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

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 displays low anticoagulation activity while retaining activity against key HSPG-binding proteins.

<p>(A) Left panel, anti-factor Xa activity: BALB/c mice were injected subcutaneously with 10 mg/kg dalteparin, M402 or M-ONC 202. Plasma was collected at different time points and analyzed for anti-Factor Xa activity (Mean±SD). Experiments were performed three times with similar results. Right panel, aPTT measured in normal human plasma. (B) The binding affinity (Mean±SD) of M402, M-ONC 202 and dalteparin to different heparin binding proteins was determined by Surface Plasmon Resonance (SPR) with a competitive inhibition assay on a Biacore T100 instrument. Different heparin-derived compounds were captured on the sensor chip with a fixed amount of growth factors including FGF2, HGF, VEGF, and SDF-1α (4, 5, 25, and 50 nM, respectively) mixed with dilution series of each heparin-derived compound flow through. The IC₅₀ (µg/mL) was calculated for each interaction. (C) Inhibition of the P-selectin/PSGL1 interaction (Mean±SD) by heparin-derived molecules was determined by an inhibition assay. A dilution series of each heparin-derived compound was mixed with 25 nM P-selectin and flowed over the sensor surface coated with PSGL1 and the response at equilibrium was measured and the K_i was calculated. (D) Heparanase activity (Mean±SD). Heparanase activity was measured using Cisbio Bioassays technology based on TR-FRET. (E) Murine melanoma B16F10 experimental metastasis model. Groups (n = 12–13) of female mice were treated with saline or different heparin-derived compounds prior to iv inoculation of 2×10⁵ B16F10 cells. Tumor colonization to the lung was quantified by lung weight (Mean±SEM.) on day 20. Statistics were performed with One-way ANOVA using Bonferroni's multiple comparison test. (F) Human C170HM2 colon carcinoma experimental metastasis model. Groups of male MF1 nude mice (n = 10) were injected intraperitoneally with 1.5×10⁶ C170HM2 human colon carcinoma cells. Daily subcutaneous injection of saline or M402 at different doses started 1 day later until the experiment was terminated 35 days after tumor inoculation. At necropsy, liver tumors were excised, weighed (Mean±SD, left panel) and cross-sectional areas (Mean±SD, right panel) measured.</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

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

M402 inhibits tumor and host cell functions <i>in vitro</i>.

<p>(A) The SDF-1α-mediated migration assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021106#s2" target="_blank">Materials and Methods</a>. Results are presented as % inhibition of migrated cell numbers. (B) M402 inhibited VEGF and FGF2 induced HUVEC sprouting. Upper panels: Representative images taken 24 hrs after incubation with VEGF-A in the presence and absence of M402 or M-ONC 202 at 30 µg/ml. Lower panels: Cumulative sprout length (CSL, Mean±SD) at different doses of M402 or M-ONC 202 in the presence of VEGF (left panel) or FGF2 (right panel). (C) M402 inhibited tumor platelet rosettes. Activated platelets were incubated for 30 min at RT with murine WEHI-3 leukemia cells (CD45⁺) pre-mixed with M402 (500 µg/ml). Platelets were stained with anti-CD41-PE and WEHI cells with anti-CD45-FITC antibodies. Platelet-WEHI-3 aggregates were imaged by immunofluorescence microscopy (upper panels) or quantitated by flow cytometry (lower panels).</p