Abstract Accumulation of Hyaluronan (HA), an important glycosaminoglycan of the extracellular matrix (ECM), has been detected in about 25% of human tumors, and 56% of breast cancers1-2. HA is produced by malignant and stromal cells and acts to cross-link ECM proteins, contributing to the desmoplastic phenotype. Aberrant accumulation of HA in tumors has been associated with more aggressive malignancy1-2. We have observed that >50% of human epidermal growth factor receptor 2 (HER2) triple-positive breast tumors have a high accumulation of HA (HA3+). Monoclonal antibodies (MAbs) have been developed successfully as part of the anticancer armamentarium. However, even in cases where the target of the antibody is stably and abundantly expressed, efficacy often falls below expectations. In this work, we present a novel HA-dependent ECM-mediated mechanism of resistance to antibody-dependent cell-mediated cytotoxicity (ADCC) and this resistance can be reduced by PEGPH20 (a pegylated human PH20) treatment. Human hyaluronan synthase 2 (HAS2)-overexpressing tumor cells formed an HA-rich pericellular matrix which can restrict human NK cells from accessing tumor cells. This HA-dependent ECM-mediated physical barrier in HAS2- overexpressing tumor cells contributed to resistance to trastuzumab/cetuximab-dependent ADCC. Removal of HA from ECM by PEGPH20 treatment sensitized HAS2-overexpressing tumor cells to trastuzumab/cetuximab-mediated ADCC in vitro. HAS2-overexpressing ovarian cancer cell SKOV3 showed more aggressive growth pattern (median survival on 42 days) compared to parental SKOV3 (median survival of 61 days) in an intra-peritoneal xenograft model. In the same model with HAS2-overexpressing SKOV3, PEGPH20 in combination with trastuzumab and NK cell treatment (to simulate ADCC in vivo) resulted in 70% tumor growth inhibition (TGI) in comparison to 40% TGI with trastuzumab and NK cell treatment, indicating that PEGPH20 enhanced in vivo ADCC by trastuzumab and NK cell. Our results demonstrated a mechanism of high HA-content pericellular matrix-mediated resistance to MAb-mediated ADCC. These results may help explain why tumors with high levels of HA (HA3+ phenotype) are more aggressive, and suggest that removal of HA by PEGPH20 treatment may be an effective combinatorial therapy together with anti-cancer MAbs. 1. Kultti A, Li X, Jiang P, Thompson C.B., Frost G.I., Shepard H.M. 2012. Therapeutic targeting of Hyaluronan in the tumor stroma. Cancers, 4:873-903; doi:10.3390/cancers4030873. 2. Sironen, R.K., Tammi, M., Tammi, R., Auvinen, P.K., Anttila, M., and Kosma, V.M. 2011. Hyaluronan in human malignancies. Exp. Cell. Res. 317:383-391. doi: 10.1016/j.yexcr.2010.11.017 Citation Format: Netai C. Singha, Tara Nekoroski, Susan Zimmerman, Chunmei Zhao, Ping Jiang, Robert Connor, Gregory I. Frost, Zhondong Huang, Michael H. Shepard. Hyaluronan-rich ECM contributes to resistance to antibody-dependent cell-mediated cytotoxicity in solid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4999. doi:10.1158/1538-7445.AM2013-4999
The synthesis and assembly of ribosomal subunits take place in the nucleolus. The nucleolus forms in the nucleus around the repeated ribosomal gene clusters and undergoes cyclic changes during the cell cycle. Although the nucleolus is easily visualized by light microscopy of cells in vitro, the nucleolus has not been imaged in cells in vivo. We report here development of a mouse model to visualize the nucleolus cycle of cancer cells in live mice. HT-1080 human fibrosarcoma cells were labeled in the nucleus with histone H2B-GFP and with retroviral RFP in the cytoplasm. The nucleolus was visualized by contrast to the fluorescence of GFP expressed in the nucleus. HT-1080 dual-color cells were seeded on the surface of a skin-flap of nude mice. The inside surface of the skin-flap was directly imaged with a laser scanning microscope 24 hours after seeding. The nucleoli of the cancer cells were clearly imaged in real-time. The appearance of the nucleoli changed dramatically during the cell cycle. During mitosis, the nucleolus disappeared. After mitosis, the nucleoli decreased in number and increased in size. The nucleolus appears to have a major role in cell cycle regulation. Nucleolar imaging could be used for more precise determination of cancer-cell position in the cell cycle in vivo.
Despite tremendous progress in cancer immunotherapy for solid tumors, clinical success of monoclonal antibody (mAb) therapy is often limited by poorly understood mechanisms associated with the tumor microenvironment (TME). Accumulation of hyaluronan (HA), a major component of the TME, occurs in many solid tumor types, and is associated with poor prognosis and treatment resistance in multiple malignancies. In this study, we describe that a physical barrier associated with high levels of HA (HA(high)) in the TME restricts antibody and immune cell access to tumors, suggesting a novel mechanism of in vivo resistance to mAb therapy. We determined that approximately 60% of HER2(3+) primary breast tumors and approximately 40% of EGFR(+) head and neck squamous cell carcinomas are HA(high), and hypothesized that HA(high) tumors may be refractory to mAb therapy. We found that the pericellular matrix produced by HA(high) tumor cells inhibited both natural killer (NK) immune cell access to tumor cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. Depletion of HA by PEGPH20, a pegylated recombinant human PH20 hyaluronidase, resulted in increased NK cell access to HA(high) tumor cells, and greatly enhanced trastuzumab- or cetuximab-dependent ADCC in vitro. Furthermore, PEGPH20 treatment enhanced trastuzumab and NK cell access to HA(high) tumors, resulting in enhanced trastuzumab- and NK cell-mediated tumor growth inhibition in vivo. These results suggest that HA(high) matrix in vivo may form a barrier inhibiting access of both mAb and NK cells, and that PEGPH20 treatment in combination with anticancer mAbs may be an effective adjunctive therapy for HA(high) tumors.
We previously developed PC-3 human prostate cancer cells expressing red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) linked to histone H2B expressed in the nucleus. We demonstrate in the present report the use of these dual-color cells for early detection of apoptosis in the presence of cancer chemotherapy agents.Induction of apoptosis was observed by real-time imaging of cytoplasmic and nuclear size and shape changes and nuclear fragmentation using fluorescence microscopy. Apoptosis was also detected by measuring DNA fragmentation. The cancer chemotherapy agents paclitaxel and vinblastine were used for induction of apoptosis.When the PC-3 dual-color cells were treated with paclitaxel or vinblastine, cytoplasmic and nuclear size and shape changes and nuclear fragmentation were observed by 24 hours. The paclitaxel-treated PC-3 dual-color cells exhibited ring-like structures formed by the fragmented nuclei, which could be brightly visualized by H2B-GFP fluorescence. Apoptosis was also detected by the dual-color PC-3 cells by 24 hours when treated with vinblastine. However, no nuclear ring-like structures were formed in the PC-3 cells by vinblastine treatment. In contrast, DNA fragmentation could not be observed in PC-3 cells until 48 hours after exposure to paclitaxel.Dual-color PC-3 cells can serve as a simple real-time early reporter of apoptosis and as a screen for novel cancer therapeutics or genotoxic agents. The dual-color cell real-time imaging assay is a more sensitive and earlier reporter for apoptosis than the DNA fragmentation assay.
Abstract In order to visualize nuclear-cytoplasmic dynamics during intravascular cancer cell-proliferation and extravasation, green fluorescent protein (GFP) was expressed in the cytoplasm of HT-1080 human fibrosarcoma cells, and red fluorescent protein (m-Cherry), linked to histone H2B, was expressed in the nucleus. Nuclear m-Cherry expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic GFP expression enabled visualization of nuclear-cytoplasmic ratios as well as simultaneous cell and nuclear shape changes. Thus, total cellular dynamics can be visualized in the living dual-color cells in real time. The cell cycle position of individual living cells was readily visualized by the nuclear-cytoplasmic ratio and nuclear morphology. Real-time induction of apoptosis was observed by nuclear size changes and progressive nuclear fragmentation. Intra- and extra-vascular mitotic cells were visualized by imaging after injection of the cancer cells in the epigastric cranials vein in an abdominal flap. After one hour, round and elongated cancer cells were observed in the vessel. Three hours after injection, invadopodia of the cancer cells was observed. Five hours after injection, dual-color cancer cells began to divide within the vessel. By 10 hours, some intra-vascular cancer cells underwent apoptosis. Deformed new blood vessels in the tumor were observed 10 days later. Extravascular cancer cells were dividing in the tumor at day 14. The subcellular in vivo imaging approach described here provides new visual targets for trafficking, extravasating and invading cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2687. doi:1538-7445.AM2012-2687
e15749 Background: Ventana Medical Systems, Inc. is developing in collaboration with Halozyme Therapeutics an affinity histochemical companion diagnostic to aid in selecting patients with pancreatic ductal adenocarcinoma (PDA) that may benefit from PEGPH20 based therapy. Methods: A recombinant hyaluronan (HA) binding probe was optimized for use with the VENTANA OptiView DAB IHC Detection Kit on the automated BenchMark ULTRA platform (HA CDx Assay). HA is a non-sulfated glycosaminoglycan polymer that often accumulates in extra-cellular tumor matrices. The HA CDx Assay was developed for use in formalin-fixed, paraffin-embedded tissue samples of PDA in a series of studies addressing sensitivity, specificity, robustness, and precision. PEGPH20 is a pegylated form of recombinant human PH20 hyaluronidase degrading tumor-associated HA with high affinity and selectivity. The scoring algorithm was defined using statistical analysis of clinical outcome data and HA CDx Assay staining pattern observed in a set of HALO109-202 Phase 2 clinical trials samples. Inter-reader precision was established by 3 pathologists evaluating 100 PDA samples across the range of HA expression levels. Results: The Ventana HA CDx Assay met all pre-defined acceptance criteria. PDA are considered HA-high when the HA CDx Assay staining in the tumor extra cellular matrix (ECM) is ≥ 50% of the tumor surface. Inter-reader precision in determining HA status resulted in agreement rates greater than 94%. Conclusions: These results highlight the robustness and reproducibility of the Ventana HA CDx Assay. In HALO 109-202, patients identified by the Ventana HA CDx Assay algorithm as HA high demonstrated clinically meaningful improvements when treated with nab-paclitaxel and gemcitabine (AG) + PEGPH20 compared to those treated with AG alone. The clinical utility of the HA CDx assay will be further validated in additional patients in subsequent PEGPH20 studies, including a planned Phase 3 study beginning in 2016.
322 Background: Pancreatic ductal adenocarcinoma (PDA) is characterized by marked stromal fibrosis and hyaluronan (HA) accumulation. Degradation of stromal HA using PEGylated recombinant human hyaluronidase (PEGPH20) in combination with anti-cancer therapeutics has demonstrated increased efficacy in preclinical models. This study used an exploratory prototype HA assay to assess HA and to explore the association of HA status with clinical and pathological variables. Methods: Sixty-four PDA samples from 49 patients treated with gemcitabine and nab-paclitaxel were stained for HA using a prototype histochemical binding assay. The tumor extracellular matrix staining for HA at any intensity above background as a proportion of the total tumor surface area was recorded. Cases were categorized as HA-high (HA score of ≥50%) or HA-low (HA score of <50%). Subgroup analyses were also performed in paired pre- and post-chemotherapy samples. Results: Twenty-six of 49 (53%) patients were determined to be HA-high. HA-high status was significantly associated with pN1 (positive node) status (p<0.001) and well/moderate differentiation (p<0.001). No correlation of HA status was observed with sex, race, primary tumor location, pT (tumor) stage, lymph-vascular invasion, pathologic stage, or initial CA19-9 levels. No trend in HA status was observed comparing pre- and post-chemotherapy specimens (n=5). Conclusions: HA status was significantly associated with nodal stage and grade.
Abstract Hyaluronan (HA), which accumulates in the tumor microenvironment of many solid tumors, is associated with tumor progression and negative clinical outcomes. Preclinical studies have demonstrated that PEGPH20-mediated HA removal from HA-rich xenograft tumors in mice decreases tumor interstitial fluid pressure and water content, resulting in decompression of tumor vasculature, increased tumor perfusion and enhanced chemotherapeutic activity. Accordingly, the HA degrading enzyme, pegylated recombinant human hyaluronidase PH20 (PEGPH20), is currently being evaluated in clinical trials with selected treatment regimens in several malignancies, including metastatic pancreatic adenocarcinoma, gastric cancer, breast cancer and non-small cell lung cancer. Advances in treatment for pediatric Wilms’ tumor patients have significantly increased survival, but salvage chemotherapy for relapsed pediatric patients remains challenging. To evaluate the role of HA in this disease, 19 Wilms’ tumor histological specimens were surveyed for HA status using a novel probe (Jadin 2014). 79% (15/19) of the samples stained strongly for HA when compared to non-tumor kidney tissue, prompting additional preclinical studies. In brief, a human Wilms’ tumor cell line, WT-CLS1, was transduced with hyaluronan synthase-3 (HAS3). The subsequent WT-CLS1/HAS3 cells produced more HA and WT-CLS1/HAS3 xenograft tumors grew significantly faster than parental WT-CLS1 tumors. To evaluate the efficacy of PEGPH20 in combination with Wilms’ tumor chemotherapy (CTX) combinations, WT-CLS1/HAS3 cells were inoculated adjacent to the tibial periosteum of nude mice and tumor growth was monitored via ultrasonography. When tumors reached ∼250 mm3, mice were staged into treatment groups: vehicle control, PEGPH20, vincristine (VIN) plus Dactinomycin (DACT), and PEGPH20+VIN+DACT at different dose/frequency combinations. Average tumor growth inhibition (TGI) and overall survival for PEGPH20+VIN+DACT-treated mice were superior to TGI and survival of VIN+DACT treatment alone. In separate studies WT-CLS1/HAS3 tumor-bearing mice were staged into three groups: vehicle control, low dose PEGPH20 (0.0375 mg/kg, iv) and high dose PEGPH20 (1 mg/kg, iv). Animals were administered the hypoxyprobe pimonidazole (60 mg/kg, ip) three hours prior to sacrifice, and whole tumors were removed and processed via immunofluorescence. High dose PEGPH20 reduced this measure of tumor hypoxia by 5% (p = 0.023). Taken together, these data demonstrate that HA accumulation in Wilms’ tumor is common, and treatment with PEGPH20 hyaluronidase can increase CTX efficacy and reduce tumor hypoxia in a preclinical model of this disease. These results support further studies with PEGPH20 in combination with chemotherapy in preclinical models of Wilms’ tumor and suggest investigation in this pediatric patient population may be warranted. Citation Format: Jessica Cowell, Susan J. Zimmerman, Mathieu Marrela, Ping Jiang, Peter J. Houghton, Michael J. LaBarre, Daniel C. Maneval, Curtis B. Thompson, Xiaoming Li. PEGPH20 increases the anticancer activity of standard chemotherapy combinations, vincristine (VIN) and D actinomycin (DACT), in a Wilms’ xenograft model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2463.
Extensive accumulation of the glycosaminoglycan hyaluronan is found in pancreatic cancer. The role of hyaluronan synthases 2 and 3 (HAS2, 3) was investigated in pancreatic cancer growth and the tumor microenvironment. Overexpression of HAS3 increased hyaluronan synthesis in BxPC-3 pancreatic cancer cells. In vivo, overexpression of HAS3 led to faster growing xenograft tumors with abundant extracellular hyaluronan accumulation. Treatment with pegylated human recombinant hyaluronidase (PEGPH20) removed extracellular hyaluronan and dramatically decreased the growth rate of BxPC-3 HAS3 tumors compared to parental tumors. PEGPH20 had a weaker effect on HAS2-overexpressing tumors which grew more slowly and contained both extracellular and intracellular hyaluronan. Accumulation of hyaluronan was associated with loss of plasma membrane E-cadherin and accumulation of cytoplasmic β -catenin, suggesting disruption of adherens junctions. PEGPH20 decreased the amount of nuclear hypoxia-related proteins and induced translocation of E-cadherin and β -catenin to the plasma membrane. Translocation of E-cadherin was also seen in tumors from a transgenic mouse model of pancreatic cancer and in a human non-small cell lung cancer sample from a patient treated with PEGPH20. In conclusion, hyaluronan accumulation by HAS3 favors pancreatic cancer growth, at least in part by decreasing epithelial cell adhesion, and PEGPH20 inhibits these changes and suppresses tumor growth.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)