iPSC-NK cells) have a typical NK cell phenotype and demonstrate improved anti-tumor activity compared with T-CAR-expressing iPSC-derived NK cells (T-CAR-iPSC-NK cells) and non-CAR-expressing cells. In an ovarian cancer xenograft model, NK-CAR-iPSC-NK cells significantly inhibited tumor growth and prolonged survival compared with PB-NK cells, iPSC-NK cells, or T-CAR-iPSC-NK cells. Additionally, NK-CAR-iPSC-NK cells demonstrate in vivo activity similar to that of T-CAR-expressing T cells, although with less toxicity. These NK-CAR-iPSC-NK cells now provide standardized, targeted "off-the-shelf" lymphocytes for anti-cancer immunotherapy

Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity.

Chimeric antigen receptors (CARs) significantly enhance the anti-tumor activity of immune effector cells. Although most studies have evaluated CAR expression in T cells, here we evaluate different CAR constructs that improve natural killer (NK) cell-mediated killing. We identified a CAR containing the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain to mediate strong antigen-specific NK cell signaling. NK cells derived from human iPSCs that express this CAR (NK-CAR-iPSC-NK cells) have a typical NK cell phenotype and demonstrate improved anti-tumor activity compared with T-CAR-expressing iPSC-derived NK cells (T-CAR-iPSC-NK cells) and non-CAR-expressing cells. In an ovarian cancer xenograft model, NK-CAR-iPSC-NK cells significantly inhibited tumor growth and prolonged survival compared with PB-NK cells, iPSC-NK cells, or T-CAR-iPSC-NK cells. Additionally, NK-CAR-iPSC-NK cells demonstrate in vivo activity similar to that of T-CAR-expressing T cells, although with less toxicity. These NK-CAR-iPSC-NK cells now provide standardized, targeted "off-the-shelf" lymphocytes for anti-cancer immunotherapy

Nonviral genome engineering of natural killer cells

Abstract Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Given their ability to rapidly and effectively recognize and kill aberrant cells, especially transformed cells, NK cells represent a unique cell type to genetically engineer to improve its potential as a cell-based therapy. NK cells do not express a T cell receptor and thus do not contribute to graft-versus-host disease, nor do they induce T cell-driven cytokine storms, making them highly suited as an off-the-shelf cellular therapy. The clinical efficacy of NK cell-based therapies has been hindered by limited in vivo persistence and the immunosuppressive tumor microenvironment characteristic of many cancers. Enhancing NK cell resistance to tumor inhibitory signaling through genome engineering has the potential to improve NK cell persistence in the tumor microenvironment and restore cytotoxic functions. Alongside silencing NK cell inhibitory receptors, NK cell killing can be redirected by the integration of chimeric antigen receptors (CARs). However, NK cells are associated with technical and biological challenges not observed in T cells, typically resulting in low genome editing efficiencies. Viral vectors have achieved the greatest gene transfer efficiencies but carry concerns of random, insertional mutagenesis given the high viral titers necessary. As such, this review focuses on nonviral methods of gene transfer within the context of improving cancer immunotherapy using engineered NK cells

The Molecular Imaging of Natural Killer Cells

The recent success of autologous T cell-based therapies in hematological malignancies has spurred interest in applying similar immunotherapy strategies to the treatment of solid tumors. Identified nearly 4 decades ago, natural killer (NK) cells represent an arguably better cell type for immunotherapy development. Natural killer cells are cytotoxic lymphocytes that mediate the direct killing of transformed cells with reduced or absent major histocompatibility complex (MHC) and are the effector cells in antibody-dependent cell-mediated cytotoxicity. Unlike T cells, they do not require human leukocyte antigen (HLA) matching allowing for the adoptive transfer of allogeneic NK cells in the clinic. The development of NK cell-based therapies for solid tumors is complicated by the presence of an immunosuppressive tumor microenvironment that can potentially disarm NK cells rendering them inactive. The molecular imaging of NK cells in vivo will be crucial for the development of new therapies allowing for the immediate assessment of therapeutic response and off-target effects. A number of groups have investigated methods for detecting NK cells by optical, nuclear, and magnetic resonance imaging. In this review, we will provide an overview of the advances made in imaging NK cells in both preclinical and clinical studies

Bioinformatics-Based Identification of Human B-Cell Receptor (BCR) Stimulation-Associated Genes and Putative Promoters

Genome engineered B-cells are being developed for chronic, systemic in vivo protein replacement therapies and for localized, tumor cell-actuated anticancer therapeutics. For continuous systemic engineered protein production, expression may be driven by constitutively active promoters. For actuated payload delivery, B-cell conditional expression could be based on transgene alternate splicing or heterologous promotors activated after engineered B-cell receptor (BCR) stimulation. This study used a bioinformatics-based approach to identify putative BCR-stimulated gene promoters. Gene expression data at four timepoints (60, 90, 210, and 390 min) following in vitro BCR stimulation using an anti-IgM antibody in B-cells from six healthy donors were analyzed using R (4.2.2). Differentially upregulated genes were stringently defined as those with adjusted p-value 2FoldChange > 1.5. The most upregulated and statistically significant genes were further analyzed to find those with the lowest unstimulated B-cell expression. Of the 46 significantly upregulated genes at 390 min post-BCR stimulation, 6 had average unstimulated expression below the median unstimulated expression at 390 min for all 54,675 gene probes. This bioinformatics-based identification of 6 relatively quiescent genes at baseline that are upregulated by BCR-stimulation (“on-switch”) provides a set of promising promotors for inclusion in future transgene designs and engineered B-cell therapeutics development