Not all tumor vessels are equal. Tumor-associated vasculature includes immature vessels, regressing vessels, transport vessels undergoing arteriogenesis and peritumor vessels influenced by tumor growth factors. Current techniques for analyzing tumor blood flow do not discriminate between vessel subtypes and only measure average changes from a population of dissimilar vessels. We have developed methodologies for simultaneously quantifying blood flow (velocity, flux, hematocrit and shear rate) in extended networks at single capillary resolution in vivo. Our approach relies on deconvolution of signals produced by labeled red blood cells as they move relative to the scanning laser of a confocal or multiphoton microscope and provides fully-resolved three-dimensional flow profiles within vessel networks. Using this methodology, we show that blood velocity profiles are asymmetric near intussusceptive tissue structures in tumors in mice. Furthermore, we show that subpopulations of vessels, classified by functional parameters, exist in, around a tumor and in normal brain.
Key Points
AT1413 is a monoclonal antibody isolated from a cured patient with AML that recognizes CD43s, a novel epitope expressed by AML and MDS blasts. AT1413 eliminates CD43s-expressing leukemic blasts in vitro and in vivo and may have potential as a therapeutic antibody.
Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay.
Most patients with acute myeloid leukemia (AML) can only be cured when allogeneic hematopoietic stem-cell transplantation induces a graft-versus-leukemia immune response (GVL). Although the role of T cells and natural killer cells in tumor immunology has been established, less is known about the contribution of B cells. From B cells of high-risk patients with AML with potent and lasting GVL responses, we isolated monoclonal antibodies directed against antigens expressed on the cell surface of AML cells but not on normal hematopoietic and nonhematopoietic cells. A number of these donor-derived antibodies recognized the U5 snRNP200 complex, a component of the spliceosome that in normal cells is found in the cell. In AML however, the U5 snRNP200 complex is exposed on the cell membrane of leukemic blasts. U5 snRNP200 complex-specific antibodies induced death of AML cells in an Fc receptor-dependent way in the absence of cytotoxic leukocytes or complement. In an AML mouse model, treatment with U5 snRNP200 complex-specific antibodies led to significant tumor growth inhibition. Thus, donor-derived U5 snRNP200 complex-recognizing AML-specific antibodies may contribute to antitumor responses.
Acute myeloid leukemia (AML) is a high-risk disease with a poor prognosis, particularly in elderly patients. Because current AML treatment relies primarily on untargeted therapies with severe side effects that limit patient eligibility, identification of novel therapeutic AML targets is highly desired. We recently described AT1413, an antibody produced by donor B cells of a patient with AML cured after allogeneic hematopoietic stem cell transplantation. AT1413 binds CD43s, a unique sialylated epitope on CD43, which is weakly expressed on normal myeloid cells and overexpressed on AML cells. Because of its selectivity for AML cells, we considered CD43s as a target for a bispecific T-cell-engaging antibody (bTCE) and generated a bTCE by coupling AT1413 to two T-cell-targeting fragments using chemo-enzymatic linkage. In vitro, AT1413 bTCE efficiently induced T-cell-mediated cytotoxicity toward different AML cell lines and patient-derived AML blasts, whereas endothelial cells with low binding capacity for AT1413 remained unaffected. In the presence of AML cells, AT1413 bTCE induced upregulation of T-cell activation markers, cytokine release, and T-cell proliferation. AT1413 bTCE was also effective in vivo. Mice either coinjected with human peripheral blood mononuclear cells or engrafted with human hematopoietic stem cells [human immune system (HIS) mice] were inoculated with an AML cell line or patient-derived primary AML blasts. AT1413 bTCE treatment strongly inhibited tumor growth and, in HIS mice, had minimal effects on normal human hematopoietic cells. Taken together, our results indicate that CD43s is a promising target for T-cell-engaging antibodies and that AT1413 holds therapeutic potential in a bTCE-format. Significance: These findings offer preclinical evidence for the therapeutic potential of a bTCE antibody that targets a sialylated epitope on CD43 in AML.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.