Autonomous Cell Migration to CSF1 Sources - ACS Publications

May 8, 2017 - potentially serve as the basis of engineering a cell to target inflammatory .... with 50 ng/mL of CSF1, showing dim (before) and bright ...
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Research Article pubs.acs.org/synthbio

Autonomous Cell Migration to CSF1 Sources via a Synthetic ProteinBased System Anam Qudrat† and Kevin Truong*,†,‡ †

Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S 3G9, Canada ‡ Edward S. Rogers, Sr. Department of Electrical and Computer Engineering, University of Toronto, 10 King’s College Circle, Toronto, Ontario M5S 3G4, Canada S Supporting Information *

ABSTRACT: Inflammatory lesions, often seen in diseases such as rheumatoid arthritis, atherosclerosis and cancer, feature an acidic (i.e., low pH) microenvironment rampant with cytokines, such as CSF1. For potential therapeutic intervention targeted at these CSF1 sources, we have assembled a system of four proteins inside a cell (i.e., HEK293) that initially had no natural CSF1seeking ability. This system included a newly engineered CSF1 chimera receptor (named CSF1Rchi), the previously engineered Ca2+ activated RhoA (i.e., CaRQ), vesicular stomatitis virus glycoprotein G (VSVG) and thymidine kinase (TK). The binding of CSF1 to the CSF1Rchi generated a Ca2+ signal that activated CaRQ-mediated cellular blebbing, allowing autonomous cell migration toward the CSF1 source. Next, the VSVG protein allowed these engineered cells to fuse with the CSF1 source cells, upon low pH induction. Finally, these cells underwent death postganciclovir treatment, via the TK suicide mechanism. Hence, this protein system could potentially serve as the basis of engineering a cell to target inflammatory lesions in diseases featuring a microenvironment with high levels of CSF1 and low pH. KEYWORDS: CSF1 receptor, CSF1, chimeras, Ca2+ signaling, blebbing, migration, protein engineering, synthetic biology

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cells could be engineered to seek CSF1 sources (e.g., inflammatory lesions) for local therapeutic intervention, it can potentially avoid these risks. Lastly, since low pH is a defining characteristic of the microenvironments of these inflammatory lesions, it can be used as a distinguishing feature to identify these sites.2,10 Innate immune cells (e.g., monocytes, mast cells, macrophages) naturally migrate toward CSF1 sources through chemoattractant signaling, but this process implicates many proteins including chemoattractant receptors (e.g., CXCR1−6, PAFR) as well as adhesion molecules (e.g., selectins and integrins).11 In some cell types, the endogenous expression of CSF1R alone can promote migration to CSF1, but these cell types may already express both known as well as unknown proteins which allow endogenous CSF1R expression to trigger downstream signaling that ultimately leads to the activation of Cdc42, Rac1, and RhoA.12 For cell types such as HEK293 that do not have a natural propensity for migration to CSF1, it is difficult to determine if it has all the necessary associated proteins for CSF1R-mediated migration to CSF1. Previously, we showed that simply expressing the VEGFR2 receptor in HEK293 does not endow migration to VEGF.13 Thus, it is not generally true that the expression of the receptor alone will allow cell migration to the target ligand. Migration to chemical

nflammatory or autoimmune diseases such as rheumatoid arthritis, nephritis, atherosclerosis, cancer, obesity and inflammatory bowel disease, to name a few, feature the formation of inflammatory lesions with low pH microenvironments and local secretion of pro-inflammatory cytokines.1−3 Macrophage-colony stimulating factor (M-CSF or CSF1) serves both as a cytokine that regulates the secretion of proinflammatory chemokines and a growth factor that promotes the proliferation and differentiation of hematopoetic stem cells (i.e., monocytes) to macrophages and osteoclasts involved in innate immunity and bone metabolism, respectively.4 To potentiate its effect, active CSF1 homodimers are released and bind the CSF1 receptor (CSF1R), a ligand-inducible protein tyrosine kinase, found on the plasma membrane. Binding of CSF1 causes CSF1R dimerization5 and autophosphorylation that facilitates the docking of various effector proteins on the cytoplasmic tail, initiating downstream signaling to modulate diverse processes including cytoskeletal rearrangements and cell cycle progression.4 Due to its potent role in autoimmune inflammatory diseases (e.g., rheumatoid arthritis), CSF1 has been targeted by several tyrosine kinase inhibitors6,7 and monoclonal antibodies8 to achieve anti-inflammatory and antiosteolytic effects. However, aside from unwanted side effects from systemic drug delivery (e.g., opportunistic infections, nausea, etc.), the administration of monoclonal antibodies poses the risk of severe immune reactions (e.g., acute anaphylaxis, serum sickness, cytokine release syndrome).9 If © XXXX American Chemical Society

Received: March 7, 2017 Published: May 8, 2017 A

DOI: 10.1021/acssynbio.7b00076 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Research Article

ACS Synthetic Biology

Figure 1. CSF1R chimera (i.e., CSF1Rchi) triggers a Ca2+ signal in response to extracellular CSF1. (A) Cartoon describing the activation of the CSF1Rchi. (B) Representative images showing CSF1 detector cells (i.e., HEK293 cells stably expressing PM-labeled RCaMP1.07 and CSF1Rchi) that are stimulated with 50 ng/mL of CSF1, showing dim (before) and bright (after) outline of cell periphery. Images are false colored: mCherry, red. Scale bars are 20 μm. Representative Ca2+ trace observed with (C) 50 ng/mL CSF1 stimulating CSF1 detector cells or (D) 2× dilution of media stimulating CSF1 detector cells near a cluster of CSF1 source cells. (E) Box plot showing range of signal duration. Bars show s.d.; n.s. not significant. (F) Percent cell response seen in CSF1 detector cells compared with cells transfected only with the VEGFR2tail or the PM-labeled RCaMP1.07 stimulated with [ATP]f = 10 μM, [CSF1]f = 50 ng/mL, [CSF2]f = 10 ng/mL or [STS]f = 50 nM. Error bars show standard deviation. Samples compared with one-factor ANOVA followed by a Tukey−Kramer post hoc test. Star indicates significance: p-value