Sensitizing Protective Tumor Microenvironments to Antibody-Mediated Therapy

Therapy-resistant microenvironments represent a major barrier toward effective elimination of disseminated malignancies. Here, we show that select microenvironments can underlie resistance to antibody-based therapy. Using a humanized model of treatment refractory B cell leukemia, we find that infiltration of leukemia cells into the bone marrow rewires the tumor microenvironment to inhibit engulfment of antibody-targeted tumor cells. Resistance to macrophage-mediated killing can be overcome by combination regimens involving therapeutic antibodies and chemotherapy. Specifically, the nitrogen mustard cyclophosphamide induces an acute secretory activating phenotype (ASAP), releasing CCL4, IL8, VEGF, and TNFα from treated tumor cells. These factors induce macrophage infiltration and phagocytic activity in the bone marrow. Thus, the acute induction of stress-related cytokines can effectively target cancer cells for removal by the innate immune system. This synergistic chemoimmunotherapeutic regimen represents a potent strategy for using conventional anticancer agents to alter the tumor microenvironment and promote the efficacy of targeted therapeutics.Massachusetts Institute of Technology. Ludwig Center for Molecular OncologyKathy and Curt Marble Cancer Research FundSingapore-MIT Alliance for Research and TechnologyGerman Research Foundation (KFO286)German Research Foundation (Fellowship)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051

Human CD34+ CD133+ Hematopoietic Stem Cells Cultured with Growth Factors Including Angptl5 Efficiently Engraft Adult NOD-SCID Il2rγ−/− (NSG) Mice

Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34+ CD133+ cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg−/− (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34+ CD133+ fraction of expanded cells and that CD34+ CD133+ cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.Singapore-MIT Alliance for Research and Technology ( Infectious Diseases research grant

Phosphodiesterase efficiency: reconciling biochemical and electrophysiological measurements of the phototransduction cascade in the rod cells of the bullfrog Rana catesbeiana

The signal generated when a rod cell captures a photon is amplified via the phototransduction cascade. Specifically, two steps of the cascade are responsible for the observed amplification: the activation of numerous transducin G-proteins by one rhodopsin photoreceptor, and the hydrolysis of numerous cyclic GMP molecules by one molecule of phosphodiesterase (PDE). Direct biochemical measurements of the rate of the first step, combined with the literature efficiency value (k_cat/K_m) of the second step, fail to account for the amplification observed in electrophysiological recordings. In this work, the long-standing contradiction between electrophysiological and biochemical measurements of the amplification of rod phototransduction is resolved. Previous indirect estimates of the rate of PDE activation by the cascade are first confirmed by direct measurements to be ~120/s. The turnover number (k_cat) and the Michaelis constant (K_m) of rod PDE are then measured within disrupted rod outer segments while varying the level of PDE activation. While the turnover number remained rather constant throughout, the apparent Km of PDE declined with decreasing levels of PDE activation, finally stabilizing at ~10 µM when only 1-2% of PDE were activated. This last, stable value, 10 µM, was concluded to be the true Michaelis constant of rod PDE. At least 10-fold lower than published estimates, this true K_m value finally explains on a molecular level the efficiency of amplification seen in electrophysiological recordings

Densely Interconnected Transcriptional Circuits Control Cell States in Human Hematopoiesis

Though many individual transcription factors are known to regulate hematopoietic differentiation, major aspects of the global architecture of hematopoiesis remain unknown. Here, we profiled gene expression in 38 distinct purified populations of human hematopoietic cells and used probabilistic models of gene expression and analysis of cis-elements in gene promoters to decipher the general organization of their regulatory circuitry. We identified modules of highly coexpressed genes, some of which are restricted to a single lineage but most of which are expressed at variable levels across multiple lineages. We found densely interconnected cis-regulatory circuits and a large number of transcription factors that are differentially expressed across hematopoietic states. These findings suggest a more complex regulatory system for hematopoiesis than previously assumed.National Institutes of Health (U.S.). Pioneer AwardBurroughs Wellcome Fund (Career Award at the Scientific Interface)Landon and Lavinia ClayHoward Hughes Medical Institut