Hyaluronan Accumulation Research Articles

1931P - Hyaluronan (HA) accumulation in the tumor microenvironment (TME) is increased in colorectal cancer (CRC) and associated with consensus molecular subtypes (CMS) 4 molecular subtype

BackgroundHA, a secreted large, megadalton glycosaminoglycan, is a major component of the extracellular matrix. Its accumulation in the TME increases interstitial pressure, induces vascular collapse and hypoperfusion, and may be a potential barrier to therapeutic access and immune infiltration. Enzymatic breakdown of HA by PEGPH20 alters the TME. PEGPH20 + chemotherapy or checkpoint inhibitor combinations are currently in clinical development. We examined the prevalence profile of HA in a large CRC cohort and report the relationship between HA accumulation and CRC molecular subtypes. MethodsHA accumulation was quantified in 115 CRC tumor samples using the Ventana Class I CDx HA assay, which scores the % of stromal HA at any intensity greater than background over the entire tumor tissue surface area. RNAseq transcriptome analysis was carried out and CMSs were determined, implemented by using the CMScaller R package. ResultsCRC samples had increased HA accumulation (HA score 20%-90%) compared with normal colon tissue (HA score 20%). HA localized predominantly to stroma in CRC samples and to lamina propria in normal colon tissue. Increased HA accumulation in CRC tumors was accompanied by increased desmoplastic stroma. We classified the 115 CRC tumors into 4 CMSs with distinct underlying biology and clinical outcomes (table). The CMS4 subtype, which has the worst prognosis, had significantly higher HA content compared with the other CMS subtypes as determined by Tukey’s honest significance test (p=0.0052 CMS4 vs CMS1; p<0.0001 CMS4 vs CMS2 and CMS3).Table: 1931PCMS Type and OutcomesTable: 1931PCMS TypePercent (Out of 115 Total SamplesBiological Characteristics and Clinical OutcomesCMS118%- Highly immunogenic - HypermutatedCMS 239%- Wingless/integrated (WNT)-β-catenin pathway activation - Good prognosisCMS314%- Metabolic cancer phenotypeCMS429%- Strong stromal gene signature - Worst prognosis ConclusionsHA accumulation is increased in CRC pts, suggesting PEGPH20 may be a promising therapeutic agent in this indication. There are potential links between HA accumulation, CRC disease biology, and clinical outcomes. Investigation of HA accumulation in association with additional molecular and clinical features of CRC is ongoing. Editorial acknowledgementElizabeth L. Pham of Halozyme Therapeutics, Inc. provided editorial support for this abstract. Legal entity responsible for the studyHalozyme Therapeutics, Inc. and Genentech, Inc. FundingHalozyme Therapeutics, Inc. and Genentech Inc. DisclosureB. Blouw: Shareholder / Stockholder / Stock options, Full / Part-time employment: Halozyme Therapeutics, Inc.. L. Ryner: Shareholder / Stockholder / Stock options, Full / Part-time employment: Genentech, Inc.. R. Johnson: Shareholder / Stockholder / Stock options, Full / Part-time employment: Genentech, Inc.. D. Taverna: Shareholder / Stockholder / Stock options, Full / Part-time employment: Halozyme Therapeutics, Inc.. Y. Wang: Shareholder / Stockholder / Stock options, Full / Part-time employment: Genentech, Inc.

Contrast-enhanced ultrasound imaging of acute changes in pancreatic cancer following targeted hyaluronan treatment.

The purpose of this study was to monitor acute changes in pancreatic tumor perfusion with contrast-enhanced ultrasound (CEUS) imaging following targeted hyaluronan (HA) treatment. Intratumoral accumulation of HA is one of contributing factors that can lead to an increased tumor interstitial pressure (TIP). These elevated TIP levels can hinder delivery of chemotherapeutic drugs and cause treatment failure. For this study, pancreatic cancer-bearing mice were imaged at baseline and again at 2 h after intravenous administration of physiological saline (control group) or PEGPH20, which targets HA (therapy group). CEUS data were collected for 5 min and the temporal sequence was first analyzed using a singular value filter (SVF) to remove any background clutter signal. Given the time history of contrast agent flow, a tumor perfusion parametric analysis was performed. A series of morphological image operations was applied to quantify structural features of the tumor angiogenic network including vessel count, density, length, diameter, tortuosity, and branching points. After imaging, animals were euthanized, and tumors excised for histological processing. Acute microvascular changes were found at 2 h after drug administration as confirmed by CEUS imaging. Further, histologic analysis of tumor sections revealed lower HA accumulation in the therapy group animals. Overall, these findings suggest that CEUS imaging of acute changes in tumor perfusion may help identify an optimal window whereby follow-up chemotherapeutic drug dosing would be more effective.

Requirement of Hyaluronan Synthase-2 in Craniofacial and Palate Development

Cleft palate is a common major birth defect resulting from disruption of palatal shelf growth, elevation, or fusion during fetal palatogenesis. Whereas the molecular mechanism controlling palatal shelf elevation is not well understood, a prevailing hypothesis is that region-specific accumulation of hyaluronan, a predominant extracellular glycosaminoglycan in developing palatal mesenchyme, plays a major role in palatal shelf elevation. However, direct genetic evidence for a requirement of hyaluronan in palate development is still lacking. In this study, we show that Has2, 1 of 3 hyaluronan synthases in mammals, plays a major role in hyaluronan synthesis in the neural crest–derived craniofacial mesenchyme during palatogenesis in mice. We analyzed developmental defects caused by tissue-specific inactivation of Has2 throughout the cranial neural crest lineage or specifically in developing palatal or mandibular mesenchyme, respectively, using Wnt1-Cre, Osr2-Cre, and Hand2-Cre transgenic mice. Inactivation of Has2 either throughout the neural crest lineage or specifically in the developing palatal mesenchyme caused reduced palatal shelf size and increased palatal mesenchyme cell density prior to the time of normal palatal shelf elevation. Whereas both Has2f/f;Wnt1-Cre and Has2f/f;Osr2-Cre mutant mice exhibit cleft palate at complete penetrance, the Has2f/f; Wnt1-Cre fetuses showed dramatically reduced mandible size and complete failure of palatal shelf elevation, whereas Has2f/f;Osr2-Cre fetuses had normal mandibles and delayed palatal shelf elevation. All Has2f/f;Hand2-Cre pups showed reduced mandible size and about 50% of them had cleft palate with disruption of palatal shelf elevation. Results from explant culture assays indicate that disruption of palatal shelf elevation in Has2f/f;Hand2-Cre mutant fetuses resulted from physical obstruction by the malformed mandible and tongue. Together, these data indicate that hyaluronan plays a crucial intrinsic role in palatal shelf expansion and timely reorientation to the horizontal position above the tongue as well as an important role in mandibular morphogenesis that secondarily affects palatal shelf elevation.

Remodeling the Tumor Microenvironment Sensitizes Breast Tumors to Anti-Programmed Death-Ligand 1 Immunotherapy.

Immunotherapies targeting immune checkpoint inhibitors have changed the landscape of cancer treatment, however, many patients are resistant or refractory to immunotherapy. The sensitivity of tumor cells to immunotherapy may be influenced by hyaluronan (HA) accumulation in the tumor microenvironment (TME). Enzymatic degradation of HA by pegvorhyaluronidase alfa (PEGPH20; PVHA) remodels the TME. This leads to reduced tumor interstitial pressure and decompressed tumor blood vessels, which are both associated with increased exposure of tumor cells to chemotherapy drugs. Here, we demonstrate PVHA increased the uptake of anti-programmed death-ligand 1 (PD-L1) antibody in HA-accumulating animal models of breast cancer. The increased levels of anti-PD-L1 antibody were associated with increased accumulation of T cells and natural killer cells and decreased myeloid-derived suppressor cells. PD-L1 blockade significantly inhibited tumor growth when combined with PVHA, but not alone. Our results suggest that PVHA can sensitize HA-accumulating tumors to anti-PD-L1 immunotherapy. SIGNIFICANCE: These findings show removal of hyaluronan in the tumor microenvironment improves immune cells and checkpoint inhibitors access to tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4149/F1.large.jpg.

Abstract 823: The Extracellular Matrix Component, Hyaluronan, Provokes a Pro-Inflammatory Phenotype in Macrophages

Background: The extracellular matrix (ECM) provides structural and functional support for the myocardium, but myocardial infarction (MI) changes the composition of the ECM. One of the chief components of the ECM, hyaluronan (HA), is elevated after MI; however, specific biological actions of HA—particularly at the level of infiltrating immune cells and implications of such interactions on ventricular remodeling—have not been explored. Goals: Because upregulation of HA coincides with macrophage infiltration after MI, we determined whether hyaluronan interacts with macrophages and investigated the implication of such interactions on macrophage function. Methods: WT mice were subjected to non-reperfused MI to determine changes in hyaluronan synthases (HAS), hyaluronidases (HYAL), and HA levels in the heart. Interaction of HA with macrophages was studied by polarizing bone marrow derived macrophages and analyzing cells by flow cytometry. Next, we characterized the ability of macrophages to metabolize HA by profiling polarized macrophages for HA-metabolizing enzymes, HA receptors, HA-binding proteins, hyaluronidase activity, and phagocytosis. Results: Compared to Sham hearts, MI (n=5/group) augmented the expression of HAS-2 (10-fold, p=0.002) and HYAL-2 (2 to 4-fold, p=0.0004) in the infarct and remote regions of the heart at 5 d post-MI. HA levels (n=8/group) were elevated in the infarct (1 to 2-fold, p=0.0200) and remote (2 to 3-fold, p=0.0007) regions of the heart compared to sham hearts. Polarizing macrophages (n=3/group) in the presence fluorescein-conjugated HA (HA-FL) showed that naïve (M0), pro-inflammatory (M1), and pro-resolving (M2) macrophages interact with HA-FL; M1 showed the highest FITC intensity. Interestingly, exposing macrophages (n=5/group) to HA provoked an inflammatory phenotype, as reflected by enhanced expression of TNFα (4-fold, p=0.0001) and IL-1β (7-fold, p=0.0094) mRNA; HA also enhanced macrophage phagocytosis (0.5-fold, p= 0.0476). Conclusion: Hyaluronan is elevated following MI and can influence macrophage function. Because of the accumulation of hyaluronan and macrophages in the post-MI heart, macrophage-hyaluronan interactions may be a nexus regulating ventricular remodeling.

Peritumoral ADC values in breast cancer: region of interest selection, associations with hyaluronan intensity, and prognostic significance

ObjectivesWe aimed to evaluate the differences in peritumoral apparent diffusion coefficient (ADC) values by four different ROI selection methods and to validate the optimal method. Furthermore, we aimed to evaluate if the peritumor-tumor ADC ratios are correlated with axillary lymph node positivity and hyaluronan accumulation.MethodsAltogether, 22 breast cancer patients underwent 3.0-T breast MRI, histopathological evaluation, and hyaluronan assay. Paired t and Friedman tests were used to compare minimum, mean, and maximum values of tumoral and peritumoral ADC by four methods: (M1) band ROI, (M2) whole tumor surrounding ROI, (M3) clockwise multiple ROI, and (M4) visual assessment of ROI selection. Subsequently, peritumor/tumor ADC ratios were compared with hyaluronan levels and axillary lymph node status by the Mann-Whitney U test.ResultsNo statistically significant differences were found among the four ROI selection methods regarding minimum, mean, or maximum values of tumoral and peritumoral ADC. Visual assessment ROI measurements represented the less time-consuming evaluation method for the peritumoral area, and with sufficient accuracy. Peritumor/tumor ADC ratios obtained by all methods except the clockwise ROI (M3) showed a positive correlation with hyaluronan content (M1, p = 0.004; M2, p = 0.012; M3, p = 0.20; M4, p = 0.025) and lymph node metastasis (M1, p = 0.001; M2, p = 0.007; M3, p = 0.22; M4, p = 0.015), which are established factors for unfavorable prognosis.ConclusionsOur results suggest that the peritumor/tumor ADC ratio could be a readily applicable imaging index associated with axillary lymph node metastasis and extensive hyaluronan accumulation. It could be related to the biological aggressiveness of breast cancer and therefore might serve as an additional prognostic factor.Key Points• Out of four different ROI selection methods for peritumoral ADC evaluation, measurements based on visual assessment provided sufficient accuracy and were the less time-consuming method.• The peritumor/tumor ADC ratio can provide an easily applicable supplementary imaging index for breast cancer assessment.• A higher peritumor/tumor ADC ratio was associated with axillary lymph node metastasis and extensive hyaluronan accumulation and might serve as an additional prognostic factor.

Abstract 97: The effect of hyaluronan degradation on the uptake and cytotoxic activity of liposome-encapsulated doxorubicin in 3D tumor spheroid models

Abstract The accumulation of hyaluronan (HA) in the solid tumor microenvironment (TME) can be associated with poor prognosis in several human cancers. Accumulation of HA in the TME can increase interstitial pressure that compresses local blood vessels and impairs the access of anti-cancer therapy. In preclinical models, enzymatic degradation of HA by pegvorhyaluronidase alfa (PEGPH20; PVHA), a PEGylated form of recombinant human hyaluronidase PH20 (rHuPH20), is associated with decreased tumor interstitial pressure, improved tumor vascular perfusion, and increased access and anti-tumor efficacy of cytotoxic and immunotherapies. The functional effect of HA degradation on the TME has been mostly characterized in animal tumor models. Here, we describe the utilization of 3D tumor spheroid models to evaluate the effects of HA degradation on the cytotoxic effect of anti-cancer therapies. A number of commonly used human colorectal cancer (CRC) cell lines were assessed for HA accumulation and tumor spheroid morphology: HCT 116, HT-29 parental, and HT-29/HAS3 (HT-29 cells engineered to over-express hyaluronan synthase 3). CRC 3D tumor spheroid models were developed to evaluate the uptake and cytotoxic activity of liposome-encapsulated doxorubicin (DOX) using the IncuCyte® S3 Live-Cell Analysis System. The effect of HA degradation on DOX uptake and cytotoxic activity was initially tested with rHuPH20 treatment in all three models, and further evaluated with PVHA treatment in HT-29 parental and HT-29/HAS3 models. A dose-dependent uptake and cytotoxic activity of DOX was observed in HCT 116 tumor spheroids. HA degradation after rHuPH20 treatment enhanced DOX uptake by an average of 6.1% (up to 7.7%) and cytotoxicity by an average of 37.9% (up to 58.2%) over the 5-day duration of the experiment compared with DOX alone. Dose-dependent uptake and cytotoxic activity of DOX in HT-29/HAS3 tumor spheroids were enhanced with rHuPH20 treatment to a greater extent than in HT-29 parental tumor spheroids. Specifically, rHuPH20 treatment led to an average increase in DOX uptake of 9.2% (up to 14.6%) with HT-29/HAS3 spheroids and 2.6% (up to 5.1%) with HT-29 parental spheroids. The addition of rHuPH20 resulted in an average increase in DOX cytotoxicity of 101% (up to 227.6%) with HT-29/HAS3 spheroids and 24.9% (up to 63.9%) with HT-29 parental spheroids. Enhanced uptake and cytotoxic activity of DOX was also observed with PVHA treatment in HT-29 and HT-29/HAS3 models. These data corroborate our previous findings with in vivo tumor models, in which the combination of PVHA-mediated HA degradation and other anti-cancer therapies display enhanced anti-tumor activity compared with either agent alone. Further, 3D tumor spheroid models may be used to evaluate the effect of extracellular matrix remodeling such as HA degradation and serve as a bridge between 2D cell-based assays and in vivo tumor models. Citation Format: Feng Gao, Susan Zimmerman, Kelly Chen, Daniel C. Maneval, Chunmei Zhao. The effect of hyaluronan degradation on the uptake and cytotoxic activity of liposome-encapsulated doxorubicin in 3D tumor spheroid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 97.

Stromal hyaluronan accumulation is associated with low tumor grade and nodal metastases in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma is an aggressive malignancy characterized by abundant desmoplastic stroma. Hyaluronan is a prominent stromal component of pancreatic ductal adenocarcinoma and is associated with unique clinical-pathological profiles in other tumor types. The current study aimed to delineate clinical and pathological features associated with hyaluronan accumulation in pancreatic ductal adenocarcinoma using a novel hyaluronan-binding assay currently being used in a clinical trial targeting hyaluronan. Sixty-four formalin-fixed, paraffin-embedded samples of pancreatic ductal adenocarcinomas from 49 patients treated at a single tertiary care hospital were stained. Fifty-two percent of tumors had high levels of hyaluronan. High levels were associated with low tumor grade and lymph node metastases, novel associations not previously seen in pancreatic ductal adenocarcinoma. This study has elucidated a novel clinical-pathological profile in pancreatic ductal adenocarcinomas using a new assay, suggesting hyaluronan may act as a biomarker for a subset of pancreatic tumors that could be targeted by hyaluronan-degrading agents.

Cardiac fibroblast activation and hyaluronan synthesis in response to hyperglycemia and diet-induced insulin resistance

Diabetic patients are at a greater risk of heart failure due to diabetic cardiomyopathy and worsened outcome post-myocardial infarction. While the molecular mechanisms remain unclear, fibrosis and chronic inflammation are common characteristics of both conditions. Diabetes mellitus (types I and II) results in excessive hyaluronan (HA) deposition in vivo, and hyperglycemia stimulates HA synthesis for several cell types in vitro. HA-rich extracellular matrix contributes to fibrotic, hyperplastic and inflammatory disease progression. We hypothesized that excessive hyperglycemia-driven HA accumulation may contribute to pathological fibroblast activation and fibrotic remodelling in diabetic patients. Therefore, we analysed the impact of both hyperglycemia and diet-induced obesity and insulin resistance on HA matrix formation and cardiac fibroblast activation. Here we report that cardiac fibroblasts isolated from mice on a diabetogenic diet acquire pro-fibrotic gene expression without a concomitant increase in HA matrix deposition. Additionally, hyperglycemia alone does not stimulate HA synthesis or cardiac fibroblast activation in vitro, suggesting that the direct effect of hyperglycemia on fibroblasts is not the primary driver of fibrotic remodelling in cardiac diabetic maladaptation.

Plasma hyaluronan is a predictive marker for pegvorhyaluronidase alfa (PEGPH20; PVHA) response in a phase II study of pancreatic ductal adenocarcinoma (PDAC).

404 Background: Hyaluronan (HA) can be a constituent of the tumor microenvironment (TME). Accumulation of HA in the TME may result in elevated interstitial pressure, vascular compression, hypoxia, and reduced drug delivery and immune cell access. Pegvorhyaluronidase alfa (PEGPH20; PVHA) enzymatically degrades HA, potentially increasing access and antitumor efficacy of cytotoxic and immuno-therapies. Liquid biopsies may provide a non-invasive approach to predict therapeutic efficacy of PVHA. Methods: Total plasma HA was analyzed as a pharmacodynamic biomarker for PVHA activity in HALO 109-202 (NCT01839487), a Phase 2, open-label, two-stage randomized study of PVHA + nab-paclitaxel + gemcitabine (PAG) vs AG only, in previously untreated patients with Stage IV metastatic PDAC. HA was measured with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) disaccharide assay using baseline plasma samples from Stage 1 (n = 132) and Stage 2 (n = 128) of the study. Results: Lower baseline plasma HA values were significantly correlated with survival outcomes, predicting progression-free survival (PFS) benefit in PVHA-treated patients in Stages 1 and 2, and overall survival (OS) benefit in PVHA-treated patients in Stage 2 (Table). Conclusions: These results support further exploration of a liquid biopsy-based companion diagnostic (CDx) for predicting the therapeutic efficacy of PVHA. [Table: see text]

Low Molecular Mass Myocardial Hyaluronan in Human Hypertrophic Cardiomyopathy.

During the development of hypertrophic cardiomyopathy, the heart returns to fetal energy metabolism where cells utilize more glucose instead of fatty acids as a source of energy. Metabolism of glucose can increase synthesis of the extracellular glycosaminoglycan hyaluronan, which has been shown to be involved in the development of cardiac hypertrophy and fibrosis. The aim of this study was to investigate hyaluronan metabolism in cardiac tissue from patients with hypertrophic cardiomyopathy in relation to cardiac growth. NMR and qRT-PCR analysis of human cardiac tissue from hypertrophic cardiomyopathy patients and healthy control hearts showed dysregulated glucose and hyaluronan metabolism in the patients. Gas phase electrophoresis revealed a higher amount of low molecular mass hyaluronan and larger cardiomyocytes in cardiac tissue from patients with hypertrophic cardiomyopathy. Histochemistry showed high concentrations of hyaluronan around individual cardiomyocytes in hearts from hypertrophic cardiomyopathy patients. Experimentally, we could also observe accumulation of low molecular mass hyaluronan in cardiac hypertrophy in a rat model. In conclusion, the development of hypertrophic cardiomyopathy with increased glucose metabolism affected both hyaluronan molecular mass and amount. The process of regulating cardiomyocyte size seems to involve fragmentation of hyaluronan.

Increased Hyaluronan and TSG-6 in Association with Neuropathologic Changes of Alzheimer's Disease.

Little is known about the extracellular matrix (ECM) during progression of AD pathology. Brain ECM is abundant in hyaluronan (HA), a non-sulfated glycosaminoglycan synthesized by HA synthases (HAS) 1-3 in a high molecular weight (MW) form that is degraded into lower MW fragments. We hypothesized that pathologic severity of AD is associated with increases in HA and HA-associated ECM molecules. To test this hypothesis, we assessed HA accumulation and size; HA synthases (HAS) 1-3; and the HA-stabilizing hyaladherin, TSG-6 in parietal cortex samples from autopsied research subjects with not AD (CERAD = 0, Braak = 0- II, n = 12-21), intermediate AD (CERAD = 2, Braak = III-IV, n = 13-18), and high AD (CERAD = 3, Braak = V-VI, n = 32-40) neuropathologic change. By histochemistry, HA was associated with deposits of amyloid and tau, and was also found diffusely in brain parenchyma, with overall HA quantity (measured by ELSA) significantly greater in brains with high AD neuropathology. Mean HA MW was similar among the samples. HAS2 and TSG-6 mRNA expression, and TSG-6 protein levels were significantly increased in high AD and both molecules were present in vasculature, NeuN-positive neurons, and Iba1-positive microglia. These results did not change when accounting for gender, advanced age (≥ 90 years versus <90 years), or the clinical diagnosis of dementia. Collectively, our results indicate a positive correlation between HA accumulation and AD neuropathology, and suggest a possible role for HA synthesis and metabolism in AD progression.

Mitogen-activated Protein Kinase-activated Protein Kinase 2 Inhibition Attenuates Fibroblast Invasion and Severe Lung Fibrosis.

Severe pulmonary fibrosis such as idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix and fibroblast activation. Targeting fibroblast activation has contributed to the development of antifibrotic therapeutics for patients with IPF. Mitogen-activated protein kinase-activated protein kinase 2 (MK2), downstream in the transforming growth factor-β/p38 mitogen-activated protein kinase pathway, has been implicated in inflammatory and fibrosing diseases. Increased concentrations of activated MK2 were expressed in IPF lung and in the mouse bleomycin model of lung fibrosis. The aim of the present study was to determine the role and the mechanisms of MK2 in fibroblast invasion and lung fibrosis. Our results showed that an MK2 inhibitor (MMI-0100) was able to inhibit the invasive capacity of lung fibroblasts isolated from patients with IPF, as well as fibroblasts isolated from both wild-type mice and mice with overexpressing hyaluronan synthase 2 (HAS2) in the myofibroblast compartment. We previously showed that hyaluronan and HAS2 regulate fibroblast invasion and lung fibrosis in vivo. The results of the present study showed that MMI-0100 reduced transforming growth factor-β-induced hyaluronan production in human and mouse fibroblasts in vitro and that HAS2 mediated MK2 activation, suggesting a feed-forward loop in fibroblast activation. More importantly, MK2 inhibition attenuated hyaluronan accumulation and reduced collagen content in bleomycin-injured mouse lungs in vivo. Conditional deletion of MK2 in fibroblasts attenuated bleomycin-induced lung fibrosis. These data provide evidence that MK2 has a role in fibroblast invasion and fibrosis and may be a novel therapeutic target in pulmonary fibrosis.

Intracameral Delivery of Layer-by-Layer Coated siRNA Nanoparticles for Glaucoma Therapy.

Glaucoma is the second leading cause of blindness worldwide, often associated with elevated intraocular pressure. Connective tissue growth factor (CTGF) is a mediator of pathological effects in the trabecular meshwork (TM) and Schlemm's canal (SC). A novel, causative therapeutic concept which involves the intracameral delivery of small interfering RNA against CTGF is proposed. Layer-by-layer coated nanoparticles of 200-260 nm with a final layer of hyaluronan (HA) are developed. The HA-coating should provide the nanoparticles sufficient mobility in the extracellular matrix and allow for binding to TM and SC cells via CD44. By screening primary TM and SC cells in vitro, in vivo, and ex vivo, the validity of the concept is confirmed. CD44 expression is elevated in glaucomatous versus healthy cells by about two- to sixfold. CD44 is significantly involved in the cellular uptake of HA-coated nanoparticles. Ex vivo organ culture of porcine, murine, and human eyes demonstrates up to threefold higher accumulation of HA compared to control nanoparticles and much better penetration into the target tissue. Gene silencing in primary human TM cells results in a significant reduction of CTGF expression. Thus, HA-coated nanoparticles combined with RNA interference may provide a potential strategy for glaucoma therapy.

Parallel Accumulation of Tumor Hyaluronan, Collagen, and Other Drivers of Tumor Progression.

Purpose: The tumor microenvironment (TME) evolves to support tumor progression. One marker of more aggressive malignancy is hyaluronan (HA) accumulation. Here, we characterize biological and physical changes associated with HA-accumulating (HA-high) tumors.Experimental Design: We used immunohistochemistry, in vivo imaging of tumor pH, and microdialysis to characterize the TME of HA-high tumors, including tumor vascular structure, hypoxia, tumor perfusion by doxorubicin, pH, content of collagen. and smooth muscle actin (α-SMA). A novel method was developed to measure real-time tumor-associated soluble cytokines and growth factors. We also evaluated biopsies of murine and pancreatic cancer patients to investigate HA and collagen content, important contributors to drug resistance.Results: In immunodeficient and immunocompetent mice, increasing tumor HA content is accompanied by increasing collagen content, vascular collapse, hypoxia, and increased metastatic potential, as reflected by increased α-SMA. In vivo treatment of HA-high tumors with PEGylated recombinant human hyaluronidase (PEGPH20) dramatically reversed these changes and depleted stores of VEGF-A165, suggesting that PEGPH20 may also diminish the angiogenic potential of the TME. Finally, we observed in xenografts and in pancreatic cancer patients a coordinated increase in HA and collagen tumor content.Conclusions: The accumulation of HA in tumors is associated with high tIP, vascular collapse, hypoxia, and drug resistance. These findings may partially explain why more aggressive malignancy is observed in the HA-high phenotype. We have shown that degradation of HA by PEGPH20 partially reverses this phenotype and leads to depletion of tumor-associated VEGF-A165. These results encourage further clinical investigation of PEGPH20. Clin Cancer Res; 24(19); 4798-807. ©2018 AACR.

Highlights of This Issue

Sipuleucel-T is an FDA-approved autologous cellular immunotherapy with established efficacy in metastatic castration-resistant prostate cancer (mCRPC); elucidation of its mechanism of action continues to evolve. Peripheral cellular and humoral immune responses to PAP and the immunogen PA2024 demonstrated long-lived, antigen-specific immune responses to sipuleucel-T that correlate with overall survival (OS). The current study shows persistent antigen-specific peripheral CD4+ T-cell responses and the induction of antigen specific CD8+ T cells with potential lytic function following sipuleucel-T. Moreover, a positive correlation between OS and PAP- as well as PA2024-specific cytotoxic T lymphocytes (CTLs) phenotype was found. Sipuleucel-T–induced antigen-specific CTL activity could provide a biologic basis for the OS benefit observed in mCRPC.Radiotherapy is an integral component in the adjuvant treatment of breast cancer, but reliable biomarkers predictive of radiotherapy benefit have been lacking. In this work, Cui and colleagues developed gene expression signatures reflecting two distinct biological processes: intrinsic radiosensitivity and antitumor immunity. Both radiosensitivity and immunological signatures were shown to be predictive of radiotherapy benefit, and integration of the two further improved patient stratification. The integrated gene signatures may be used to select patients who are most likely to benefit from radiotherapy, or to identify patients who may not respond well to standard radiotherapy and will need alternative therapies.LncRNAs are promising diagnostic markers, prognostic factors and therapeutic targets in tumorigenesis. Bian and colleagues revealed that LncRNA—FEZF1-AS1 is frequently upregulated in colorectal cancer (CRC) and was associated with poor prognosis. The in vitro and in vivo functional assays demonstrated that FEZF1-AS1 promotes CRC cell growth and metastasis. Mechanistically, FEZF1-AS1 could bind and increase the stability of PKM2 protein, resulting in the activating of STAT3 signaling and the increasing of aerobic glycolysis. Targeting FEZF1-AS1/PKM2 signaling appears to be a promising treatment for CRC.The glycosaminoglycan hyaluronan (HA) accumulates in the tumor microenvironment (TME) of many solid tumors, with HA accumulation linked to aggressive malignancy. To further elucidate the biological and physical changes associated with tumor HA accumulation, Li and colleagues used two different methods to modulate HA in mouse tumor models. Experiments compared xenografts from parental cells with cells engineered to overexpress hyaluronan synthase. Additionally, a PEGylated hyaluronidase (PEGPH20) was used to enzymatically degrade HA. Higher tumor HA was accompanied by increased collagen, reduced perfusion, increased hypoxia, and increased α-SMA. PEGPH20 treatment reversed these changes and depleted tumor-associated VEGF-A165. Data support continued investigation of strategies to target HA in the TME.

Human recombinant hyaluronidase injections for upper limb muscle stiffness in individuals with cerebral injury: A case series

Introduction: Spasticity, muscle stiffness, and contracture cause severe disability after central nervous system injury. However, current treatment options for spasticity produce muscle weakness which can impede movement and do not directly address muscle stiffness. Here we propose that the accumulation of hyaluronan within muscles promotes the development of muscle stiffness and report that treatment with the enzyme hyaluronidase increases upper limb movement and reduces muscle stiffness without producing weakness.

Abstract B38: Increasing tumor-infiltrating CD8+ T cell response and checkpoint inhibitor efficacy by enzymatic reduction of tumor hyaluronan in a murine syngeneic pancreatic cancer model

Abstract Inducing an efficient CD8+ T-cell-mediated antitumor immune response at the tumor site is critical for successful immunotherapy. The trafficking and activation of these effector CD8+ T cells to tumors are often inhibited by various immunosuppressive mechanisms in the tumor microenvironment (TME). Hyaluronan (HA), a glycosaminoglycan that accumulates in the TME of many solid tumors, is associated with rapid tumor progression, poor prognosis and increased immunosuppression. We have previously demonstrated in preclinical models that enzymatic degradation of TME HA by intravenous PEGylated recombinant human hyaluronidase PH20 (PEGPH20) enhances anti-Programmed death-ligand 1 (anti-PD-L1) efficacy and increases access of immune cells to the tumor (Clift, AACR 2017 poster #641). In this study, we aimed to extend these findings to a murine syngeneic pancreatic cancer model to explore the pan-tumor potential of combining PEGPH20 and anti-PD-L1 to improve anti-tumor immune response. To test this hypothesis in a pancreatic tumor model, Pan02 murine pancreatic adenocarcinoma cells were engineered to over-express hyaluronan synthase-3 (Pan02/Has3). Peritibial Pan02/Has3 tumors accumulated 10-fold higher levels of HA (1668 ng/mg) compared with Pan02 parental tumors (166 ng/mg) as determined by HA ELISA. HA accumulating Pan02/Has3 tumors were treated with combinations of PEGPH20 and/or anti-PD-L1, and tumor growth was monitored. Treatment with PEGPH20 (1mg/kg, biweekly) resulted in nearly complete removal of HA at 24hrs, the time point at which anti-PD-L1 treatment (5mg/kg, biweekly) was initiated. Tumor burden growth was reduced by 50% with combination of PEGPH20 and anti-PD-L1 versus 21% with PEGPH20 alone (p&amp;lt;0.01) or 5% with anti-PD-L1 alone (p&amp;lt;0.01). To elucidate the underlying mechanism, tumor infiltrating immune cells were analyzed by flow cytometry. We observed a significant increase in the numbers of cytotoxic CD8+ tumor infiltrating lymphocytes (TILs) in tumors treated with PEGPH20 alone compared with the vehicle group. Similarly, tumors treated with PEGPH20 in combination with anti-PD-L1 contained higher numbers of CD8+ TILs, CD4+ TILs, and Natural killer (NK) cells, and had increased CD8:Treg ratios compared to anti-PD-L1 treatment alone. Taken together, our data suggest that tumor HA accumulation may act as a barrier to immune cell access in a mouse model of pancreatic cancer, and that enzymatic reduction of HA can facilitate access of CD8+ T cells and enhance anti-PD-L1 efficacy. These findings are consistent with prior studies and support the ongoing clinical evaluation of combining PEGPH20 with immune checkpoint inhibitors to improve anti-tumor efficacy in HA-accumulating tumors. Citation Format: Benjamin Thompson, Trevor Kimbler, Barbara Blouw, Renee Clift, Yujun Huang, Sanna Rosengren, Curtis B. Thompson, Jisook Lee. Increasing tumor-infiltrating CD8+ T cell response and checkpoint inhibitor efficacy by enzymatic reduction of tumor hyaluronan in a murine syngeneic pancreatic cancer model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B38.

Asthmatic bronchial epithelial cells promote the establishment of a Hyaluronan-enriched, leukocyte-adhesive extracellular matrix by lung fibroblasts

BackgroundAirway inflammation is a hallmark of asthma. Alterations in extracellular matrix (ECM) hyaluronan (HA) content have been shown to modulate the recruitment and retention of inflammatory cells. Bronchial epithelial cells (BECs) regulate the activity of human lung fibroblasts (HLFs); however, their contribution in regulating HLF production of HA in asthma is unknown. In this study, we tested the hypothesis that BECs from asthmatic children promote the generation of a pro-inflammatory, HA-enriched ECM by HLFs, which promotes the retention of leukocytes.MethodsBECs were obtained from well-characterized asthmatic and healthy children ages 6–18 years. HLFs were co-cultured with BECs for 96 h and samples were harvested for analysis of gene expression, synthesis and accumulation of HA, and subjected to a leukocyte adhesion assay with U937 monocytes.ResultsWe observed increased expression of HA synthases HAS2 and HAS3 in HLFs co-cultured with asthmatic BECs. Furthermore, we demonstrated greater total accumulation and increased synthesis of HA by HLFs co-cultured with asthmatic BECs compared to healthy BEC/HLF co-cultures. ECM generated by HLFs co-cultured with asthmatic BECs displayed increased HA-dependent adhesion of leukocytes in a separate in vitro binding assay.ConclusionsOur findings demonstrate that BEC regulation of HA production by HLFs is altered in asthma, which may in turn promote the establishment of a more leukocyte-permissive ECM promoting airway inflammation in this disease.

Accumulation of hyaluronan in reared Japanese eel Anguilla japonica during early ontogeny

Hyaluronan (HA) is a main component of the highly hydrated extracellular body matrix of the larval stage of eels (leptocephalus). To determine the physiological and nutritional significance of HA in artificially reared larvae of the Japanese eel Anguilla japonica, HA and water content were measured at various larval stages in captivity. HA accumulated in the larval body with growth and water content also increased in parallel with HA. Almost all the accumulated HA was consumed during metamorphosis, suggesting HA plays a significant role in eel growth and metamorphosis. However, the amounts of HA and water were significantly lower in reared than in wild-caught larvae at the young stage, indicating a poorer nutritional condition of reared larvae, probably due to the deficient performance of currently used artificial diets. HA accumulation in reared larvae, however, eventually attained a level sufficient for metamorphosis by 250 days or more, and the glass eels of reared larvae had the same nutritional condition as that of wild glass eels. These findings are discussed with respect to the development of more effective artificial diets for rearing eel larvae.