Lipid Cross-Linking of Nanolipoprotein Particles Substantially Enhances Serum Stability and Cellular Uptake

Nanolipoprotein particles (NLPs) consist of a discoidal phospholipid lipid bilayer confined by an apolipoprotein belt. NLPs are a promising platform for a variety of biomedical applications due to their biocompatibility, size, definable composition, and amphipathic characteristics. However, poor serum stability hampers the use of NLPs for in vivo applications such as drug formulation. In this study, NLP stability was enhanced upon the incorporation and subsequent UV-mediated intermolecular cross-linking of photoactive DiynePC phospholipids in the lipid bilayer, forming cross-linked nanoparticles (X-NLPs). Both the concentration of DiynePC in the bilayer and UV exposure time significantly affected the resulting X-NLP stability in 100% serum, as assessed by size exclusion chromatography (SEC) of fluorescently labeled particles. Cross-linking did not significantly impact the size of X-NLPs as determined by dynamic light scattering and SEC. X-NLPs had essentially no degradation over 48 h in 100% serum, which is a drastic improvement compared to non-cross-linked NLPs (50% degradation by ∼10 min). X-NLPs had greater uptake into the human ATCC 5637 bladder cancer cell line compared to non-cross-linked particles, indicating their potential utility for targeted drug delivery. X-NLPs also exhibited enhanced stability following intravenous administration in mice. These results collectively support the potential utility of X-NLPs for a variety of in vivo applications

Diagnostic Microdosing Approach to Study Gemcitabine Resistance

Gemcitabine metabolites cause the termination of DNA replication and induction of apoptosis. We determined whether subtherapeutic “microdoses” of gemcitabine are incorporated into DNA at levels that correlate to drug cytotoxicity. A pair of nearly isogenic bladder cancer cell lines differing in resistance to several chemotherapy drugs were treated with various concentrations of ¹⁴C-labeled gemcitabine for 4–24 h. Drug incorporation into DNA was determined by accelerator mass spectrometry. A mechanistic analysis determined that RRM2, a DNA synthesis protein and a known resistance factor, substantially mediated gemcitabine toxicity. These results support gemcitabine levels in DNA as a potential biomarker of drug cytotoxicity

Supplemental Figure S1-S2 from Niclosamide and Bicalutamide Combination Treatment Overcomes Enzalutamide- and Bicalutamide-Resistant Prostate Cancer

Supplemental Fig.1. Bicalutamide and AR-V7 knockdown combination suppressed PSA luciferase activity; Supplemental Fig.2. Niclosamide and bicalutamide combination treatment suppressed AR and AR-V7 target genes expression in C4-2B MDVR cells.</p

Supplementary Figure S1 from Oxaliplatin–DNA Adducts as Predictive Biomarkers of FOLFOX Response in Colorectal Cancer: A Potential Treatment Optimization Strategy

Supplementary Figure S1 showing correlation between CRC cell line oxaliplatin sensitivity and oxaliplatin-DNA area under the adduct curve</p

Expression status of genes encoding therapeutic targets in this study.

<p>At least 18 genes encoded direct or functional targets of signal transduction that have inhibitory agents approved by the FDA or in clinical trials. (A) Absolute expression values (FPKM), mutations and copy numbers for each of the indicated genes is shown in the bar graph. (B) Comparison of protein expression of selected target genes studied here. Protein expression levels were determined by IHC in HER2, HER3, FGFR3 and SRC, and shown in-~+++. The expression of EphB4 was determined by immunofluorescence staining and shown as positive or negative. N/T: not tested. The expression data for therapeutic targets is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134346#pone.0134346.s010" target="_blank">S7</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134346#pone.0134346.s012" target="_blank">S9</a><b>Tables</b>. (C) Validation of target protein expression with IHC. Only selected imagings were shown. PDX BL0440 was stained 3+ for <i>HER2</i> with chicken wire pattern of positive staining at cell membrane while BL0515 was stained negative. BL0293 was stained positive for FGFR3 both on cell membrane and cytoplasm. BL0269 was stained positive for Src on cell membrane, but more on nucleus. (D) Immunofluorescence staining of tissue microarray for EphB4 of BL0293. Both patient specimen from cystectomy (left) and PDX were stained positive for EphB4.</p

Conservation of genomic variants between patient bladder cancer specimens and PDXs.

<p>WES analysis was performed on genomic DNA samples isolated from parental patient tumors and engrafted PDX P0 tumors. WES data was filtered for variants occurring at a frequency of >0.5 and then compared between samples on the basis of variant types. The number and type of variants occurring in each parental tumor and in the P0 PDX tumor were quantified and depicted as percentage of conservation in the graph. For all variants, 91.8% and 97.6% were conserved in BL0429 and BL0440, respectively.</p

Expression levels and mutation status of tumor suppressor pathway genes.

<p>(A) Integrated analysis of <i>mRNA Expression</i>, <i>Mutations</i>, and <i>Copy Number</i> was performed for genes in the <i>TP53</i> and <i>RB1</i> tumor suppressor pathways. Gene-level expression (FPKM) values for pathway member genes are presented and relative gene expression for each gene across the panel of PDX models is indicated by the heatmap (green = lower than the median, red = greater than the median). Mutations (amino acid changes) in <i>TP53</i> and <i>RB1</i> are indicated. Gene copy numbers are presented, and variants indicated as losses (<i>i</i>.<i>e</i>., < 2) or gains (<i>i</i>.<i>e</i>., > 2) shown by darker shades of red and green, respectively. (B) Expression levels (FPKM) of tumor suppressor pathway genes in each PDX are shown in the bar graph.</p

RNAseq and qRT-PCR show similar trends in gene expression levels for selected resistance-realated genes.

<p>Four genes (TSPAN7, AKR1C2, AKR1C1, and CYR61) have increased levels in 5637R cells and two genes (HTRA1 and AQP3) have decreased levels in the resistant cells. (A) Fold changes in chemoresistance gene levels relative to the 5637 parental cells as determined by RNA-seq. (B) Fold change in chemoresistance gene transcript levels relative to the 5637 parental cells as determined by qRT-PCR.</p

Morphology of PDX.

<p>(A) Comparison of morphology between patient specimens and PDXs, subcutaneous and orthotopic PDXs, of PDX BL0293 and BL0440. Hematoxylin and eosin stain (H & E stain) showed that cell morphology was maintained during establishment of PDX and during passaging in mice, both at the subcutaneous site (S.C., at passage 6) and at the orthotopic bladder wall (at passage 4). However, more mitotic cells were observed in PDXs, especially at passage six. (B) Staining of human Ki67. Both PDXs were stained positive with anti-human Ki67, supporting that these PDX cells were indeed of human origin. In PDX BL0440, more cells were stained positive with Ki67 at passage six PDX compared to the human bladder cancer specimen, suggesting more cells were in cell proliferation, and this finding was consistent with the observation of more mitotic cells in Panel A of H & E staining. (C) Staining of human vimentin. Some human bladder cancer cells (left panel) and stromal cells (middle panel) were stained positive for human vimentin in the patient specimens. In the PDX specimen at passage 0, only a few bladder cancer cells were stained positive for human vimentin, suggesting that the stromal cells in PDX were not derived from human stromal cells.</p

Efficacy test of molecularly guided targeted therapy matched with aberrations.

<p>(A) Efficacy studies of molecularly guided targeted therapy. BL0269 overexpressed HER2 and Src. Compared to the control group of median progression-free survival (PFS) of 13 days, a HER2 inhibitor lapatinib and a Src inhibitor ponatinib had little effect in suppressing tumor growth with a median PFS of 12 (p = 0.16) and 18 (p = 0.11) days, respectively. In contrast, lapatinib was very effective in BL0440 that expressed both HER2 and HER3 with PFS of 25.4 days versus 18.4 days in the control group (p = 0.0007). In BL0269 which also harbored a PIK3CA activation mutation H1047R, a PIK3CA inhibitor BEZ235 significantly suppressed tumor growth (p<0.0001). (B) Studies of efficacy and secondary resistance mechanisms of BGJ398 in BL0293. BL0293 overexpressed FGFR3. Compared to the control group of PFS at 9.5 days, an FGFR3 inhibitor BGJ398 significantly prolonged PFS to 18.5 days (p = 2. 61 X 10⁻⁶). Mice were sacrificed and PDXs were harvested (red arrows) before treatment, at Day 3 and at Day 17 (time of resistance) for western blot. Low levels of p-Akt and p-Erk at the baseline and at Day 3, suggesting low downstream signaling activity of FGFR3. Upon the development of resistance to BGJ398 at Day 17, both p-Akt and p-Erk levels increased, suggesting re-activation of the downstream signaling activity. BGJ398-resistant PDX BL0293 was re-implanted in NSG mice to form xenografts. Mice carrying BGJ398-resistant BL0293 were treated with PBS control, BGJ398, or a Raf inhibitor sorafenib plus a PIK3CA inhibitor BEZ235 combination. Compared to the BGJ398 group, treatment of BGJ398-resistant PDX with sorafenib and BEZ235 significantly prolonged PFS from 12 days to 22 days (p = 0.001). (C) Screening for effective chemotherapeutic agents. The first six PDXs were tested for sensitivity to cisplatin, gemcitabine or combination of both drugs. Only BL0440 was sensitive to cisplatin while only BL0269 and BL0479 were resistant to gemcitabine. Resistance to cisplatin could be overcome by gemcitabine, leaving four of these six PDXs sensitive to the combination therapy.</p