Transitioning Cellular and Molecular Observations through Computational Models:

Transitioning Cellular and Molecular Observations through Computational Models:

Transitioning Cellular and Molecular Observations through Computational Models: Impact of Paracrine Signaling on SelfRenewal and Differentiation of Stem Cells ANS/HPS 208 Conference Applicability of Radiation-Response Models to Low-Dose Protection Standards Guidelines September 30 October 3, 2018 Nicholas Dainiak, MD, FACP Department of Therapeutic Radiology Yale University School of Medicine New Haven, CT Ronald Goans, MD, PhD, MPH MJW Corporation Amherst, NY 1 Objectives 1. Review the spatial distribution of hematopoietic stem cell (HSC) niches in the

normal bone marrow, and the role of paracrine signaling in maintaining homeostasis of the hematopoietic system. 2. Understand the importance of microvesicles (MV) in trafficking HSC differentiation signals and bystander signals, including radiation-induced death signals (FasL; TNFSF6). 3. Apply a mathematical model to describe MV diffusion and calculate diffusion length for MV to results obtained in a full-thickness human skin model exposed to -particles, using the Columbia University charged-particle microbeam (Belyakov OV, et al. Proc Natl Acad Sci USA 1982; 102:14203-14208). 350 m Bone Marrow Anatomy 350 m Kiel MJ and Morrison SJ, Nature Rev Immunol 2008; 8: 290

Arrows: arterioles that empty into larger sinusoids Morrison SJ and Scadden DT, Nature 2014; 505: 327 Spatially Distinct Marrow Niches with Different Functions Endosteal Niche T-cells B-cells NK cells Macs Is there one HSC niche that is multifunctional or multiple HSC niches, each with a separate

function? Perivascular Niche Kiel MJ and Morrison SJ, Nature Rev Immunol 2008; 8: 290 HSCs release MV that carry signals, suggesting that bidirectional communication occurs via MV. Projected Distances Needed to Traverse a Niche Venous sinusoid Fewer than 20% of HSCs are within 10

m of the endosteum. In some cases, a signal must travel > 100 m to reach a HSC target. HSCs are CD150+ (red), CD48-, CD41Kiel MJ and Morrison SJ, Nature Rev Immunol 2008; 8: 290 Intercellular Signaling Exocytosis (hormones, cytokines, neural transmitters) Proteolytic cleavage of surface molecules (EGFR) Juxtacrine signaling at plasma membrane (pro-TGF, Boss-sevenless, PAF) Gap junctions (ROS, DDR mediators, ions, cAMP) Exfoliation (growth/death factors, MHC-I,-II, FcR, TfR) Some signals may be transmitted by more than one

mechanism: CSF, SCF, IL-1, Flt-3 Ligand, TNF, Fas and FasL Intercellular Signaling Exocytosis (hormones, cytokines, neural transmitters) Proteolytic cleavage of surface molecules (EGFR) Juxtacrine signaling at plasma membrane (pro-TGF, Boss-sevenless, PAF) Gap junctions (ROS, DDR mediators, ions, cAMP) Exfoliation (growth/death factors, MHC-I,-II, FcR, TfR) Some signals may be transmitted by more than one mechanism: CSF, SCF, IL-1, Flt-3 Ligand, TNF, Fas and FasL Model of Radiation-Induced Bystander Effects (RIBE) Klammer H et al. Cancer Lett 2015; 356: 58 Propagation Distance of Gap Junction

Communication The stress-responsive protein p21Waf1 was used to assess propagation distance within a 100-m radius around the intranuclear particle impact point in confluent human fibroblast cultures. Mean distance ranged from 20 40 m. Gaillard S et al. Radiat Res 2009; 171: 513 Green: p21Waf1 induction Red x: signalemitting cells eliminated from analysis

Proximal Intercellular Associations in Hematopoiesis RC: red cell precursors; CM central macrophage Tavassoli and Shaklai. (1979). Brit J Haematol 43:235 Bar - 0.5 m (500 nm) Porvaznik and MacVittie, J. (1999). Cell Biol 82:555 LCM Pellets Contain PM-Derived SVs Freeze Fracture Electron Micrographs Arrows: pits thought to represent integral membrane proteins Mean size of 0.25 m, range of 0.1-0.4 m Dainiak N. and Cohen CM (1982) Blood 60:583

Vesicles Shed from Lymphocytes Spun to Equilibrium on Dextran Gradients CM pellets were layered on dextran T70 in isotonic saline (A) or 0.3mM Na phosphate (B). Dainiak N and Cohen CM. (1985). Ann NY Acad Sci. 459: 129 12 MV Express Growth Factors Lymphocyte-derived BPA

Tip of Protrusion Shed Vesicle (SV) Base of Protrusion COS-1 cells: M-CSF Dainiak N and Cohen CM, Blood 1982; 60: 583 SV PM Tuck D et al. Blood 1994: 84: 2182 13

IR Induces Fas (TNFRSF6) Expression on MV Surface of SW620 Cells Vesicles A: Western blots: PMs were isolated from control and irradiated SW620 cells, solubilized and analyzed B: RT-PCR

C: Flow Cytometry Albanese J and Dainiak N Radiat Res 2000; 153:49 SV released into serum-free medium were isolated, exposed to 0, 4 or 10 Gy, solubilized, resolved on SDS-PAGE and reacted with antiTNFSF6 ab. Arscott WT et al. Transl Oncol 2013; 6: 638 Comparison of Microvesicles (MV) Characteristic Shed Vesicles Exosomes

PM Multivesicular Bodies (MVBs) Mechanism of Release PM Budding Exocytosis of MVBs Size (nm) 100 - 1, 000 40 - 100 1.02 - 1.03 (lymphocytes)

1.10 - 1.20 Abundant Minimal Tetraspanins (CD63) No Yes Tumor Susceptibility Gene Protein (TSG101) No Yes

GFs, death factors, metalloproteins, integrins, immune molecules, cytoskeletal proteins GF receptors, PM fusion proteins, adaptor proteins, lipid rafts, HSPs, immune molecules, cytoskeletal proteins Site of Origin Density in Sucrose (r) Annexin V (PS) Some Other Proteins

Albanese J and Dainiak N Exp Hematol 2003; 31:455 15 Horizontal Transfer of DNA and RNA via MV Transfer to fibroblasts of DNA (green) to nuclei and RNA (yellow) to cytoplasm by MV isolated from cardiac myocytes Waldenstrom et al. PLoS ONE 2012; 7: e34653 MV Mediate the Bystander Effect MV Characterization Requirement of MV for Bystander Effect

ICCM: Irradiated cell-conditioned medium prepared in DMEM with 10% Serum. ICCM-EXO: medium supernatants after removal of MV by 100,000 x g, 1 hr. MV were visualized by scanning EM in STEM mode (top), and their size distribution was assessed with the Nanosight LM10 system (B, C). MV were positive for the tumor susceptibility gene (TSG-101) protein marker for exosomes. Jella KK et al. (2014) Radiat Res 181:138 17 -Particle Damage to Unirradiated Keratinocytes/Fibroblasts Protocol Skin

Model Apoptotic Cells Columbia University charged-particle microbeam: 7.2-MeV -particles scatter < 1 m range 60 m stopping power 80 keV/m 18 Belyakov OV et al, PNAS (2005) 102:14203 Analysis of MN Data MN and broken neoplasmic bridge SigmaPlot v. 11.0

(Systat Software, Inc., San Jose, CA 95131): curve-fitting software Model to Determine MV Diffusion Length Assumptions: 1. MN number correlates with radiation dose 2. Average MV size is 0.25 m 3. MV diffuse in culture medium with a diffusion length Diffusion equation in a 3-dimensional absorbing medium is described by: Laplacian Operator MV Flux L = Diffusion Length

Calculation of Diffusion Length (L) by General Diffusion Equations Diffusion Length is calculated by: MV Diffusion Coefficient Macroscopic Cross-Section for MV absorption in a cell Columbia microbeam for MV transport is configured as 1-dimensional system where MN formation decreases as a function of . Solution reduces to: MV Flux at =0 where flux is # cells interacting with MV cm2 - sec Determination of L for MV Best exponential fit of MN formation by MV is linear for the fraction of cells with MN relative to the distance from irradiated cells: Bystander effects in an in vivo human skin model. Belyakov et al. PNAS 102, 14203-7 (2005) r = 0.95

r2 =r =0.90 0.95 Fraction of Cells with micronuclei 0.10 r2 = 0.90 Slope = 1 / L, and L = 1270 160 m Unirradiated Bystanders Control 0.01 200

400 600 800 1000 1200 Distance from Irradiated Cells, microns Conclusions: The diffusion length of 1.27 1.6 mm for MV is sufficient to transfer signals throughout HSC niches in the bone marrow and beyond. Modeling IR effects can be used to enhance our understanding of normal biophysical processes. Thank You! Nicholas Dainiak, MD, FACP

Clinical Professor Director, CT Biodosimetry Laboratory at Yale Department of Therapeutic Radiology Yale University School of Medicine PO Box 08040 333 Cedar Street New Haven, CT 06520 [email protected] [email protected] Ronald Goans, MD, PhD, MPH MJW Corporation, Inc 15 Hazelwood Drive Amherst, NY 14228 [email protected] www.mjwcorp.com

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