Cancer is a complex disease involving changes to multiple pathways 
and microenvironments. Wels et al have proposed a complex interplay between the primary tumor, sites of metastases, and the bone marrow (illustrated in ) that is mediated by soluble factors 
. In this model, tumor cells secrete soluble factors that can modify the microenvironment of distal tissues (e.g. sites of metastases) or induce mobilization of bone marrow-derived cells that are recruited to the primary tumor or to metastatic lesions 
. We believe that heparan sulfate proteoglycans could play a central role in orchestrating these signals by binding and presenting growth factors and chemokines that are involved in remodeling microenvironments and recruiting distal cells. Indeed, many of the key soluble factors implicated in defining tumor-associated niches, such as VEGF, PlGF, SDF-1α, S100A8, S100A9, SAA3, TNF-α, and TGF-β 
are HSPG-binding proteins. M402, a novel HSPG mimetic, was designed to inhibit multiple pathways involved in tumor progression and metastasis.
Role of HSPG-binding factors in connecting tumor niches.
The anti-tumor effects of heparin or related molecules are well-documented 
. A number of retrospective and prospective clinical studies have indicated that heparin therapy may prolong survival of cancer patients across a variety of solid tumor types 
. Several heparin-derived molecules and synthetic carbohydrate compounds are undergoing preclinical and clinical development 
. Despite repeated efforts, definitive data supporting anti-tumor activity of heparins in randomized clinical trials has been lacking 
possibly because such studies have been constrained by the dose limiting anti-coagulant properties of heparin. Non-anticoagulant heparin-related molecules have been generated by various methods, such as desulfation of the 2O- and 3O-positions or N
-desulfation followed by glycol-splitting 
. Recently, two novel heparan sulfate mimetics, PG545 and SST0001, have been described in the literature as potential anti-angiogenic/anti-metastatic agents 
. PG545 is a synthetic, fully-sulfated tetrasaccharide functionalized with a cholestanyl aglycon that shows potent inhibition of heparanase with low anticoagulant properties. PG545 exhibits a long plasma half-life and shows activity in multiple models of cancer 
. On the other hand, SST0001 is an N
-acetylated, glycol-split high molecular weight heparin that also exhibits low anticoagulant activity, selectively inhibits heparanase and has shown activity in an in vivo
model of multiple myeloma 
. However, N
-acetylation of heparin sequences has been shown to also reduce binding affinity against many heparin-binding proteins, such as VEGF, FGF2, and SDF-1α 
. These activities may be important for the anti-tumor activity of heparin. M402 has been specifically engineered to substantially reduce anticoagulant activity while retaining or improving its heparan sulfate-like binding properties to multiple targets involved in tumor progression and metastasis.
It is difficult to define a single ‘smoking gun’ with respect to M402's mechanism of action. This is likely due to the fact that M402 affects multiple pathways that regulate tumor cell interactions with its microenvironment. Studies investigating the mechanism of action of heparin in cancer have primarily focused on a few pathways. Folkman was the first to describe the anti-angiogenic properties of heparin, which is mediated by multiple factors including VEGF and FGF2 
. Varki and colleagues described the inhibitory effects of heparin on P-selectin-mediated cloaking of tumors cells by platelets, which promote tumor dissemination 
. Vlodavsky and colleagues have published extensively on the role of heparanase, a heparin sulfate-cleaving enzyme, in tumor progression 
. M402 inhibits these pathways and others with similar or increased potency with respect to low molecular weight heparin, but with greatly reduced anti-Factor Xa activity.
The current armamentarium of targeted therapies aimed at specific factors, such as VEGF, have shown great promise in a variety of solid tumors. However the beneficial activity of anti-VEGF therapies, such as bevacizumab, is generally short lived. Tumors adapt by exploiting alternative angiogenic factors and recruiting EPCs and MDSCs to promote angiogenesis 
, resulting in resistance to cancer therapies and often the emergence of tumor cells with a more aggressive phenotype 
. It is notable that M402 inhibits endothelial sprouting in response to FGF2 and HB-EGF as well as VEGF. In addition, M402-treated animals showed reduced numbers of circulating myeloid derived suppressor cells (MDSCs or CD11b+
immature myeloid cells). MDSCs have been associated with tumor progression and angiogenesis 
, and may contribute to the development of tumor resistance to anti-VEGF treatment 
.The multi-targeted nature of M402 suggests the possibility that it may confer protection against the development of resistance to VEGF-specific therapies.
There has been an increased effort in identifying combinations of targeted drugs that can be used together in cancer treatments. However, combining potent single-targeted drugs is often limited by their safety profile, and can result in unexpected adverse activity 
. Moreover the expense of combining targeted therapies has put significant pressure on the healthcare system. Targeting HSPGs is an attractive approach to targeting multiple pathways because of the versatility of HSPG functions and their importance in facilitating tumor progression 
. Potent antibody- or small molecule-based therapies represent a digital or ‘on-off’ approach to inhibiting a specific pathway. Because of its relatively low affinity in binding to different key proteins, M402 represents an analog approach to attenuate, rather than completely shutting off, multiple pathways. This broad based attenuation of multiple pathways may confer a safety advantage over combination of current drugs that completely shut down critical pathways.
Studies interrogating heparin antitumor activity in vivo
have been primarily restricted to experimental metastasis models wherein tumor cells are injected i.v. concurrently with a single injection of heparin. Varki and colleagues have demonstrated that heparin inhibition of P-selectin accounts for a major portion of heparin's activity in experimental models of metastasis by inhibiting platelet-tumor cell binding, which is thought to protect tumor cells in circulation and facilitate tumor cell binding to vascular endothelial cells 
. We have shown that M402 also inhibits experimental metastasis in two models, the B16F10 i.v. model and the human C170HM2 colon carcinoma model. The fact that a single dose of heparin or M402, administered concurrently with B16F10 cells, inhibits lung colonization indicates that this model primarily reflects tumor seeding. The anticoagulant properties of heparin may have constrained the ability to interrogate the effects of chronic dosing of heparin on tumor progression in spontaneous models of metastases.
To investigate the effects of M402 on metastatic processes beyond tumor dissemination, we tested M402 in a highly aggressive model of spontaneous metastasis utilizing orthotopically implanted syngeneic 4T1 mammary carcinoma cells. This model recapitulates multiple aspects of tumor dissemination, progression and clinical outcome similar to that of Stage IV human breast cancer 
. The aggressive nature of this model is indicated by the following aspects: 1) inoculation of a single 4T1 tumor cell has the potential to develop into a tumor 
; 2) the doubling time of the tumor cells in vivo
is approximately 24 hours as measured by bioluminescent imaging, which means that a 75% reduction in tumor load would translate into an extension of median survival by only 2 days; and 3) standard chemotherapeutic agents show weak activity in this model. We demonstrate that M402, in combination with chemotherapeutic agents, significantly decreased metastatic tumor burden and increased survival compared to mice treated with chemotherapy alone. The effect of M402 on tumor progression beyond dissemination is reflected in reduced microvessel density, in both the primary tumor and in metastatic lesions. These results are consistent with our observation that M402 selectively accumulates in tumor tissue. Recent studies have also demonstrated the superior efficacy of M402 in combination with gemcitabine over gemcitabine alone in a genetic model (KrasLSL-G12D/+
) of pancreatic cancer (AACR Annual Meeting 2010 Proceedings). These studies indicate that M402 affects tumor progression beyond tumor dissemination. Moreover these studies show that activity of M402, which targets multiple host microenvironmental factors, can be complementary to the direct antitumor activity of conventional chemotherapeutics agents.
In summary, our data demonstrate that targeting HSPG biology represents a potential novel strategy to treat multiple facets of cancer. Heparin, from which M402 is derived, has been used clinically for decades with an acceptable and well understood risk-benefit profile. M402 has been specifically engineered to substantially reduce anticoagulant activity while retaining or improving its other heparan sulfate-like binding properties. Here we demonstrated that M402 successfully interfered with various pathways essential for tumor progression, as well as targeted different tumor compartments. The efficacy, safety, and multimodal activity of M402 will need to be established in human clinical trials.