Jennifer H.E. Baker

Microregional Effects of Gemcitabine in HCT-116 Xenografts

To examine the tumor microregional effects after gemcitabine administration to mice, we mapped the location of proliferating and hypoxic cells relative to vasculature in human colon cancer xenografts. The S-phase marker bromodeoxyuridine was used as a surrogate of drug effect and administered 2 hours before tumor excision, whereas vessel position and perfusion were assessed via staining for CD31 and intravenous injection of carbocyanine, respectively. Hypoxia was detected using pimonidazole. Images of the four markers were overlaid to reveal the spatial relationship between proliferation, vasculature, and hypoxia and to examine the microregional effects. Within 1 day after administration of 240 mg/kg of gemcitabine, proliferation throughout the tumor was completely inhibited. Over time, a reemergence of dividing cells occurred in relation to the distance from vasculature. Microregional analysis revealed that cells located distal to vasculature commenced cycling sooner than cells located proximal to vasculature. A similar trend was seen after multiple doses of gemcitabine (40 mg/kg on days 1, 4, 7, and 10). The possibility that the effect of gemcitabine could be attributed to changes in oxygenation was discounted after examining the vessel perfusion and patterns of hypoxia. The effect of gemcitabine was examined in multilayered cell culture, and at doses <30 μmol/L, a gradient in proliferation between the exposed and unexposed sides was observed. We show a differential effect on cell proliferation in relation to vasculature and conclude that cells distal to blood vessels are less affected by gemcitabine probably because of limited penetration.

Direct Visualization of Heterogeneous Extravascular Distribution of Trastuzumab in Human Epidermal Growth Factor Receptor Type 2 Overexpressing Xenografts

Purpose: The high molecular weight and binding affinity of trastuzumab, a monoclonal antibody in use for treatment of breast cancers overexpressing human epidermal growth factor receptor type 2 (HER2), in combination with microenvironmental factors, may limit its distribution and efficacy. We assessed and mapped the distribution of systemically given, unlabeled trastuzumab at micrometer resolution in tumor xenografts using immunohistochemistry. Experimental Design: Mice bearing MDA-435/LCC6HER2 xenografts were given single doses of 4 or 20 mg/kg unlabeled trastuzumab with tumor harvest at various time points thereafter; bound trastuzumab was imaged directly in tumor cryosections using fluorescently tagged antihuman secondary antibodies. Combinations of additional markers, including HER2, 5-bromo-2-deoxyuridine, CD31, DioC7(3), desmin, and collagen IV were also mapped on the same tumor sections. Results: Distribution of trastuzumab in MDA-435/LCC6HER2 tumors is found to be heterogeneous, with tumor margins saturating more thoroughly in doses and times analyzed. Considerable intervessel heterogeneity is also seen. For example, in unsaturated tissues, there remain perfused vessels without any trastuzumab in addition to vessels with a few layers of positively stained perivascular cells, in addition to vessels with bound drug up to 150 μm away. This heterogeneity is independent of HER2 expression, microvessel density, and perfusion. A slightly greater proportion of vessels were associated with pericytes in sections with greater trastuzumab saturation, but this would not adequately account for observed heterogeneous trastuzumab distribution. Conclusions: Complete penetration of trastuzumab in tumor tissue was not seen in our study, leaving the possibility that inadequate distribution may represent a mechanism for resistance to trastuzumab.

Translational Activation of HIF1α by YB-1 Promotes Sarcoma Metastasis

Metastatic dissemination is the leading cause of death in cancer patients, which is particularly evident for high-risk sarcomas such as Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma. Previous research identified a crucial role for YB-1 in the epithelial-to-mesenchymal transition (EMT) and metastasis of epithelial malignancies. Based on clinical data and two distinct animal models, we now report that YB-1 is also a major metastatic driver in high-risk sarcomas. Our data establish YB-1 as a critical regulator of hypoxia-inducible factor 1α (HIF1α) expression in sarcoma cells. YB-1 enhances HIF1α protein expression by directly binding to and activating translation of HIF1A messages. This leads to HIF1α-mediated sarcoma cell invasion and enhanced metastatic capacity in vivo, highlighting a translationally regulated YB-1-HIF1α axis in sarcoma metastasis.

Metronomic gemcitabine suppresses tumour growth, improves perfusion, and reduces hypoxia in human pancreatic ductal adenocarcinoma.

Background:The current standard of care for pancreatic cancer is weekly gemcitabine administered for 3 of 4 weeks with a 1-week break between treatment cycles. Maximum tolerated dose (MTD)-driven regimens as such are often associated with toxicities. Recent studies demonstrated that frequent dosing of chemotherapeutic drugs at relatively lower doses in metronomic regimens also confers anti-tumour activity but with fewer side effects.Methods:Herein, we evaluated the anti-tumour efficacy of metronomic vs MTD gemcitabine, and investigated their effects on the tumour microenvironment in two human pancreatic cancer xenografts established from two different patients.Results:Metronomic and MTD gemcitabine significantly reduced tumour volume in both xenografts. However, Ktrans values were higher in metronomic gemcitabine-treated tumours than in their MTD-treated counterparts, suggesting better tissue perfusion in the former. These data were further supported by tumour-mapping studies showing prominent decreases in hypoxia after metronomic gemcitabine treatment. Metronomic gemcitabine also significantly increased apoptosis in cancer-associated fibroblasts and induced greater reductions in the tumour levels of multiple pro-angiogenic factors, including EGF, IL-1α, IL-8, ICAM-1, and VCAM-1.Conclusion:Metronomic dosing of gemcitabine is active in pancreatic cancer and is accompanied by pronounced changes in the tumour microenvironment.

Irinophore C, a Novel Nanoformulation of Irinotecan, Alters Tumor Vascular Function and Enhances the Distribution of 5-Fluorouracil and Doxorubicin

Purpose: To examine the antitumor effects of Irinophore C, a nanopharmaceutical formulation of irinotecan, on the tissue morphology and function of tumor vasculature in HT-29 human colorectal tumors. Experimental Design: Fluorescence microscopy was used to map and quantify changes in tissue density, tumor vasculature, hypoxia, and the distribution of Hoechst 33342, a perfusion marker, and the anticancer drug, doxorubicin. Noninvasive magnetic resonance imaging was used to quantify Ktrans, the volume transfer constant of a solute between the blood vessels and extracellular tissue compartment of the tumor, as a measure of vascular function. Following treatment with Irinophore C, 19F magnetic resonance spectroscopy was used to monitor the delivery of 5-fluorouracil (5-FU) to the tumor tissue, whereas scintigraphy was used to quantify the presence of bound [14C]5-FU. Results: Irinophore C decreased cell density (P = 8.42 × 10−5), the overall number of endothelial cells in the entire section (P = 0.014), tumor hypoxia (P = 5.32 × 10−9), and Ktrans (P = 0.050). However, treatment increased the ratio of endothelial cells to cell density (P = 0.00024) and the accumulation of Hoechst 33342 (P = 0.022), doxorubicin (P = 0.243 × 10−5), and 5-FU (P = 0.0002) in the tumor. Vascular endothelial growth factor and interleukin-8, two proangiogenic factors, were down-regulated, whereas the antiangiogenic factor TIMP-1 was up-regulated in Irinophore C-treated tumors. Conclusions: Irinophore C treatment improves the vascular function of the tumor, thereby reducing tumor hypoxia and increasing the delivery and accumulation of a second drug. Reducing hypoxia would enhance radiotherapy, whereas improving delivery of a second drug to the tumor should result in higher cell kill.

Hyperbranched Polyglycerols as Trimodal Imaging Agents: Design, Biocompatibility, and Tumor Uptake

Combining various imaging modalities often leads to complementary information and synergistic advantages. A trimodal long-circulating imaging agent tagged with radioactive, magnetic resonance, and fluorescence markers is able to combine the high sensitivity of SPECT with the high resolution of MRI over hours and days. The fluorescence marker helps to confirm the in vivo imaging information at the microscopic level, in the context of the tumor microenvironment. To make a trimodal long-circulating probe, high-molecular-weight hyperbranched polyglycerols (HPG) were modified with a suitable ligand for (111)In radiolabeling and Gd coordination, and additionally tagged with a fluorescent dye. The resulting radiopharmaceutical and contrast agent was nontoxic and hemocompatible. Measured radioactively, its total tumor uptake increased from 2.6% at 24 h to 7.3% at 72 h, which is twice the increase expected due to tumor growth in this time period. Both in vivo MRI and subsequent histological analyses of the same tumors confirmed maximum HPG accumulation at 3 days post injection. Furthermore, Gd-derivatized HPG has an excellent contrast enhancement on T1-weighted MRI at 10× lower molar concentrations than commercially available Galbumin. HPG derivatized with gadolinium, radioactivity, and fluorescence are thus long-circulating macromolecules with great potential for imaging of healthy and leaky blood vessels using overlapping multimodal approaches and for the passive targeting of tumors.

In vivo Evaluation of Mucoadhesive Nanoparticulate Docetaxel for Intravesical Treatment of Non–Muscle-Invasive Bladder Cancer

Purpose: The present work describes the development and in vitro and in vivo evaluation of a mucoadhesive nanoparticulate docetaxel (DTX) formulation for intravesical bladder cancer therapy. Experimental Design: Mucoadhesive formulations based on hyperbranched polyglycerols (HPG), hydrophobically derivatized with C8/C10 alkyl chains in the core and modified with methoxy-polyethylene glycol (MePEG) and amine groups in the shell (HPG-C8/10-MePEG-NH2) were synthesized and DTX was loaded into these by a solvent evaporation method. Both low-grade (RT4, MGHU3) and high-grade (UMUC3) human urothelial carcinoma cell lines were treated with various concentrations of DTX formulations in vitro. KU7 cells that stably express firefly luciferase (KU7-luc) were inoculated in female nude mice by intravesical instillation and quantified using bioluminescence imaging. Mice with established KU7-luc tumors were given a single intravesical instillation with PBS, Taxotere (DTX from Sanofi-aventis), and DTX-loaded HPG-C8/10-MePEG and/or HPG-C8/10-MePEG-NH2. Drug uptake was conducted using LC/MS-MS (liquid chromatography/tandem mass spectrometry) and tumor microenvironment and uptake of rhodamine labeled HPGs was assessed. Results:In vitro, all DTX formulations potently inhibited bladder cancer proliferation. However, in vivo, DTX-loaded HPG-C8/10-MePEG-NH2 (mucoadhesive DTX) was the most effective formulation to inhibit tumor growth in an orthotopic model of bladder cancer. Furthermore, mucoadhesive DTX significantly increased drug uptake in mouse bladder tissues. In addition, rhodamine labeled HPG-C8/10-MePEG-NH2 showed enhanced uptake of these nanoparticles in bladder tumor tissues. Conclusions: Our data show promising in vivo antitumor efficacy and provide preclinical proof of principle for the intravesical application of mucoadhesive nanoparticulate DTX formulation in the treatment of bladder cancer. Clin Cancer Res; 17(9); 2788–98. ©2011 AACR.

Targeting Quiescent Tumor Cells via Oxygen and IGF-I Supplementation

Conventional chemotherapy targets proliferating cancer cells, but most cells in solid tumors are not in a proliferative state. Thus, strategies to enable conventional chemotherapy to target noncycling cells may greatly increase tumor responsiveness. In this study, we used a 3-dimensional tissue culture system to assay diffusible factors that can limit proliferation in the context of the tumor microenvironment, with the goal of identifying targets to heighten proliferative capacity in this setting. We found that supraphysiologic levels of insulin or insulin-like growth factor I (IGF-I) in combination with oxygen supplementation were sufficient to initiate proliferation of quiescence cells in this system. At maximal induction with IGF-I, net tissue proliferation increased 3- to 4-fold in the system such that chemotherapy could trigger a 3- to 6-fold increase in cytotoxicity, compared with control conditions. These effects were confirmed in vivo in colon cancer xenograft models with demonstrations that IGF-I receptor stimulation was sufficient to generate a 45% increase in tumor cell proliferation, along with a 25% to 50% increase in chemotherapy-induced tumor growth delay. Although oxygen was a dominant factor limiting in vitro tumor cell proliferation, we found that oxygen supplementation via pure oxygen breathing at 1 or 2 atmospheres pressure (mimicking hyperbaric therapy) did not decrease hypoxia in the tumor xenograft mouse model and was insufficient to increase tumor proliferation. Thus, our findings pointed to IGF-I receptor stimulation as a rational strategy to successfully increase tumor responsiveness to cytotoxic chemotherapy.

Tissue uptake of docetaxel loaded hydrophobically derivatized hyperbranched polyglycerols and their effects on the morphology of the bladder urothelium.

Recently, we have reported that docetaxel (DTX) loaded, amine terminated hyperbranched polyglycerol (HPG-C(8/10)-MePEG-NH(2)) nanoparticles significantly increased drug uptake in mouse bladder tissues and was the most effective formulation to significantly inhibit tumor growth in an orthotopic model of bladder cancer. The objective of this study was to investigate the effects of HPG-C(8/10)-MePEG-NH(2) nanoparticles on bladder urothelial morphology and integrity, DTX uptake and permeability in bladder tissue and the extent of bladder urothelial recovery following exposure to, and then washout of, HPG-C(8/10)-MePEG-NH(2) nanoparticles. HPG-C(8/10)-MePEG-NH(2) nanoparticles significantly increased the uptake of DTX in both isolated pig bladder as well as in live mouse bladder tissues. Furthermore, HPG-C(8/10)-MePEG-NH(2) nanoparticles were demonstrated to increase the permeability of the urinary bladder wall by causing changes to the urothelial barrier function and morphology through opening of tight junctions and exfoliation of the superficial umbrella cells. These data suggest that exfoliation may be triggered by an apoptosis mechanism, which was followed by a rapid recovery of the urothelium within 24 h post-instillation of HPG-C(8/10)-MePEG-NH(2) nanoparticles. HPG-C(8/10)-MePEG-NH(2) nanoparticles cause significant but rapidly recoverable changes in the bladder urothelial morphology, which we believe may make them suitable for increasing drug permeability of bladder tissue and intravesical drug delivery.

Opposing Roles for CD34 in B16 Melanoma Tumor Growth Alter Early Stage Vasculature and Late Stage Immune Cell Infiltration

Tumor growth and metastasis are determined by the complex interplay of factors, including those intrinsic to tumor cells and extrinsic factors associated with the tumor microenvironment. Our previous work demonstrated key roles for CD34 in the maintenance of vascular integrity and eosinophil and mast cell homing. Since both of these functions affect tumor development, we characterized the effect of CD34 ablation on tumor growth using the B16F1 melanoma model. Intriguingly, we found that CD34 plays a biphasic role in tumor progression. In early growth, both subcutaneous-injected tumors and intravenous-injected lung metastases grew more slowly in Cd34−/− mice. This correlated with abnormal vessel morphology and increased vascular permeability in these mice. Bone marrow transplantation experiments confirmed that this reflects a non-hematopoietic function of CD34. At later stages, subcutaneous tumor growth was accelerated in Cd34−/− mice and surpassed growth in wildtype mice. Bone marrow chimera experiments demonstrated this difference was due to a hematopoietic function for CD34 and, correspondingly we found reduced intra-tumor mast cell numbers in Cd34−/− mice. In aggregate, our analysis reveals a novel role for CD34 in both early and late tumor growth and provides novel insights into the role of the tumor microenvironment in tumor progression.