A solid tumor is a highly specialized organ-like structure in disequilibrium with the rest of the body. For the purpose of this review, we have conceptually divided the tumor into two functional parts, malignant cells and the tumor stroma, and will focus this review on the role of the glycosaminoglycan hyaluronan (HA) in the interaction between the tumor stroma and the associated malignant cells. We will also discuss recent preclinical evidence supporting therapeutic targeting of tumor stroma HA via depletion of HA.

The tumor stroma, a supportive framework for malignant cells, consists of various non-malignant cell types and the associated tumor extracellular matrix (ECM), with its multitude of embedded growth factors and protumorigenic elements [1]. Specialized cell types found in the tumor stroma include fibroblasts, myofibroblasts, endothelial cells, pericytes, tumor-associated macrophages and varying numbers of other immune cells [2,3]. More than just a passive bystander to disease progression, the tumor stoma undergoes massive remodeling in response to signals derived from both malignant and host cells, evolving from a barrier to tumor growth to an active contributor of malignant progression [3].

While the communication between the malignant cells and stromal cells may help drive the chronicity of the malignancy, it also makes the tumor stroma an emerging source of targets for the treatment of malignant disease. Indeed, a number of candidate cancer therapeutics have been identified from studies focused on malignant cell:stromal cell interactions (see recent reviews [4,5]). Probably the best known cancer therapeutic to come from these studies is trastuzumab (Herceptin^®), an antibody inhibitor of the p185^HER2 receptor tyrosine kinase (RTK). It was discovered in a screen for monoclonal antibodies that reversed the macrophage-resistant phenotype of tumor cells, making them more sensitive to killing by macrophage-elaborated tumor necrosis factor-alpha (TNF-α) [6]. Additional examples of therapeutic targets identified from malignant cell:stromal cell interaction studies include: bevacizumab, which inhibits vascular endothelial growth factor A (VEGF-A), an angiogenesis-related growth factor, preventing stromal vessel formation [7]; vismodegib, a small molecule inhibitor of the hedgehog pathway which targets both tumor stroma and cancer stem cells and has shown very promising results in preclinical studies, and which is currently being tested in several clinical trials of pancreatic adenocarcinoma (PDA) [8]; and TH-302, a hypoxia-activated prodrug which has also been shown to increase the antitumor activity of a broad range of conventional chemotherapy agents in human xenograft models and for which clinical testing is ongoing [9,10]. Similarly, and the focus of this review, the accumulation of HA in the tumor stroma is now recognized as a stromal target for the experimental treatment of malignancies [11,12,13,14], and is the subject of ongoing clinical investigations using an intravenously delivered HA-depleting enzyme, specifically a pegylated human recombinant PH20 hyaluronidase (PEGPH20) [15,16].

HA, a large, unbranched, non-sulfated glycosaminoglycan composed of repeating disaccharide units of D-glucuronic acid and N-acetylglucosamine, is a major constituent of the ECM of most tissues. While simple in composition, HA polymers can reach up to 25,000 disaccharide units, with molecular weights in the 10⁷ Dalton range, and are capable of organizing into complex structures and interacting directly or indirectly with many components of the ECM. The enzymes that produce HA are hyaluronan synthases (HAS). There are three HA synthases (HAS1-3) in mammalian cells, each of which possesses the two glycosyltransferase activities responsible for the transfer of N-acetylglucosamine and D-glucuronic acid from their uridine 5'-diphosphate (UDP) precursors to the growing HA chains [17]. The three isoforms are independently regulated and are thought to produce distinct molecular weight forms of HA [18,19,20,21,22]. HAS2 deficiency has been shown to be embryonic lethal, whereas the other synthases may play more subtle roles in development [23,24]. Unlike most other glycosaminoglycans, which are processed and extensively modified in the Golgi, HA is synthesized at the inner face of the plasma membrane and secreted as a linear undecorated polysaccharide directly into the extracellular space. The kinetics controlling HA synthesis in vivo are largely unknown but are likely dependent on available cytosolic UDP-monosaccharide concentrations [25]. In malignancy, HAS activity generates large intact linear molecules of HA that are either rapidly incorporated into the ECM surrounding the tumor cells, or retained at the cell surface through HA binding receptors and interacting glycoproteins and proteoglycans. HA may also be tethered to the cell surface by interactions with HAS proteins themselves [26,27,28]. Transgenic studies have shown that while their expression is not itself transforming, HA synthases can cooperate with oncogenes to promote tumor growth and desmoplastic transformation of the tumor stroma [29].

HA degradation is carried out by a family of enzymes called hyaluronidases (HYAL). Genomic analysis reveals that mammals possess multiple hyaluronidase genes, including HYAL-1 through HYAL-5, PH20, and in humans a pseudogene designated HYALP-1 [30]. None of the degradative hyaluronidase enzymes have a lethal phenotype [31,32,33,34,35,36]. HYAL-5 appears to have enzymatic activity redundant to PH20, except for a difference in pH optima, and is not expressed in humans. PH20 is encoded by the sperm adhesion molecule-1 (SPAM-1) gene, and has been studied mostly in the testes where it is localized to the heads of mature sperm. An engineered therapeutic hyaluronidase, a human recombinant form of PH20 (rHuPH20) has been developed, and is approved for use in the local dispersion of co-injected drugs, and is currently being tested in clinical trials to facilitate the subcutaneous delivery of macromolecules [37,38,39].

Due to its high negative charge, HA is capable of coordinating up to 10,000 times its weight in water, which contributes to its ability to increase interstitial fluid pressure at those sites where it accumulates [40]. Overall, about 25−30% of human tumors overexpress HA, and of special interest for this review, up to 87% of PDAs express high levels of this glycosaminoglycan (Figure 1) [11]. In particular, in tumors that originate from epithelia, which normally express low levels of HA (e.g., breast, prostate, bladder and colorectal cancer), cancer cell-associated HA accumulation is a negative prognostic indicator for cancer patients [41]. The aberrant accumulation of HA in tumors likely occurs via dysregulation of HA synthases and hyaluronidases during disease progression [42,43]. Indeed, it has been suggested that different HAS isoforms might be expressed in different stages of tumor growth to maximize survival of the cancer cells [43]. Whether the constant degradation and resynthesis of HA in tumor foci characterized by high levels of accumulation of this glycosaminoglycan may contribute to the state of immune activation within a tumor is the subject on ongoing research [44].

The ability of HA to imbibe water molecules coupled to its overexpression in the tumor foci contributes to the high interstitial fluid pressure observed in tumors. Indeed, high concentrations of HA in the ECM cause tissue spaces to become hydrated and to swell due to the HA-mediated increase in tissue osmotic pressure [45]. As compared to normal tissues, tumor interstitial fluid pressure (tIFP) can be elevated more than 30-fold, resulting in compression of blood vessels, hypoxia and drug resistance [46]. While overexpression of HA alone in the absence of other cross-linking matrix proteins does not appear to be capable of causing elevated interstitial fluid pressure [47], when sequestered in the tumor stroma together with other ECM components it can significantly contribute to elevated tIFP.

High tIFP may also contribute to metastasis [48]. Several instances of malignant disease that link accumulation of HA with poor prognosis in cancer patients have been reported, and are discussed in detail below. The cross-linking of the ECM by HA not only has the potential to trap protumorigenic growth factors and cytokines, it can also physically interfere in the ability of immune cells to access malignant cells. A more simplistic, but equally intriguing explanation may be that elevated tIFP may in of itself generate a mobility signal for tumor cells to disseminate towards low pressure environments [49].

HA binds directly to molecules collectively referred to as hyaladherins. These hyaladherins include proteins with diverse molecular functions, such as versican and CD44 [50,51]. Here, we categorize hyaladherins into two groups: (1) matrix proteoglycans and glycoproteins; and (2) cell surface receptors. A summary of all hypothetical HA molecular interactions, the HA interactome, illustrating HA’s binding partners and associated downstream effectors, is presented in Figure 2. Both direct and indirect interactions as reported in the literature are shown, and it is understood that there are varying degrees of evidence to support the interactions shown in Figure 2.

Literature reports from the early 1950s have associated overproduction of HA with malignancy [101,102]. Later work [103,104,105] provided preliminary evidence that the spreading factor activity of hyaluronidase enhanced clinical efficacy of chemotherapy in doses up to 200,000 U/day. In addition, previous preclinical studies [106,107,108] have attributed chemopotentiation by hyaluronidase to a reduction in tIFP, which facilitates enhanced drug penetration into HA-depleted tumors. Increased production of HA in tumor cells, via ectopic HAS expression, has been shown to induce microvillus structures, suppress contact inhibition, and increase tumor growth rates in vivo [13,29,69,109,110]. Conversely, reduction of tumor HA levels in preclinical studies, either by addition of hyaluronidase or by inhibition of HAS activity, has been reported to reduce in vitro tumor cell proliferation, motility and invasion, and to reduce the growth of implanted tumors [13,14,21,111,112,113,114,115]. Similarly, inhibition of HA synthesis by 4-methylumbelliferone has been shown to inhibit cancer cell adhesion, breast cancer, and melanoma cell invasion and melanoma metastasis in vivo [116,117,118,119]. Recent work which helps to define the utility of PEGPH20 in the treatment of human cancer will be the subject of discussion later in this review.

Further exploration of the role of HA accumulation in tumor progression in patients has included clinical work with several solid and hematopoietic tumors [41,120]. In breast adenocarcinoma, five-year survival deteriorated as a function of increasing stromal HA levels; for low, moderate, and high HA levels, respectively, the five-year overall survival was 45%, 39%, and 26% (p = 0.002) and recurrence-free survival was 66%, 56%, and 40% (p = 0.008). The presence of HA-positive carcinoma cells correlated significantly with axillary lymph node positivity and poor differentiation. The five-year overall survival of patients exhibiting HA-positive carcinoma cells was significantly lower compared to patients without HA-positive carcinoma cells (54% versus 81% respectively, p = 0.01) [120].

In gastric carcinoma, the HA profile of 215 Stage I–IV gastric carcinoma patients was examined. A high proportion of HA-positive cells was found that was significantly associated with deep tumor invasion, nodal metastasis, positive lymphatic invasion, poor differentiation grade, as well as inferior prognosis in univariate survival analysis. Forty-four percent of the tumors evaluated had an HA labeling index of 30–100% HA-positive cells [121]. In colorectal carcinomas, the cellular association of HA to overall survival and recurrence-free survival in 202 colorectal carcinoma samples was followed for a mean of 14 years. Both high HA intensity and labeling indices were frequently found and significantly associated with poorer overall survival, shorter recurrence-free survival, and elevated Dukes classification for 187 evaluable patients [122].

HA levels were studied in 309 epithelial ovarian cancers and 45 matched metastatic lesions. While in 73% (227 of 309) of the cases the fraction of HA-positive cancer cells was <10%, high stromal HA levels were significantly correlated with poor differentiation, serous histologic type, advanced stage, and large primary residual tumors [123]. Accumulation of HA is similarly reported to predict worsened outcomes in prostate, bladder and squamous cell carcinoma-type non-small cell lung cancers (NSCLC) [124,125,126]. Recent data suggest that pancreatic cancer has a high frequency of HA overexpression at about 87% (Figure 1) [11]. In other complementary studies, the expression of HA receptors CD44 and RHAMM have been found to predict more aggressive disease in hematologic neoplasias [127,128] and colon cancer [129]. Both of these receptors have been shown to be dysregulated by inactivation of the P53 tumor suppressor gene [130,131].

While early clinical investigations of hyaluronidase in cancer patients [103,104,105] demonstrated potential therapeutic activity, these studies were curtailed because of concerns of potential anaphylactic responses to the foreign proteins that are found in hyaluronidase products from animal sources and a remarkably short plasma half-life. In order to pursue the potential therapeutic utility of the hyaluronidases in cancer therapy, a pegylated form of recombinant human PH20 hyaluronidase was developed, PEGPH20. Whereas the non-pegylated recombinant human PH20 enzyme, rHuPH20, has a very short serum residence time (t_1/2 = 2.3 min), the polyethylene glycol (PEG)-modified rHuPH20 (PEGPH20) exhibited an increased in vivo half-life in mice of ∼270-fold (t_1/2 = 10.3 h), making it well suited for subsequent studies evaluating the effect of HA removal and the subsequent stromal remodeling of HA-overexpressing tumors [14].

Characterization of multiple tumor cell lines for HA expression, followed by examination of their response to the exogenous administration of PEGPH20 as transplanted tumors in animal models, demonstrated that tumors which express elevated levels of HA in vivo were more sensitive to the antitumor effects of PEGPH20 [13,14]. To test whether higher HA-expressing tumors are more sensitive to HA depletion, a set of NSCLC patient explants was analyzed for HA content using a semiquantitative scoring method that identified squamous cell lung cancers with HA⁺¹ (low), HA⁺² (medium), or HA⁺³ (high) phenotypes. Patient explants were used for this study in order to best imitate the genetic heterogeneity of tumors in situ, while hopefully maintaining some stromal elements that might be important to HA production [132] and response to PEGPH20. The results showed dramatic differences in the degree of response, depending upon the degree of tumor-associated HA (Figure 3) [13]. Tumor growth inhibition responses were 97% for HA⁺³ (LUM697), 44% for HA⁺² (LUM330), and 16% for HA⁺¹ (LUM858) NSCLC patient explants grown in nude mice. This suggests that in clinical trials testing stromal HA depletion using PEGPH20, pre-selection for an HA⁺³ phenotype may enhance the response rate in treated patients.

The exogenous administration of hyaluronidase has been shown to enzymatically degrade HA both in vitro and in vivo, removing HA pericellular matrices from cultured cancer cells [13,14,69] and depleting tumor HA in preclinical animal models [11,12,13,14,111].

In studies of PC3 prostate cancer xenograft tumors, administration of a single intravenous bolus dose of PEGPH20 effectively removed HA from the tumor stroma within 2 h following administration, compared with tumor-bearing mice treated with vehicle alone, and the HA removal was concomitant with the appearance of PEGPH20 in the tumor (Figure 4). Further, following enzymatic treatment with PEGPH20, tumor HA was essentially absent for 3 days, gradually appearing by Day 10 [14].

The degraded HA immediately has access to extracellular spaces and the circulatory system, including the lymphatics. The appearance of HA in the blood is a convenient pharmacodynamic marker for the hyaluronidase activity of PEGPH20 in animal models and patients (Figure 5), and can assist in monitoring HA degradation in vivo.

Recently, several papers have been published which shed light on the mechanism of antitumor activity mediated by the exogenous addition of PEGPH20 [11,12,13,14], and these provide additional rationale for HA-depleting reagents in the treatment of cancer. At the macrophysiological level, the hyaluronidase PEGPH20 induces massive structural changes in the tumor. These changes are translated into altered gene expression patterns. The most striking biomechanical impact of PEGPH20-mediated HA depletion from an HA⁺³ tumor is rapid loss of tIFP (Figure 6) [14]. Depending on dose and time, interstitial fluid pressures comparable to normal tissue can be achieved in vivo within 2 h of treatment. The reduction in tIFP leads to expansion of tumor blood vessels, and vascular re-perfusion [14]. Perhaps paradoxically, this increased tumor perfusion is associated with a decrease in DNA synthesis [13].

While there are numerous sequelae to these initial changes induced by PEGPH20 alone, it is now clear that these changes alone can result in inhibition of tumor cell DNA synthesis, and reduction in the expression of collagen (collagen 1-alpha-1 [Col1α1]; collagen 5-alpha-1 [Col5α1]) and tenascin-C (TNC) (Figure 7) [13], none of which directly interacts with HA. Thus, depletion of HA alone appears sufficient to induce broad remodeling of the tumor stroma. Depletion of HA, collagen and other stromal components from the ECM has raised the question of whether these changes can impact the metastatic behavior of the malignant cells resident within the stroma.

In addition to the xenograft models which provided initial proof of concept for the antitumor activity of PEGPH20, a novel model of pancreatic cancer using a genetically engineered mouse model, called the KPC (LSL-Kras^G12D/+; LSL-Trp53^R172H/+; Pdx-1-Cre) mouse has recently been employed [133]. The advantage of this model is that it develops autochthonous tumors that appear to mimic structural aspects and clinical symptoms of human PDA [134]. PDAs that occur in this model commonly accumulate HA and express high tIFP [11,12]. Treatment of KPC tumors with PEGPH20 depletes tumors of HA, leads to the expansion of intratumoral blood vessels, and, similarly to xenograft models [11,12,14], increases delivery of chemotherapy (doxorubicin, gemcitabine) to the tumor [11]. While similar effects have been reported by other agents that target stromal targets, e.g., the hedgehog pathway [134], the mechanism by which this occurs has not been defined. In the case of PEGPH20, the rapid expansion of blood vessels as HA is depleted leads to ultrastructural changes in tumor endothelium, characterized in part as a significant increase in endothelial fenestrae (Figure 8) [11]. No similar changes in the non-tumor-associated endothelium are observed [11], which suggests that increased fenestrae result from the abrupt change in vessel structure once tIFP is reduced and blood vessels rapidly expand.

As a single agent, hyaluronidases routinely show therapeutic efficacy in preclinical tumor xenografts that accumulate HA [13,14,111]. Shuster and co-workers reported that purified testicular hyaluronidase suppresses tumor growth of breast cancer xenografts [111], while removal of HA using PEGPH20 has been efficacious in mouse xenograft models of human prostate, breast, lung and pancreatic cancer and in human NSCLC explant models (see Figure 3) [11,13,14]. It is likely that this antitumor effect is related to the major structural alterations induced in the tumor (stroma, including vasculature) resulting from stromal remodeling, and subsequent changes in gene expression. These structural changes, especially vascular expansion and formation of fenestrae, which occur only in the impacted tumor stroma and not in normal tissues, are among the alterations observed in tumor vasculature following treatment with PEGPH20. This outcome allows for the possibility of synergistic antitumor activity of hyaluronidases and chemotherapy. Hyaluronidase has been shown to enhance antitumor activity of adriamycin and vinblastine in breast cancer and melanoma mouse models, respectively [106,135]. There are also early clinical studies reporting that co-treatment of hyaluronidase and chemotherapy or radiation therapy added efficacy of the treatment compared to monotherapy in Kaposi’s sarcoma and malignant pediatric tumors [103,136]. On the other hand, in a large randomized study of high-grade astrocytomas, hyaluronidase failed to show a synergstic effect with chemotherapy or radiation therapy upon survival, which was speculated to be due to the intrinsic resistance of high grade astrocytomas to chemotherapeutic agents [104].

Enhanced activity of chemotherapy by co-treatment with PEGPH20 was initially shown in transplantable mouse tumor models [14]. Synergistic activity with the combination of PEGPH20 with chemotherapy was also observed in the transgenic mouse model (KPC) of PDA (Table 1) [11,12]. In this model, PEGPH20 administration (4.5 mg/kg; every 3 days) had no significant effect compared to vehicle alone (~10 days); there was a 43% increase in survival time when animals were treated with gemcitabine (15 days) (administered 30 min post-PEGPH20), and an almost three-fold increase in survival time when animals were treated with a combination of PEGPH20 and gemcitabine (28.5 days) [11].

Given the observations that endogenous hyaluronidase expression can act as a tumor promoter (Section 9.1), we aimed to evaluate whether the enzymatic removal of tumoral HA from HA-rich tumors promotes or inhibits metastasis using the highly metastatic human hormone-refractory prostate cancer cell line PC3 [168] which expresses high levels of HA both in vitro and in vivo [14]. First, to assess in vitro whether PEGPH20-mediated removal of extracellular HA influences the attachment (adhesion) of cells to basement membrane, potentially by exposing previously HA-hidden cell adhesion receptors such as integrins, or similarly, the invasion of cells, we used established cell adhesion [169] and Matrigel™ cell invasion assays. PEGPH20 treatment, at a concentration previously shown to completely remove all extracellular HA (1,000 U/mL) [14], reduced cell adhesion by 29% compared to vehicle alone (Figure 9a, left panel); whereas the number of PC3 cells invading downward through the Matrigel toward the chemoattractant EGF was reduced by >50% in cells treated with PEGPH20 compared to vehicle (Figure 9a, right panel). These in vitro results indicate that HA depletion may result in anti-metastatic activity, as suggested by recent laminar flow assays which demonstrated that blockade of HA-CD44 interaction by CD44 antibody reduced tumor cell adhesion to HA in several CD44-expressing cancer cell lines [170].

Orthotopic implantation of human prostate cancer PC3 cells into nude mice was used to evaluate the effect of PEGPH20 on metastasis in vivo. This model has been shown to lead to the spontaneous formation of adjacent para-aortic lymph node (PLN) metastases [171], and is known to reproducibly generate PLN metastases in 100% of PC3 tumor-bearing mice within 43 days [168]. Accordingly, PC3 cells expressing the enhanced green fluorescence protein (GFP) were injected into the left lobe of prostates. When tumors reached approximately 100 mg (Day 25), the absence of PLN metastasis was confirmed in satellite animals and mice were staged into two treatment groups, vehicle control or PEGPH20. Although it is ideal to remove the primary tumor in most orthotopic spontaneous metastasis models, physical proximity of prostate tumors to the urethra and bladder makes this difficult in mice; additionally, Stephenson and colleagues [171] have previously demonstrated that orchiectomy has little effect on metastasis in this orthotopic model. Three doses of PEGPH20 (4.5 mg/kg; q3d × 3) inhibited tumor growth of the primary tumor by >59% within 9 days, in agreement with our earlier work which showed that higher doses of PEGPH20 reduced tumor growth in peritibially-implanted prostate PC3 cells by up to 70% by Day 27 [14]. At study conclusion (Day 34), primary tumor and metastasis to prostate-adjacent PLNs were evaluated (Figure 9b,c). Primary tumor growth was inhibited by >59% within 9 days (Figure 9b, left panel).

Metastasis occurred in 100% (15 of 15) of vehicle-treated animals; whereas only 62.5% (10 of 16) of the PEGPH20-treated animals were positive for PLN metastatic lesions (Figure 9b, right panel; Figure 9c). Similarly, in the KPC model of PDA, combination treatment of PEGPH20 and gemcitabine decreased metastatic tumor burden compared to gemcitabine monotherapy [12]. Taken together, these studies suggest that PEGPH20-mediated HA removal, following exogenous administration, reduces metastatic incidence. This is further supported by the finding that HA fragments, similar to degradation products of PEGPH20 (Figure 5), decrease metastasis to bone in a mouse bone metastasis model of breast cancer [160].

In recent years, with the increased recognition of the role of the tumor microenvironment in disease progression, stromal components of the tumor have emerged as attractive targets for therapeutic intervention. One such emerging target is the large glycosaminoglycan HA, a major constituent of the ECM of most tissues, and a molecule which accumulates in many solid tumors [41]. HA actively interacts with its binding molecules, the hyaladherins, which consist of ECM proteoglycans and glycoproteins, such as versican, and cell surface receptors, such as CD44 and RHAMM [29,77,80,172]. The outcome of these interactions, and the associated downstream effectors, the biological complexity of which can be graphically illustrated as a HA interactome, highlight HA’s complex role in tumor cell adhesion, motility, proliferation and invasion [26,42,43,72].

Accordingly, enzymatic depletion of HA can significantly impact tumor behavior by inducing a complete reorganization of the ECM, and in preclinical studies, intravenous administration of a pegylated hyaluronidase is associated with decreased tIFP, expansion of tumor blood vessels, increased delivery of chemotherapeutics, tumor growth suppression and improved survival [11,12,13,14]. The anti-tumor effects observed following the systemic administration of hyaluronidases to HA-overexpressing tumors appears to be in contrast to studies where local endogenous hyaluronidase, secreted within the tumor itself, acts predominantly as a tumor promoter [150,152]. These contradictory findings may be a hyaluronidase “concentration-dependent phenomenon”, with tumor growth inhibition only observed following treatment at the pharmacological levels seen with exogenous administration [150]. Similarly, HA fragments have been shown to both suppress and stimulate tumor progression, and this likely depends on the size and local concentration of the HA fragments in the tumor (i.e., the balance between high molecular weight HA and smaller HA fragments) [43,167] which can be regulated by HA synthases and/or the local tumor hyaluronidase concentration [150,167].

In the future, further knowledge about the biology of the HA interactome, as well as the tumor stroma as a whole, and, furthermore, a more complete definition of the important elements of the stroma that dictate aggressive malignancy, will enable new cancer therapies to emerge.