Intraperitoneal Administration of a Tumor-Associated Antigen SART3, CD40L, and GM-CSF Gene-Loaded Polyplex Micelle Elicits a Vaccine Effect in Mouse Tumor Models

<div><p>Polyplex micelles have demonstrated biocompatibility and achieve efficient gene transfection <i>in vivo</i>. Here, we investigated a polyplex micelle encapsulating genes encoding the tumor-associated antigen squamous cell carcinoma antigen recognized by T cells-3 (SART3), adjuvant CD40L, and granulocyte macrophage colony-stimulating factor (GM-CSF) as a DNA vaccine platform in mouse tumor models with different types of major histocompatibility antigen complex (MHC). Intraperitoneally administrated polyplex micelles were predominantly found in the lymph nodes, spleen, and liver. Compared with mock controls, the triple gene vaccine significantly prolonged the survival of mice harboring peritoneal dissemination of CT26 colorectal cancer cells, of which long-term surviving mice showed complete rejection when re-challenged with CT26 tumors. Moreover, the DNA vaccine inhibited the growth and metastasis of subcutaneous CT26 and Lewis lung tumors in BALB/c and C57BL/6 mice, respectively, which represent different MHC haplotypes. The DNA vaccine highly stimulated both cytotoxic T lymphocyte and natural killer cell activities, and increased the infiltration of CD11c⁺ DCs and CD4⁺/CD8a⁺ T cells into tumors. Depletion of CD4⁺ or CD8a⁺ T cells by neutralizing antibodies deteriorated the anti-tumor efficacy of the DNA vaccine. In conclusion, a SART3/CD40L+GM-CSF gene-loaded polyplex micelle can be applied as a novel vaccine platform to elicit tumor rejection immunity regardless of the recipient MHC haplotype.</p></div

Anti-tumor efficacy of the polyplex micelle-based DNA vaccine in mice harboring peritoneal and subcutaneous tumors.

<p>(A) Vaccination schedule of polyplex micelles encapsulating therapeutic genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101854#pone-0101854-t001" target="_blank">Table 1</a>) in the peritoneal dissemination model of CT26 tumors. (B) There were significant increases in the survival rates of SART3/CD40L+GM-CSF, GM-CSF, and SART3+GM-CSF groups (<i>P</i><0.0001, n = 18; <i>P</i><0.01, n = 10; <i>P</i><0.01, n = 7 vs. mock control, n = 19, respectively; left panel). Long-term surviving mice were only obtained by vaccination of polyplex micelles with SART3, CD40L, and GM-CSF genes. No significant improvement in survival was detected by transfection of SART3 or CD40L genes alone (n = 6 and 10; right panel). (C) Vaccination schedule of polyplex micelles with therapeutic genes in subcutaneous CT26 and LLC tumor models. (D) Polyplex micelles with SART3, CD40L, and GM-CSF genes (n = 7 and 9), but not other transgenes (n = 5−6), significantly decreased the weights of CT26 (left panel) and LLC subcutaneous tumors (right panel) compared with those in the mock control (n = 6 and 7). *<i>P</i><0.01.</p

Immunohistochemical analysis of CD11c⁺ DCs in lymph node, spleen, and tumor tissues.

<p>(A) Tissue sections of the lymph nodes, spleen, and tumors from mice that received polyplex micelles with the indicated transgenes were immunostained with an anti-CD11c antibody. Scale Bar  = 200 µm. (B) The number of CD11c⁺ DCs was significantly increased in the lymphatic organs of GM-CSF and SART3/CD40L+GM-CSF transfection groups. In tumor tissues, a significant increase of CD11c⁺ DC numbers was detected in SART3/CD40L+GM-CSF vaccine group. <i>*P<</i>0.05, <i>**P<</i>0.01 vs. mock control (n = 5).</p

Polyplex micelle-based DNA vaccine induces CTL activation and memory immunity.

<p>(A) Splenocytes (effector cells) were isolated from mice bearing CT26 and LLC subcutaneous tumors, and then co-cultured with irradiated CSFE-labeled CT26 or YAC-1 target cells at the indicated E/T cell ratios. NK cell activity (upper panel) was increased in all treatment groups with the GM-CSF transgene. In contrast, CTL activity (lower panel) was remarkably elevated by the polyplex micelle encapsulating SART3/CD40L+GM-CSF genes (DNA vaccine group) in an E/T cell ratio-dependent manner. (B) CT26 cells were re-challenged in the flank region of mice that survived for more than 80 days. The formation of subcutaneous tumors was monitored for a further 60 days. Complete rejection of re-challenged tumor cells was detected in the SART3/CD40L+GM-CSF vaccine group, but not in the control. (C) Splenocytes isolated from mice with re-challenge of CT26 cells were subjected to the CTL assay. CTL activity was increased in long-term surviving mice that received the SART3/CD40L+GM-CSF vaccine, but not in the control. (D) CTL activity against CT26 target cells with anti-MHC class 1 (H-2L and -2D) antibodies in mice that were administered the SART3/CD40L+GM-CSF vaccine was reduced to the almost same level as that in the no vaccination control (left panel; n = 2). The CTL activity against SART3-knockdown CT26 cells in mice that were administered the SART3/CD40L+GM-CSF vaccine was reduced compared with that against control CT26 cells, but the knockdown efficiency of SART3 siRNA at the protein level was only 50% (right panel; n = 2).</p

Collagen internalization by pancreatic cancer cells is dependent on Endo180 expression.

<p>(A) SUIT-2 and KP-2 cells were transfected with siEndo180-1 or siEndo180-2, and knockdown of Endo180 expression compared with control siRNA-transfected cells was confirmed by quantitative RT-PCR (upper panel) and western blotting analysis (lower panel) for at least 3 days. (B) Following EMT induction with 10 ng/ml TGF-β1, SUIT-2 and KP-2 cells were incubated with OG-gelatin for 2 h. The percentages of OG-gelatin-internalized cells were evaluated by flow cytometry. The dot plots show representative flow cytometry data for untreated SUIT-2 and KP-2 cells and cells transfected with control siRNA, siEndo180-1 or siEndo180-2 (upper panels). The collagen internalization scores represent the relative ratios of collagen-internalized cells among cells transfected with control siRNA, siEndo180-1 or siEndo180-2 to those in untreated cells. The enhanced collagen internalization abilities in SUIT-2 and KP-2 cells after TGF-β treatment are dramatically reduced by knockdown of Endo180 expression (lower panel). Each sample was analyzed in triplicate, and comparisons between TGF-β1-treated cells transfected with control siRNA and other cells were performed using Student's <i>t</i>-test. *<i>P</i><0.01; **<i>P</i><0.001.</p

Evaluation of the correlation between EMT induction and expression of collagen receptors in pancreatic cancer cell lines.

<p>(A) Relative expression levels of E-cadherin, vimentin, Endo180, β1-integrin and α2-integrin in 10 pancreatic cancer cell lines and four PSC cultures. The expression levels of all genes were normalized by the corresponding expression levels of β-actin. Each sample was analyzed in triplicate, and the data are expressed as means ± SD. One-fifth of the acquired levels for E-cadherin and Endo180 are shown in the graph for ease of reference. (B) The pancreatic cancer cell lines were divided into two groups showing an epithelial phenotype (SW1990, HS766T, BxPC-3, CAPAN-1 and AsPC-1) and a mesenchymal phenotype (SUIT-2, CFPAC-1, MIA PaCa-2, Panc-1 and KP-2) on the basis of the expression ratio of E-cadherin to vimentin. Endo180 is more highly expressed in the cell lines with the mesenchymal phenotype than in those with the epithelial phenotype, while the expressions of β1-integrin and α2-integrin are not correlated with the cell phenotypes. Statistical analyses were performed using the Mann–Whitney <i>U</i> -test. (C) SUIT-2 and KP-2 cells were incubated with or without TGF-β1 for 72 h, and the expression levels of Endo180, β1-integrin and α2-integrin were determined by quantitative RT-PCR (upper panel). The expression levels of all genes were normalized by the corresponding expression levels of 18S rRNA. The fold changes were calculated as the mRNA levels in TGF-β1-treated cells divided by the levels in untreated cells. Endo180 mRNA expression in SUIT-2 and KP-2 cells is more highly increased by EMT induction than β1-integrin and α2-integrin mRNA expression. Increased cell surface expression of Endo180 in EMT-induced SUIT-2 and KP-2 cells (red lines) compared with untreated SUIT-2 and KP-2 cells (blue lines), respectively, was confirmed by flow cytometry analyses (<i>P</i><0.01 for each; lower panels). Each sample was analyzed in triplicate, and comparisons between untreated and TGF-β1-treated cells were performed using Student's <i>t</i>-test.</p

Collagen uptake by PSCs and pancreatic cancer cells.

<p>(A) Representative microphotograph of immunofluorescence staining of α-SMA in PSCs. The PSCs have a stellate-like or spindle-shaped morphology, and express α-SMA (red). The PSCs have internalized many collagen molecules (green). Scale bar: 100 µm. Original magnification: ×200. (B) Flow cytometry analyses of PSCs, SUIT-2 cells and KP-2 cells after incubation with OG-gelatin for 2 h. Each sample was analyzed in triplicate, and the percentages of collagen-internalized cells are shown as means ± SD in the representative figures. (C) Confocal microscopy images of collagen in the cytoplasm of SUIT-2 and KP-2 cells. The cells were incubated with OG-gelatin (green), fixed and stained with Alexa Fluor 647-conjugated phalloidin (red) to visualize the cell outlines. Orthogonal sections in the XY, XZ and YZ planes are shown. The Y and Z axes illustrate the localization of the internalized collagen more clearly (arrowheads). In particular, the spindle-shaped cancer cells with a mesenchymal morphology contain many collagen molecules (arrows). Scale bars: 100 µm. Original magnification: ×400.</p

Effects of Endo180 expression by pancreatic cancer cells on their invasive ability.

<p>SUIT-2 and KP-2 cells (4×10⁴ cells/well) transected with siRNAs were suspended in 50 µg of 3D type I collagen matrix, and seeded in the upper chambers with or without TGF-β1. After gel formation by incubation at 37°C for 30 min, the upper chambers were placed in the wells of a 24-well culture dish containing 750 µl of 10%FBS/DMEM. The cells that invaded to the lower surface of each upper chamber membrane through the collagen I matrix were fixed, stained with hematoxylin and eosin, and counted. (A) Representative photomicrographs of invading SUIT-2 and KP-2 cells transfected with control siRNA, siEndo180-1 or siEndo180-2. Scale bars, 100 µm. Original magnification, ×200. (B) The invasive abilities of SUIT-2 and KP-2 cells are increased by EMT induction, but attenuated by knockdown of Endo180 expression. Each sample was analyzed in triplicate, and comparisons between two groups were performed using Student's <i>t</i>-test. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001. (C) Collagen internalization scores in SUIT-2 and KP-2 cells after coculture with PSCs for 72 h. The collagen internalization abilities are slightly enhanced in SUIT-2 and KP-2 cells via cancer–stroma interactions. The experiments were performed in triplicate, and comparisons between the data were performed using Student's <i>t</i>-test. *<i>P</i><0.05; **<i>P</i><0.01.</p

Primers used for qRT-PCR.

<p>Primers used for qRT-PCR.</p

Factors affecting walking ability in female patients with rheumatoid arthritis

<div><p>Objective</p><p>To determine the factors associated with gait parameters in female patients with rheumatoid arthritis (RA).</p><p>Methods</p><p>The gait analysis was performed in a large cohort of RA patients, and three basic gait parameters (step length, cadence and gait speed) were calculated. Clinical and laboratory data were also collected. Factors associated with gait parameters were analyzed using multivariable linear regression in the three models with forced entry. Then, we divided those patients with Health Assessment Questionnaire disability index (HAQ) scores ≤ 0.5 into two groups according to their gait speed that were compared to identify the characteristics of patients with a good HAQ score but poor walking ability.</p><p>Results</p><p>A total of 318 female patients were analyzed. Knee extension strength had the strongest positive association with all three gait parameters (P < 0.0001), while methotrexate use was also positively associated with all three gait parameters (step length: P < 0.05, cadence: P < 0.05 in model 1 and 2; P < 0.01 in model 3, gait speed: P < 0.01). The disease activity score was negatively associated with step length and gait speed (step length, gait speed: P < 0.01 in model 1 and 2; P < 0.05 in model 3). 26% of patients with good HAQ scores showed slow gait speed. Patients with good HAQ scores and slow gait speed had higher disease activity scores (P < 0.05) and lower knee extension strength (P < 0.0001) than those with good HAQ scores and normal gait speed.</p><p>Conclusions</p><p>High knee extension strength, low disease activity and administration of methotrexate were strongly associated with good walking ability in female patients with RA. And, even if patients showed good HAQ scores, about quarter of those patients had poor walking ability, and they showed higher disease activity, lower knee extension strength, compared to the patients with normal gait speed.</p></div