Faculty Bibliography

A major impeding factor in designing effective therapies against glioblastoma (GBM) is its extensive molecular heterogeneity and the diversity of microenvironmental conditions within any given tumor. To test whether heterogeneity with the GBM stem cell (GSC) population is required to ensure tumor growth in such diverse microenvironments, we used human GBM biospecimens to examine the identity of cells marked by two established GSC markers: CD133 and activation of the Notch pathway. Using primary GBM cultures engineered to express GFP upon activation of Notch signaling, we observed only partial overlap between cells expressing cell surface CD133 and cells with Notch activation (n = 3 specimens), contrary to expectations based on prior literature. To further investigate this finding, we FACS-isolated these cell populations and characterized them. While both CD133+ (CD133 + /Notch-) and Notch+(CD133-/Notch+) cells fulfill GSC criteria, they differ vastly in their transcriptome, metabolic preferences and differentiation capacity, thus giving rise to histologically distinct tumors. CD133+ GSCs have increased expression of hypoxia-regulated and glycolytic genes, and are able to expand under hypoxia by activating anaerobic glycolysis. In contrast, Notch+ GSCs are unable to utilize anaerobic glycolysis under hypoxia, leading to decreased tumorsphere formation ability. While CD133+ GSCs give rise to histologically homogeneous tumors devoid of large tumor vessels, tumors initiated by Notch+ GSCs are marked by large perfusing vessels enveloped by pericytes. Using a lineage tracing system, we showed that pericytes are derived from Notch+ GSCs. In addition, Notch+ cells are able to give rise to all tumor lineages in vitro and in vivo, including CD133 + /Notch- cells, as opposed to Notch- populations, which have restricted differentiation capacity and do not generate Notch+ lineages. Our findings demonstrate that GSC heterogeneity is a mechanism used by tumors to sustain growth in diverse microenvironmental conditions

Cancer is an important long-term risk for astronauts exposed to protons and high-energy charged particles during travel and residence on asteroids, the moon, and other planets. NASA's Biomedical Critical Path Roadmap defines the carcinogenic risks of radiation exposure as one of four type I risks. A type I risk represents a demonstrated, serious problem with no countermeasure concepts, and may be a potential "show-stopper" for long duration spaceflight. Estimating the carcinogenic risks for humans who will be exposed to heavy ions during deep space exploration has very large uncertainties at present. There are no human data that address risk from extended exposure to complex radiation fields. The overarching goal in this area to improve risk modeling is to provide biological insight and mechanistic analysis of radiation quality effects on carcinogenesis. Understanding mechanisms will provide routes to modeling and predicting risk and designing countermeasures. This white paper reviews broad issues related to experimental models and concepts in space radiation carcinogenesis as well as the current state of the field to place into context recent findings and concepts derived from the NASA Space Radiation Program.

T cells directed to endogenous tumor antigens are powerful mediators of tumor regression. Recent immunotherapy advances have identified effective interventions to unleash tumor-specific T cell activity in patients who naturally develop them. Eliciting T cell responses to a patient's individual tumor remains a major challenge. Radiation therapy can induce immune responses to model antigens expressed by tumors, but it remains unclear if it can effectively prime T cells specific for endogenous antigens expressed by poorly immunogenic tumors. We hypothesized that TGFbeta activity is a major obstacle hindering the ability of radiation to generate an in situ tumor vaccine. Here we show that antibody-mediated TGFbeta neutralization during radiation therapy effectively generates CD8+ T cell responses to multiple endogenous tumor antigens in poorly immunogenic mouse carcinomas. Generated T cells were effective at causing regression of irradiated tumors and non-irradiated lung metastases or synchronous tumors (abscopal effect). Gene signatures associated with IFNgamma and immune-mediated rejection were detected in tumors treated with radiation therapy and TGFbeta blockade in combination but not as single agents. Upregulation of programmed death (PD) ligand-1 and -2 in neoplastic and myeloid cells and PD-1 on intratumoral T cells limited tumor rejection resulting in rapid recurrence. Addition of anti-PD-1 antibodies extended survival achieved with radiation and TGFbeta blockade. Thus, TGFbeta is a fundamental regulator of radiation therapy ability to generate an in situ tumor vaccine. The combination of local radiation therapy with TGFbeta neutralization offers a novel individualized strategy for vaccinating patients against their tumors.

Potentially carcinogenic compounds may cause cancer through direct DNA damage or through indirect cellular or physiological effects. To study possible carcinogens, the fields of endocrinology, genetics, epigenetics, medicine, environmental health, toxicology, pharmacology and oncology must be considered. Disruptive chemicals may also contribute to multiple stages of tumor development through effects on the tumor microenvironment. In turn, the tumor microenvironment consists of a complex interaction among blood vessels that feed the tumor, the extracellular matrix that provides structural and biochemical support, signaling molecules that send messages and soluble factors such as cytokines. The tumor microenvironment also consists of many host cellular effectors including multipotent stromal cells/mesenchymal stem cells, fibroblasts, endothelial cell precursors, antigen-presenting cells, lymphocytes and innate immune cells. Carcinogens can influence the tumor microenvironment through effects on epithelial cells, the most common origin of cancer, as well as on stromal cells, extracellular matrix components and immune cells. Here, we review how environmental exposures can perturb the tumor microenvironment. We suggest a role for disrupting chemicals such as nickel chloride, Bisphenol A, butyltins, methylmercury and paraquat as well as more traditional carcinogens, such as radiation, and pharmaceuticals, such as diabetes medications, in the disruption of the tumor microenvironment. Further studies interrogating the role of chemicals and their mixtures in dose-dependent effects on the tumor microenvironment could have important general mechanistic implications for the etiology and prevention of tumorigenesis.

Abstract Changes in activity or levels of transforming growth factor-beta (TGF-beta) are associated with a variety of diseases; however, measurement of TGF-beta in biological fluids is highly variable. TGF-beta is biologically inert when associated with its latency-associated peptide (LAP). Most available immunoassays require exogenous activation by acid/heat to release TGF-beta from the latent complex. We developed a novel electrochemiluminescence-based multiplexed assay on the MesoScale Discovery(R) platform that eliminates artificial activation, simultaneously measures both active TGF-beta1 and LAP1 and includes an internal control for platelet-derived TGF-beta contamination in blood specimens. We optimized this assay to evaluate plasma levels as a function of activation type and clinical specimen preparation. We determined that breast cancer patients' plasma have higher levels of circulating latent TGF-beta (LTGF-beta) as measured by LAP1 than healthy volunteers (p < 0.0001). This assay provides a robust tool for correlative studies of LTGF-beta levels with disease, treatment outcomes and toxicity with a broad clinical applicability.

The interplay between host genetics, tumor microenvironment and environmental exposure in cancer susceptibility remains poorly understood. Here we assessed the genetic control of stromal mediation of mammary tumor susceptibility to low dose ionizing radiation (LDIR) using backcrossed F1 into BALB/c (F1Bx) between cancer susceptible (BALB/c) and resistant (SPRET/EiJ) mouse strains. Tumor formation was evaluated after transplantation of non-irradiated Trp53-/- BALB/c mammary gland fragments into cleared fat pads of F1Bx hosts. Genome-wide linkage analysis revealed 2 genetic loci that constitute the baseline susceptibility via host microenvironment. However, once challenged with LDIR, we discovered 13 additional loci that were enriched for genes involved in cytokines, including TGFbeta1 signaling. Surprisingly, LDIR-treated F1Bx cohort significantly reduced incidence of mammary tumors from Trp53-/- fragments as well as prolonged tumor latency, compared to sham-treated controls. We demonstrated further that plasma levels of specific cytokines were significantly correlated with tumor latency. Using an ex vivo 3-D assay, we confirmed TGFbeta1 as a strong candidate for reduced mammary invasion in SPRET/EiJ, which could explain resistance of this strain to mammary cancer risk following LDIR. Our results open possible new avenues to understand mechanisms of genes operating via the stroma that affect cancer risk from external environmental exposures.

Postnatal mammary gland development and differentiation occur during puberty and pregnancy. To explore the role of DNA methylation in these processes, we determined the genome-wide DNA methylation and gene expression profiles of CD24+CD61+CD29hi, CD24+CD61+CD29lo, and CD24+CD61-CD29lo cell populations that were previously associated with distinct biological properties at different ages and reproductive stages. We found that pregnancy had the most significant effects on CD24+CD61+CD29hi and CD24+CD61+CD29lo cells, inducing distinct epigenetic states that were maintained through life. Integrated analysis of gene expression, DNA methylation, and histone modification profiles revealed cell-type- and reproductive-stage-specific changes. We identified p27 and TGFbeta signaling as key regulators of CD24+CD61+CD29lo cell proliferation, based on their expression patterns and results from mammary gland explant cultures. Our results suggest that relatively minor changes in DNA methylation occur during luminal differentiation compared with the effects of pregnancy on CD24+CD61+CD29hi and CD24+CD61+CD29lo cells.

Several groups have reported that transforming growth factor-beta1 (TGF-beta1) regulates cellular responses to gamma-irradiation; however, the exact mechanism has not been fully elucidated. In the present study, the role of TGF-beta1 in cellular responses to gamma-irradiation was investigated in detail. The data indicate that TGF-beta1 pretreatment decreased the aftermath of ionizing radiation (IR)-induced DNA damage in a SMAD-dependent manner. To determine the underlying mechanism for these effects, the extent of IR-induced DNA repair activity in the presence or absence of TGF-beta1 was examined. Studies reveal that TGF-beta1 up-regulated DNA ligase IV (LIG4), augmented IR-induced nuclear retention of the DNA ligase, and enhanced non-homologous end-joining (NHEJ) repair activity. In addition, knockdown of LIG4 reduced the TGF-beta1-induced protection against IR. Overall, these data indicate that TGF-beta1 facilitates the NHEJ repair process upon gamma-irradiation and thereby enhances long-term survival. Implications: These findings provide new insight and a possible approach to controlling genotoxic stress by the TGF-beta signaling pathway.