M402, a Novel Heparan Sulfate Mimetic, Targets Multiple Pathways Implicated in Tumor Progression and Metastasis

Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis

The Astronomy and Space Science Concept Inventory: Development and Validation of Assessment Instruments Aligned with the K–12 National Science Standards

We report on the development of an item test bank and associated instruments based on those K–12 national standards which involve astronomy and space science. Utilizing hundreds of studies in the science education research literature on student misconceptions, we have constructed 211 unique items that measure the degree to which students abandon such ideas for accepted scientific views. Piloted nationally with 7599 students and their 88 teachers spanning grades 5–12, the items reveal a range of interesting results, particularly student difficulties in mastering the NRC Standards and AAAS Benchmarks. Teachers generally perform well on items covering the standards of the grade level at which they teach, exhibiting few misconceptions of their own. Teachers dramatically overestimate their students’ performance, perhaps because they are unaware of their students’ misconceptions. Examples are given showing how the developed instruments can be used to assess the effectiveness of instruction and to evaluate the impact of professional development activities for teachers

M402 targets primary tumors.

<p>Fluo-M402 biodistribution in 4T1 tumor bearing mice. Female Balb/c mice were implanted orthotopically into the first mammary fat pad at a concentration of 1×10⁵ 4T1 cells on Day 0. Mice were injected on Day 3 with a single subcutaneous dose of either 10 mg/kg fluorescently-labeled M402 or free dye of approximately the same intensity in saline. (A) Mice were imaged in the ventral view at various time points after injection with Fluo-M402 (upper panels) or free dye (lower panels). White circles indicate primary tumors, and yellow boxes highlight signals from the liver regions. (B) Quantification of fluorescent signals (Mean±SEM) at the primary tumor site at different times after Fluo-M402 or free dye injection. (C) CD31 immunohistology. Groups of female BALB/c mice (n = 16) were inoculated orthotopically with 8×10⁴ 4T1 cells in the 4th mammary fat pad on day 0. Saline or M402 (20 mg/kg/day) treatment delivered by s.c. implanted osmotic pumps started on day 5. Primary tumors were removed on day 9 by surgery and the tumor weights were recorded. There was no significant difference in primary tumor weight between the groups 4 days after the start of the treatments. Primary tumors were fixed in buffered-formalin, embedded in paraffin and stained for CD31 by immunohistochemistry. Representative CD31-staining is presented in the left panel where the brownish staining (arrows) indicate CD31⁺ vessels. Quantification of microvessel density as numbers of CD31⁺ vessels/40× field (Mean±SEM) is displayed on the right. *, P<0.05 (t-test) when compared with saline control group.</p

Role of HSPG-binding factors in connecting tumor niches.

<p>Abbreviations: HPC, hematopoietic progenitor cell; EPC, endothelial progenitor cell; iMC, immature myeloid cell.</p

M402 inhibits tumor progression and angiogenesis at the metastastic site and normalized circulating MDSCs <i>in vivo</i>.

<p>(A) M402 combined with cisplatin inhibited lung metastasis of orthotopically inoculated 4T1 tumors. The experimental set up is described in the legand of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021106#pone-0021106-g004" target="_blank">Figure 4C</a>. The experiment was terminated on day 32, lung tissues were isolated and lung weight (Mean±SEM) quantified (A). Fixed lungs were embedded in paraffin and % tumor (as % of total section areas, Mean±SEM) quantified under the microscope on H&E stained slides (B). Left panels show representative pictures of H&E staining where the solid darker purple stained areas (indicated by solid yellow arrow heads) represent metastatic lung tumors and the lighter-stained porous areas (indicated by white open arrows) are the normal lung tissues. Tumor areas were quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021106#s2" target="_blank">Materials and Methods</a>, and the results are displayed in the right panel. *, P<0.05 compared to saline control, Cisplatin, and M402 monotherapy groups; one-way ANOVA. (C) Lung tissues were also stained with CD31 immunohistology. Representative images from saline and combination therapy treated lung tumors are presented in the left panel where the CD31⁺ vessels are indicated by the arrows. CD31⁺ vessels (Mean±SEM) were quantified in the tumor areas and results displayed in the right panel. Statistics were performed with One-way ANOVA using Bonferroni's multiple comparison test. (D) Blood samples obtained by cardiac puncture were analyzed by flow cytometry. Left panel: representative CD11b and GR-1 staining of blood CD45⁺ cells of naïve or 4T1-tumor bearing mice. Right panel: quantification of MDSCs as % of total cells (Mean±SEM) in different treatment groups. *, P<0.05; **, P<0.01 compared to saline control group (one-way ANOVA).</p

M402 monotherapy or in combination with docetaxel shows survival benefits in the orthotopic murine mammary carcinoma 4T1 model.

<p>(A) Groups of female BALB/c mice (n = 20) were inoculated orthotopically with 1×10⁵ 4T1 cells in the 4th mammary fat pad on day 0. M402 treatment delivered by sc implanted osmotic pumps at 40 mg/kg/day started on day 1. Primary tumors were removed on day 10. Survival of the M402 treated group was significantly longer than that of the saline control group (P<0.02 by Log-Rank test). (B–D) Groups of female BALB/c mice (n = 16) were inoculated orthotopically with 5×10⁴ 4T1-luc2-1A4 cells in the 4th mammary fat pad on day 0. M402 treatment delivered by sc implanted osmotic pumps at 40 mg/kg/day started on day 1. Primary tumors were removed on day 10 by surgery. Weekly ip injection of saline or docetaxel (10 mg/kg) started on day 14. After primary tumor resection, animals were monitored twice weekly with bioluminescent imaging. (B) Whole body bioluminescence (Mean±SEM) quantified as photons/second over time. (C) Bioluminescence imaging of all the experimental animals on day 29. (D) Kaplan-Meier survival curve. Survival of the M402 and docetaxel combination group was significantly longer than that of the saline control and the docetaxel monotherapy group (P<0.0001 and P<0.05, respectively, by Log-Rank test).</p

Physiochemical characteristics of M402, M-ONC 202 and Dalteparin.

<p>Physiochemical characteristics of M402, M-ONC 202 and Dalteparin.</p