Filomicelles Deliver a Chemo-Differentiation Combination of Paclitaxel and Retinoic Acid That Durably Represses Carcinomas in Liver to Prolong Survival

Drug resistance and relapse is common in cancer treatments with chemotherapeutics, and while drug combinations with naturally occurring, differentiation-inducing retinoic acid (RA) provide remission-free cures for one type of liquid tumor, solid tumors present major problems for delivery. Here, inspired by filoviruses that can be microns in length, flexible filomicelles that self-assemble from an amphiphilic block copolymer (PEG–PCL) are shown to effectively deliver RA and paclitaxel (TAX) to several solid tumor models, particularly in the liver. These hydrophobic compounds synergistically load into the cores of the elongated micelles, and the coloaded micelles prove most effective at causing cell death, ploidy, and durable regression of tumors compared to free drugs or to separately loaded drugs. RA-TAX filomicelles also reduce mortality of human lung or liver derived cancers engrafted at liver, intraperitoneal, and subcutaneous sites in immunodeficient mice. In vitro studies show that the dual drug micelles effectively suppress proliferation while upregulating a generic differentiation marker. The results highlight the potency of dual-loaded filomicelles in killing cancer cells or else driving their differentiation away from growth

Progerin phosphorylation in interphase is lower and less mechanosensitive than lamin-A,C in iPS-derived mesenchymal stem cells

<p>Interphase phosphorylation of lamin-A,C depends dynamically on a cell's microenvironment, including the stiffness of extracellular matrix. However, phosphorylation dynamics is poorly understood for diseased forms such as progerin, a permanently farnesylated mutant of <i>LMNA</i> that accelerates aging of stiff and mechanically stressed tissues. Here, fine-excision alignment mass spectrometry (FEA-MS) is developed to quantify progerin and its phosphorylation levels in patient iPS cells differentiated to mesenchymal stem cells (MSCs). The stoichiometry of total A-type lamins (including progerin) versus B-type lamins measured for Progeria iPS-MSCs prove similar to that of normal MSCs, with total A-type lamins more abundant than B-type lamins. However, progerin behaves more like farnesylated B-type lamins in mechanically-induced segregation from nuclear blebs. Phosphorylation of progerin at multiple sites in iPS-MSCs cultured on rigid plastic is also lower than that of normal lamin-A and C. Reduction of nuclear tension upon i) cell rounding/detachment from plastic, ii) culture on soft gels, and iii) inhibition of actomyosin stress increases phosphorylation and degradation of lamin-C > lamin-A > progerin. Such mechano-sensitivity diminishes, however, with passage as progerin and DNA damage accumulate. Lastly, transcription-regulating retinoids exert equal effects on both diseased and normal A-type lamins, suggesting a differential mechano-responsiveness might best explain the stiff tissue defects in Progeria.</p

TCR Triggering by pMHC Ligands Tethered on Surfaces via Poly(Ethylene Glycol) Depends on Polymer Length

<div><p>Antigen recognition by T cells relies on the interaction between T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) at the interface between the T cell and the antigen presenting cell (APC). The pMHC-TCR interaction is two-dimensional (2D), in that both the ligand and receptor are membrane-anchored and their movement is limited to 2D diffusion. The 2D nature of the interaction is critical for the ability of pMHC ligands to trigger TCR. The exact properties of the 2D pMHC-TCR interaction that enable TCR triggering, however, are not fully understood. Here, we altered the 2D pMHC-TCR interaction by tethering pMHC ligands to a rigid plastic surface with flexible poly(ethylene glycol) (PEG) polymers of different lengths, thereby gradually increasing the ligands’ range of motion in the third dimension. We found that pMHC ligands tethered by PEG linkers with long contour length were capable of activating T cells. Shorter PEG linkers, however, triggered TCR more efficiently. Molecular dynamics simulation suggested that shorter PEGs exhibit faster TCR binding on-rates and off-rates. Our findings indicate that TCR signaling can be triggered by surface-tethered pMHC ligands within a defined 3D range of motion, and that fast binding rates lead to higher TCR triggering efficiency. These observations are consistent with a model of TCR triggering that incorporates the dynamic interaction between T cell and antigen-presenting cell.</p></div

T cell activation by IEkMCC tethered with PEG polymers of different lengths.

<p>(A) T cell IL2 production in response to IEkMCC-PEG ligands of varying coating densities after 6 hours of stimulation. Data are representative of three independent experiments. The percent of T cells producing IL2 was determined by intracellular staining and flow cytometry. Three experiments using T cells from three different mice were performed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112292#pone.0112292.s008" target="_blank">Fig. S8</a> for flow cytometry plots). The percent of T cells producing IL2 was normalized to the highest value in each experiment. The data points are averages of the normalized values with standard errors of the means. (B) The rate of T cell response to IEkMCC ligands tethered with PEG polymers of different lengths. T cell IL2 production in response to stimulation on 96 well plates coated with 110 pM IEkMC-PEG ligands. T cells were harvested every hour for 6 hours and levels of IL2 expression were assayed by flow cytometry. Three experiments using T cells from three different mice were performed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112292#pone.0112292.s009" target="_blank">Fig. S9</a> for flow cytometry plots). The percent of T cells producing IL2 was normalized to the highest value in each experiment. The data points are averages of the normalized values with standard errors of the means. (C) The rates of T cell IL2 responses to IEkMCC ligands tethered with PEG polymers were extracted from the slope of linear fitting curves in Fig. 4B and plotted against the Flory radius of the polymers. The linear regressions and equations for deriving the rates are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112292#pone.0112292.s007" target="_blank">Fig. S7</a>.</p

Characterization and separation of PEG polymer linkers and IEkMCC-PEG conjugates.

<p>(A) Compiled elution curves of nine PEG polymer linkers from a Superdex 200 10/300 GL gel filtration column. The polymers were detected through the weak UV absorption of the biotin group using a 245 nm UV detector. (B) Separation of the IEkMCC and PEG polymer reaction products. The reaction products were loaded on a Superdex 200 10/300 GL gel filtration column to separate IEkMCC-PEG conjugates, unreacted IEkMCC, and unreacted PEG polymers. The reaction products of PEG 15000, PEG 30000 and PEG 60000 were first purified with an IEk-binding affinity column to eliminate unreacted PEG polymers. The dotted vertical line indicates the elution volume of IEkMCC protein. The late elution peaks of unreacted polymers can be seen for PEGs ranging from PEG 88 to PEG 5000. In reaction with PEG 7500, unreacted IEkMCC and unreacted polymer formed a single peak that was eluted at a position between unconjugated IEkMCC and pure PEG 7500.</p

FRET between streptavidin on plastic plates and IEkMCC tethered with PEG polymers.

<p>(A) Measured FRET efficiencies of IEkMCC tethered with six different PEG polymers. The intensity of DyLight 549 was captured before and after DyLight 649 was photobleached. The measured FRET efficiency () was calculated using the intensity of DyLight 549 before () and after () DyLight 649 photobleaching ( ). The averaged values of two measurements were plotted with standard deviations. (B) After normalization, the measured FRET efficiencies match those calculated based on the Flory radius () of the PEG polymers. The of the PEG polymer of subunits and unit length was calculated using , where is 0.28 nm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112292#pone.0112292-Oesterhelt1" target="_blank">[32]</a>. Theoretical FRET efficiency () was calculated using the equation , where the Förster distance () of the DyLight 549-DyLight 649 donor-acceptor pair is 5 nm and the distance between the pMHC ligand and streptavidin is of the PEG polymer plus the pMHC radius of 2 nm. The FRET efficiencies were normalized by dividing the FRET efficiencies by the FRET efficiency of PEG 88.</p

PEG linkers and properties.

1<p>PEG contour length is calculated based on the PEO unit length of 0.28 nm in water <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112292#pone.0112292-Oesterhelt1" target="_blank">[32]</a>.</p>2<p>The Flory radius () of the PEG polymer of subunits and unit length was calculated using , where is 0.28 nm.</p><p>PEG linkers and properties.</p

RhoA is essential for two stages of platelet production.

<p>RhoA coordinates cytokinesis of promegakaryocytes and endomitosis of megakaryocytes by regulating effectors that control the actin contractile ring. The contractile ring underlies and constricts the cleavage furrow, which facilitates cell division. Another potential site of regulation is the ROCK-myosin pathway during thrombopoiesis. Actomyosin forces limit proplatelet formation, which ultimately controls platelet size.</p

Platelet production in RhoA-null megakaryocytes is impaired.

<p>(A) Mice were injected with GPIbα antibodies, and platelet levels were counted manually from blood smears. While the control mice began to recover from the depletion after two days, RhoA^fl/fl PF4CRE⁺ mice did not begin to recover until after four days. Mean ±SE is shown; * indicates P<0.05; ** indicates P<0.005. N = 5. (B) The platelet survival of infusing either RhoA-null megakaryocytes or controls into the mαIIb^−/−/hαIIb^+/+ mice was measured. Platelet production of the infused RhoA-null megakaryocytes diminished over a 24-hour period of time as compared to the controls (* indicates P<0.005). The mean ±SE is shown. N = 5. The red line shows the “best fit” trajectory of platelet depletion during the first eight hours, while the blue line approximates the platelet clearance rate from eight hours onward.</p

Mice with targeted deletion of RhoA in megakaryocytes and in platelets exhibit macrothrombocytopenia and impaired MLC phosphorylation.

<p>(A) The RhoA transgene construct contains loxP sites flanking exon three. These mice were crossed with mice expressing PF4 promoter-driven CRE recombinase. (B) Western blotting confirmed that platelets from RhoA^fl/fl PF4CRE⁺ positive mice did not express detectable RhoA protein, but had normal amounts of Rac1 and CDC42. (C) Cell blood counts were normal, except that the RhoA^fl/fl PF4CRE⁺ mice were macrothrombocytopenic. * indicates p<0.005, ** indicates p<0.05. The immunoblot of thrombin-treated platelets were probed with antibodies against the phospho-MLC2 Thr¹⁸ (D) or Ser¹⁹ (E). Phosphorylation of the MLC2 Ser¹⁹ residue was normal (D), but phosphorylation of the MLC2 Thr¹⁸ residue was undetectable in RhoA-null platelets for all stimulation times (E).</p