Biomimetic Hydrogels Incorporating Polymeric Cell-Adhesive Peptide To Promote the 3D Assembly of Tumoroids

Toward the goal of establishing physiologically relevant in vitro tumor models, we synthesized and characterized a biomimetic hydrogel using thiolated hyaluronic acid (HA-SH) and an acrylated copolymer carrying multiple copies of cell adhesive peptide (PolyRGD-AC). PolyRGD-AC was derived from a random copolymer of <i>tert</i>-butyl methacrylate (<i>t</i>BMA) and oligomeric (ethylene glycol) methacrylate (OEGMA), synthesized via atom transfer radical polymerization (ATRP). Acid hydrolysis of <i>tert</i>-butyl moieties revealed the carboxylates, through which acrylate groups were installed. Partial modification of the acrylate groups with a cysteine-containing RGD peptide generated PolyRGD-AC. When PolyRGD-AC was mixed with HA-SH under physiological conditions, a macroscopic hydrogel with an average elastic modulus of 630 Pa was produced. LNCaP prostate cancer cells encapsulated in HA-PolyRGD gels as dispersed single cells formed multicellular tumoroids by day 4 and reached an average diameter of ∼95 μm by day 28. Cells in these structures were viable, formed cell–cell contacts through E-cadherin (E-CAD), and displayed cortical organization of F-actin. Compared with the control gels prepared using PolyRDG, multivalent presentation of the RGD signal in the HA matrix increased cellular metabolism, promoted the development of larger tumoroids, and enhanced the expression of E-CAD and integrins. Overall, hydrogels with multivalently immobilized RGD are a promising 3D culture platform for dissecting principles of tumorigenesis and for screening anticancer drugs

WT1 regulates Snail expression.

<p>(A) Analysis of Snail protein (top) and mRNA (bottom) in the isogenic SN12C and ACHN cell lines. (B) HEK293T cells were transfected with scrambled oligonucleotides or VHL-specific siRNAs and Snail protein was measured. (C) HEK293T cells were transfected with GFP or GFP-WT1 and Snail protein (top) and mRNA (bottom) was measured. Graphs show mean±SD of one representative of three independent experiments. *, <i>P</i><0.05. (D) HEK293T and SN12C-VHL cells were transfected as indicated and protein expression was assessed by immunoblot.</p

WT1 transcriptionally upregulates Snail in VHL-deficient cells.

<p>(A) ChIP assays for WT1 were performed in the isogenic SN12C and ACHN cell lines. WT1 was immunoprecipitated and the bound DNA fragments were analyzed by PCR amplification for Snail. Histone H3 and rabbit IgG were used as positive and negative controls, respectively. Human RPL30 exon3 primers amplified a 160 bp fragment from immunoprecipitation of H3. Snail primers amplified a 120 bp fragment from immunoprecipitation using WT1 antibody. (B) <i>SNAI1</i> promoter luciferase activity was measured in the isogenic SN12C and ACHN cells. (C) SN12C-VHL and ACHN-VHL cells were cotransfected with either scrambled or siRNA-WT1 oligonucleotides, and <i>SNAI1</i> promoter luciferase activity was measured. Graphs depict mean±SD of one representative of three independent experiments. (D) Top, schematic representation of WT1 binding sequence within the Snail promoter with mutated residues highlighted with asterisks (***). Bottom, HEK293T cells were cotransfected with GFP or GFP-WT1 and either wild-type or mutated <i>SNAI1</i> promoter constructs and luciferase activity was measured. Graph depicts mean±SD of three independent experiments. *, <i>P</i><0.05.</p

WT1 upregulates E-cadherin expression in the presences of Snail.

<p>(A) Immunoblot analysis of E-cadherin and N-cadherin expression in the isogenic SN12C and ACHN cell lines. (B) VHL-knockdown RCC cells were transfected with scrambled or WT1-specifc siRNAs and E-cadherin expression was assessed by immunoblot. (C) Top, HEK293T cells were transfected as indicated and protein expression was assessed by immunoblot. Bottom, analysis of E-cadherin mRNA levels. (D, E) HEK293T (D) or MDCK (E) cells were cotransfected with the indicated plasmids and the <i>CDH1</i> promoter reporter construct, and luciferase activity was measured. Graphs represent mean±SD of one representative experiment. *, <i>P</i><0.05.</p

VHL-knockdown alters the expression of EMT markers in RCC cells.

<p>Representative phase-contrast and immunofluorescence images of the isogenic SN12C and ACHN cells. Scale bars = 100 µm in the phase-contrast images. Scale bars = 7.5 µm and 10 µm in the immunofluorescence images of the SN12C and ACHN cells, respectively.</p

CD19-Targeted Nanodelivery of Doxorubicin Enhances Therapeutic Efficacy in B‑Cell Acute Lymphoblastic Leukemia

Nanomedicine has advanced to clinical trials for adult cancer therapy. However, the field is still in its infancy for treatment of childhood malignancies such as acute lymphoblastic leukemia (ALL). Nanotherapy offers multiple advantages over conventional therapy. It facilitates targeted delivery and enables controlled release of drugs to reduce treatment-related side effects. Here, we demonstrate that doxorubicin (DOX) encapsulated in polymeric nanoparticles (NPs) modified with targeting ligands against CD19 (CD19-DOX-NPs) can be delivered in a CD19-specific manner to leukemic cells. The CD19-DOX-NPs were internalized via receptor-mediated endocytosis and imparted cytotoxicity in a CD19-dependent manner in CD19-positive ALL cells. Leukemic mice treated with CD19-DOX-NPs survived significantly longer and manifested a higher degree of agility, indicating reduced apparent systemic toxicity during treatment compared to mice treated with free DOX. We suggest that targeted delivery of drugs used in childhood cancer treatment should improve therapeutic efficacy and reduce treatment-related side effects in children

WT1 preserves epithelial junctions and suppresses motility in renal cells.

<p>(A) MDCK cells were transfected as indicated and protein expression was assessed by immunoblot. (B) Representative immunofluorescene images of MDCK cells transfected with Snail alone or Snail and WT1. (C) Electron microscopy images of MDCK cells transfected with WT1, Snail, or WT1 and Snail. Red boxes indicate cell-cell contact regions showing intercellular junctions junctions enlarged in the inset (arrow). Note the absence of intercellular contacts and spindle morphology of Snail-transfected cells. (D) HEK293T cells were transfected with either GFP or GFP-WT1 and a scratch motility assay was performed. (E) Quantification of the width of the wound at the indicated time points. Graph depicts mean±SD of three independent experiments. *, <i>P</i><0.05.</p

Dexamethasone-Loaded Block Copolymer Nanoparticles Induce Leukemia Cell Death and Enhance Therapeutic Efficacy: A Novel Application in Pediatric Nanomedicine

Nanotechnology approaches have tremendous potential for enhancing treatment efficacy with lower doses of chemotherapeutics. Nanoparticle (NP)-based drug delivery approaches are poorly developed for childhood leukemia. Dexamethasone (Dex) is one of the most common chemotherapeutic drugs used in the treatment of childhood leukemia. In this study, we encapsulated Dex in polymeric NPs and validated their antileukemic potential in vitro and in vivo. NPs with an average diameter of 110 nm were assembled from an amphiphilic block copolymer of poly­(ethylene glycol) (PEG) and poly­(ε-caprolactone) (PCL) bearing pendant cyclic ketals (ECT2). The blank NPs were nontoxic to cultured cells in vitro and to mice in vivo. Encapsulation of Dex into the NPs (Dex-NP) did not compromise the bioactivity of the drug. Dex-NPs induced glucocorticoid phosphorylation and showed cytotoxicity similar to the free Dex in leukemic cells. Studies using NPs labeled with fluorescent dyes revealed leukemic cell surface binding and internalization. In vivo biodistribution studies showed NP accumulation in the liver and spleen with subsequent clearance of the particles with time. In a preclinical model of leukemia, Dex-NPs significantly improved the quality of life and survival of mice as compared to the free drug. To our knowledge, this is the first report showing the efficacy of polymeric NPs to deliver Dex to potentially treat childhood leukemia and reveals that low doses of Dex should be sufficient for inducing cell death and improving survival

Characterization of shRNA-β clones.

<p>(A) Immunoblots showing the levels of NaK-β₁ and NaK-α₁ in Caki-1 cells stably transfected with scrambled shRNA (SCRAM) or shRNA against NaK-β₁ (shRNA-β). Two clones (cl#1 and cl#2) were further studied. Actin was used as a loading control. (B) Rubidium uptake assay showing Na,K-ATPase pump activity was comparable in SCRAM and shRNA-β cells. (C) Cells were treated with TRIAM, DEX, or FLUOR, and NaK-β₁ and NaK-α₁ expression was evaluated by immunoblotting.</p

The anti-tumor effects were reproduced in UMRC6 cells.

<p>(A) The small molecule drugs were effective in up-regulating NaK-β₁ protein levels in the human renal clear cell carcinoma line, UMRC6, as observed in the immunoblot. (B) Additionally, the drugs were efficacious in reducing the motility of UMRC6 cells as demonstrated by the wound healing assay. Drug treated cells migrated a shorter distance. (C) Quantification of the wound healing indicates that the compounds reduced the distance migrated. (D) The attenuation in motility was not due to changes in the rate of cell growth, since DMSO and drug treated cells grew comparably as revealed by a MTT assay.</p