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Supplementary Figures S1-S6 and Tables S1-S4

Supplementary Materials and Methods

Supplemental FIGS S4 - S5 - Summary of vascular density, vascular patentcy and tissue hypoxia measurements following camptothecin treatment.

Supplemental FIGS S1 & S2 - Whole tissue section images comparing drug extravasation in HCT116 and HT29 xenografts.

Supplemental FIG S3 - Whole tissue section images comparing irinotecan extravasation versus MRI based contrast agent localization.

Notch signaling is important for tumor angiogenesis induced by vascular endothelial growth factor A. Blockade of the Notch ligand Dll4 inhibits tumor growth in a paradoxical way. Dll4 inhibition increases endothelial cell sprouting, but vessels show reduced perfusion. The reason for this lack of perfusion is not currently understood. Here we report that inhibition of Notch signaling in endothelial cell using an inducible binary transgenic system limits VEGFA-driven tumor growth and causes endothelial dysfunction. Neither excessive endothelial cell sprouting nor defects of pericyte abundance accompanied the inhibition of tumor growth and functional vasculature. However, biochemical and functional analysis revealed that endothelial nitric oxide production is decreased by Notch inhibition. Treatment with the soluble guanylate cyclase activator BAY41-2272, a vasorelaxing agent that acts downstream of endothelial nitric oxide synthase (eNOS) by directly activating its soluble guanylyl cyclase receptor, rescued blood vessel function and tumor growth. We show that reduction in nitric oxide signaling is an early alteration induced by Notch inhibition and suggest that lack of functional vessels observed with Notch inhibition is secondary to inhibition of nitric oxide signaling. Coculture and tumor growth assays reveal that Notch-mediated nitric oxide production in endothelial cell requires VEGFA signaling. Together, our data support that eNOS inhibition is responsible for the tumor growth and vascular function defects induced by endothelial Notch inhibition. This study uncovers a novel mechanism of nitric oxide production in endothelial cells in tumors, with implications for understanding the peculiar character of tumor blood vessels. Cancer Res; 74(9); 2402–11. ©2014 AACR.

PDF file - 3357K, Figure S1. DnMAML-mediated EC-specific Notch blockade inhibits larger vessel function. Figure S2. Flow cytometry analysis of PDGFRβ in LLC-VEGFA tumors of control and double Tg. Figure S3. NO production in the microvasculature of LLC-VEGFA tumors in response to EC-specific Notch inhibition. Figure S4 Effect of the NO-independent soluble guanylyl cyclase agonist BAY41-2272 on tumor growth in EC-specific Notch-inhibited mice. Figure S3. NO production in the microvasculature of LLC-VEGFA tumors in response to EC-specific Notch inhibition. Figure S5. dnMAML-mediated EC Notch blockade inhibits growth, functional blood vessels and eNOS activation of B16F10-VEGFA tumors. Figure S6. Expression analysis of vasodilatory genes Nts and Adm in response to EC-specific Notch inhibition in tumors. Table S1. RT-PCR analysis of Ace2, Edn2, Agt and Ednra in bulk and sorted endothelial cells (EC) from LLC-VEGFA tumors grown in control and double Tg VE-cadherin-tTA x TetOS-dnMAML-GFP.

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Background: Preclinical drug development studies rarely consider the impact of a candidate drug on established metastatic disease. This may explain why agents that are successful in subcutaneous and even orthotopic preclinical models often fail to demonstrate efficacy in clinical trials. It is reasonable to anticipate that sites of metastasis will be phenotypically unique, as each tumor will have evolved heterogeneously with respect to gene expression as well as the associated phenotypic outcome of that expression. The objective for the studies described here was to gain an understanding of the tumor heterogeneity that exists in established metastatic disease and use this information to define a preclinical model that is more predictive of treatment outcome when testing novel drug candidates clinically. Methods: Female NCr nude mice were inoculated with fluorescent (mKate), Her2/neu-positive human breast cancer cells (JIMT-mKate), either in the mammary fat pad (orthotopic; OT) to replicate a primary tumor, or directly into the left ventricle (intracardiac; IC), where cells eventually localize in multiple sites to create a model of established metastasis. Tumor development was monitored by in vivo fluorescence imaging (IVFI). Subsequently, animals were sacrificed, and tumor tissues were isolated and imaged ex vivo. Tumors within organ tissues were further analyzed via multiplex immunohistochemistry (mIHC) for Her2/neu expression, blood vessels (CD31), as well as a nuclear marker (Hoechst) and fluorescence (mKate) expressed by the tumor cells. Results: Following IC injection, JIMT-1mKate cells consistently formed tumors in the lung, liver, brain, kidney, ovaries, and adrenal glands. Disseminated tumors were highly variable when assessing vessel density (CD31) and tumor marker expression (mkate, Her2/neu). Interestingly, tumors which developed within an organ did not adopt a vessel microarchitecture that mimicked the organ where growth occurred, nor did the vessel microarchitecture appear comparable to the primary tumor. Rather, metastatic lesions showed considerable variability, suggesting that each secondary tumor is a distinct disease entity from a microenvironmental perspective. Conclusions: The data indicate that more phenotypic heterogeneity in the tumor microenvironment exists in models of metastatic disease than has been previously appreciated, and this heterogeneity may better reflect the metastatic cancer in patients typically enrolled in early-stage Phase I/II clinical trials. Similar to the suggestion of others in the past, the use of models of established metastasis preclinically should be required as part of the anticancer drug candidate development process, and this may be particularly important for targeted therapeutics and/or nanotherapeutics.

PURPOSE There is a critical need for non-invasive imaging biomarkers of tumor oxygenation to assist in patient stratification and development of hypoxia targeting therapies. Using a cycling gas challenge and independent component analysis (ICA), we sought to improve the sensitivity and speed of existing oxygen enhanced MRI (OE-MRI) techniques to detect changes in oxygenation with dynamically acquired T1 W signal intensity images (dOE-MRI). METHODS Mice were implanted with SCCVII, HCT-116, BT-474, or SKOV3 tumors in the dorsal subcutaneous region and imaged at 7T. T1 W images were acquired during a respiratory challenge with alternating 2-minute periods of air and 100% oxygen for three cycles. Data were analyzed with ICA and oxygenation maps were generated and compared to corresponding histology sections stained for hypoxia (pimonidazole) and blood vessels (CD31). RESULTS Cycling air-oxygen-air gas challenges were well tolerated and ICA permitted extraction of the oxygen-enhancing component in all imaged tumors from four different models. Comparison with synthetic response functions showed that dOE-MRI does not require any a-priori knowledge of the physiological response. The fraction of O2 -negative dOE-MRI voxels that correlate inversely with the ICA gas-cycling component correspond well with the histological hypoxic fraction in SCCVII tumors (r = 0.91, p = 0.0016) but did not correlate in HCT-116 tumors (r = 0.13, p = 0.81). CONCLUSIONS Using ICA and adding a cycling gas challenge extends the sensitivity of OE-MRI and allows the oxygenation status of tumors to be assessed in as little as six minutes. These findings support further development of OE-MRI as a biomarker of tumor oxygenation.

Treatment strategies involving immune-checkpoint blockade (ICB) have significantly improved survival for a subset of patients across a broad spectrum of advanced solid cancers. Despite this, considerable room for improving response rates remains. The tumor microenvironment (TME) is a hurdle to immune function, as the altered metabolism-related acidic microenvironment of solid tumors decreases immune activity. Here, we determined that expression of the hypoxia-induced, cell-surface pH regulatory enzyme carbonic anhydrase IX (CAIX) is associated with worse overall survival in a cohort of 449 patients with melanoma. We found that targeting CAIX with the small-molecule SLC-0111 reduced glycolytic metabolism of tumor cells and extracellular acidification, resulting in increased immune cell killing. SLC-0111 treatment in combination with immune-checkpoint inhibitors led to the sensitization of tumors to ICB, which led to an enhanced Th1 response, decreased tumor growth, and reduced metastasis. We identified that increased expression of CA9 is associated with a reduced Th1 response in metastatic melanoma and basal-like breast cancer TCGA cohorts. These data suggest that targeting CAIX in the TME in combination with ICB is a potential therapeutic strategy for enhancing response and survival in patients with hypoxic solid malignancies.

Macromolecular gadolinium (Gd)-based contrast agents are in development as blood pool markers for MRI. HPG-GdF is a 583 kDa hyperbranched polyglycerol doubly tagged with Gd and Alexa 647 nm dye, making it both MR and histologically visible. In this study we examined the location of HPG-GdF in whole-tumor xenograft sections matched to in vivo DCE-MR images of both HPG-GdF and Gadovist. Despite its large size, we have shown that HPG-GdF extravasates from some tumor vessels and accumulates over time, but does not distribute beyond a few cell diameters from vessels. Fractional plasma volume (fPV) and apparent permeability-surface area product (aPS) parameters were derived from the MR concentration-time curves of HPG-GdF. Non-viable necrotic tumor tissue was excluded from the analysis by applying a novel bolus arrival time (BAT) algorithm to all voxels. aPS derived from HPG-GdF was the only MR parameter to identify a difference in vascular function between HCT116 and HT29 colorectal tumors. This study is the first to relate low and high molecular weight contrast agents with matched whole-tumor histological sections. These detailed comparisons identified tumor regions that appear distinct from each other using the HPG-GdF biomarkers related to perfusion and vessel leakiness, while Gadovist-imaged parameter measures in the same regions were unable to detect variation in vascular function. We have established HPG-GdF as a biocompatible multi-modal high molecular weight contrast agent with application for examining vascular function in both MR and histological modalities.

SummaryMetastatic 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.

Kulic et al. show that RBPJ, a transcriptional repressor of Notch, is frequently deleted in human cancers and can function as a tumor suppressor. Loss of RBPJ acts to derepress target gene promoters, allowing Notch-independent activation by alternate transcription factors that promote tumor growth., Aberrant Notch activity is oncogenic in several malignancies, but it is unclear how expression or function of downstream elements in the Notch pathway affects tumor growth. Transcriptional regulation by Notch is dependent on interaction with the DNA-binding transcriptional repressor, RBPJ, and consequent derepression or activation of associated gene promoters. We show here that RBPJ is frequently depleted in human tumors. Depletion of RBPJ in human cancer cell lines xenografted into immunodeficient mice resulted in activation of canonical Notch target genes, and accelerated tumor growth secondary to reduced cell death. Global analysis of activated regions of the genome, as defined by differential acetylation of histone H4 (H4ac), revealed that the cell death pathway was significantly dysregulated in RBPJ-depleted tumors. Analysis of transcription factor binding data identified several transcriptional activators that bind promoters with differential H4ac in RBPJ-depleted cells. Functional studies demonstrated that NF-κB and MYC were essential for survival of RBPJ-depleted cells. Thus, loss of RBPJ derepresses target gene promoters, allowing Notch-independent activation by alternate transcription factors that promote tumorigenesis.

The ability of a panel of camptothecin derivatives to access the tumor compartment was evaluated to determine the mechanisms by which the architecture of solid tumors may act to limit their activity. Microregional localization and activity of members of the camptothecin class of topoisomerase I targeting agents, including topotecan, irinotecan, and irinophore C, a lipid-based nanoparticulate formulation of irinotecan, were evaluated over time in HCT116 and HT29 colorectal tumor xenografts. Using native drug fluorescence, their distributions in tissue cryosections were related to the underlying tumor vasculature, tumor cell proliferation, and apoptosis. Topotecan exhibited a relatively uniform tumor distribution; in tissue 100 μm away from vessels, it reached 94% ± 5% of levels seen around blood vessels, whereas irinotecan and irinophore C were found to reach only 41% ± 10% and 5% ± 2%, respectively. Surprisingly, all three agents were able to initially inhibit proliferation uniformly throughout the tumors, and it was their rate of washout (topotecan > irinotecan > irinophore C) that correlated with activity. To explain this discrepancy, we looked at SN38, the active metabolite of irinotecan, and found it to penetrate tissue similarly to topotecan. Hence, the poor access to the tumor compartment of irinotecan and irinophore C could be offset by their systemic conversion to SN38. It was concluded that all three agents were effective at reaching tumor cells, and that despite the poor access to the extravascular compartment of irinophore C, its extended plasma exposure and systemic conversion to the diffusible metabolite SN38 enabled it to effectively target solid tumors. Mol Cancer Ther; 13(11); 2727–37. ©2014 AACR.

Multiplex Immunohistochemistry (mIHC) allows for the simultaneous detection of multiple targets of interest in a single tissue section. Using this method it is therefore possible to evaluate several effectors of a signal transduction pathway at one time, co-localization of markers, or distribution of molecules relative to one another within tissues. This method plays an important role in characterizing molecular signaling and the outcome of signal ablation when assessing mechanisms of drug action. Additionally, this approach can be used to characterize: the tumor microenvironment including vascular architecture and hypoxia, cellular proliferation, cell death as well as drug distribution. Medium to high-throughput images can be obtained via automation of slide imaging and analysis. Here we describe the basic principles of designing and implementing an mIHC protocol to create parameter tumor maps of a variety of specifically labeled markers.

Determining how a molecular targeting agent affects signaling pathways is challenging. To facilitate this exploration, we use a combination of medium-throughput methodologies such as tissue microarrays (TMA) and digital image analysis for quantification of immunohistochemistry (IHC). Because TMAs enable the simultaneous analysis of up to 1000 tissues, this tool can be used to comprehensively assess changes in signal transduction pathways over time. TMAs can be digitized and analyzed using the Aperio ScanScope imaging system and Aperio ImageScope software to deliver semiquantitative data. This chapter describes the methodologies for tissue collection and for the construction, sectioning, staining, and digital analysis of TMAs.

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. Cancer Res; 72(3); 801–9. ©2011 AACR.

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.

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.

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.

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.

We have previously reported that the hypoxic cytotoxin tirapazamine causes central vascular dysfunction in HCT-116 xenografts. Here we further extend this finding to SiHa xenografts and SCCVII murine tumors. Within 1 day after treatment with tirapazamine both tumor types develop areas of non-perfused tissue in central regions of tumors. To explore the mechanism by which the hypoxic cytotoxin tirapazamine causes vascular dysfunction we altered the blood oxygen content with carbogen (95% O 2 and 5% CO 2 ) breathing in tumor bearing mice. Carbogen treatment was able to decrease the number of tumors responding to tirapazamine but was not able to eradicate the vascular dysfunction completely. In complementary in vitro studies, immunohistochemical staining of tirapazamine-treated endothelial cells indicated that, unlike the vascular targeting agent (VTA) combretastatin-A-4-phosphate, the vascular effects caused by tirapazamine are not due to microtubule disruption. Another possible mechanism of action for tirapazamine could involve its ability to inhibit nitric oxide synthase (NOS). Studies combining other vascular targeting agents (VTAs) such as the combretastatins have shown a potentiation of vascular disruption in tumors when combined with NOS inhibitors, possibly due to vessel constriction from decreased nitric oxide (NO) levels. We propose the theory that vascular dysfunction caused by tirapazamine may be via NOS inhibition. In support of this hypothesis preliminary experiments showed NOS inhibition with l -NNA ( N -omega-nitro- l -arginine) increases tumor necrosis, 1 day after administration, in our HCT-116 tumor model.

// Svetlana Bortnik 1, 2 , Courtney Choutka 1, 3 , Hugo M. Horlings 4, 5, 6 , Samuel Leung 4, 5 , Jennifer H. Baker 7 , Chandra Lebovitz 1, 3 , Wieslawa H. Dragowska 8 , Nancy E. Go 1 , Marcel B. Bally 8, 9, 10, 11 , Andrew I. Minchinton 7 , Karen A. Gelmon 12, 13 , Sharon M. Gorski 1, 2, 3, 14 1 The Genome Sciences Centre, BC Cancer Agency, Vancouver, BC, Canada 2 Interdisciplinary Oncology Program, University of British Columbia, Vancouver, BC, Canada 3 Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada 4 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada 5 Vancouver General Hospital, BC Cancer Agency, Vancouver, BC, Canada 6 Department of Pathology, Netherlands Cancer Institute, Amsterdam, The Netherlands 7 Radiation Biology Unit - Department of Integrative Oncology, BC Cancer Agency, Vancouver, BC, Canada 8 Department of Experimental Therapeutics, BC Cancer Agency, Vancouver, BC, Canada 9 Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, Canada 10 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada 11 Centre for Drug Research and Development, Vancouver, BC, Canada 12 Medical Oncology, BC Cancer Agency, Vancouver, BC, Canada 13 Department of Medicine, University of British Columbia, Vancouver, BC, Canada 14 Centre for Cell Biology, Development, and Disease, Simon Fraser University, Burnaby, BC, Canada Correspondence to: Svetlana Bortnik, email: sbortnik@bcgsc.ca Keywords: breast cancer, autophagy, ATG4B, HER2, trastuzumab Received: March 20, 2016 Accepted: August 09, 2016 Published: August 19, 2016 ABSTRACT Autophagy, a lysosome-mediated degradation and recycling process, functions in advanced malignancies to promote cancer cell survival and contribute to cancer progression and drug resistance. While various autophagy inhibition strategies are under investigation for cancer treatment, corresponding patient selection criteria for these autophagy inhibitors need to be developed. Due to its central roles in the autophagy process, the cysteine protease ATG4B is one of the autophagy proteins being pursued as a potential therapeutic target. In this study, we investigated the expression of ATG4B in breast cancer, a heterogeneous disease comprised of several molecular subtypes. We examined a panel of breast cancer cell lines, xenograft tumors, and breast cancer patient specimens for the protein expression of ATG4B, and found a positive association between HER2 and ATG4B protein expression. We showed that HER2-positive cells, but not HER2-negative breast cancer cells, require ATG4B to survive under stress. In HER2-positive cells, cytoprotective autophagy was dependent on ATG4B under both starvation and HER2 inhibition conditions. Combined knockdown of ATG4B and HER2 by siRNA resulted in a significant decrease in cell viability, and the combination of ATG4B knockdown with trastuzumab resulted in a greater reduction in cell viability compared to trastuzumab treatment alone, in both trastuzumab-sensitive and -resistant HER2 overexpressing breast cancer cells. Together these results demonstrate a novel association of ATG4B positive expression with HER2 positive breast cancers and indicate that this subtype is suitable for emerging ATG4B inhibition strategies.

The study of angiogenesis as a therapeutic target requires reliable in vivo assays that can provide physiologically relevant data. A murine in vivo Matrigel-based angiogenesis assay is presented here which includes the quantitative assessment of vascular-specific indicators of neovascularization. Matrigel containing 175 ng/ml bFGF is encapsulated in synthetic chambers which are implanted subcutaneously in C57/B16J mice. Ex vivo implants can be imaged to qualitatively view perfused vasculature within the chambers, or histologically processed to confirm the presence of vascular-specific tissue within the Matrigel. Viable cells are recovered from the excised chambers and quantified cytometrically using endothelial cell-specific markers CD34 and CD144, and for a marker of nucleated cells, Hoechst 33342. Thalidomide, 200 mg/kg/day, was tested using the assay and was found to inhibit angiogenesis by 46%. Angiogenesis inhibitors secreted by LL/M27 tumors were also characterized, where tumor-bearing mice showed a 73% inhibition of angiogenesis compared to tumor-free controls. Analysis of the number of nucleated cells in these samples failed to show a strong correlation with the number of endothelial cells, indicating that quantification of nonvascular-specific tissue in in vivo angiogenesis assays may not be sufficient. This new assay provides an objective, comprehensive determination of the vasculature-specific response of both endogenous and exogenous angiogenesis inhibitors in vivo, and also creates new opportunities for obtaining primary murine endothelial cells.

Targeting tumor hypoxia continues to be a goal of translational radiation oncology owing to overwhelming preclinical and clinical validation of the importance of tumor oxygenation to radiation sensitivity. We describe a rational, structure-based drug development and characterization program for novel prodrugs that can target DNA-PK selectively within hypoxic tumor cells. The dual selectivity of these prodrugs has the potential to substantially improve the therapeutic index of radiation therapy used to treat numerous cancers. Using several biochemical, cell and novel tissue-based screening systems we explored the mechanistic and biologic activity of two new classes of DNA-PK inhibitors. Iterative improvements in molecular activity were driven by high-throughput assays utilizing both immunostaining and clonogenic survival endpoints. Kinase selectivity and in vitro ADME assays facilitated the identification of drug-like compounds with the potential for good oral bioavailability. 3D-tissue culture models helped explore tissue penetration and bystander effects and experiments analyzing the oxygen dependence of prodrug cleavage to the active DNA-PK inhibitors guided the selection of hypoxic trigger moieties. Novel parental compounds and hypoxia-activated prodrugs in two distinct chemical classes that are potent inhibitors of DNA-PK have been developed. Lead compounds exhibit high in vitro potency and good oral drug-like properties and show significant enhancement of radiation-induced cytotoxicity. In vivo experiments indicate excellent oral bioavailability with plasma T½‘s exceeding 30 minutes and significant antitumor activity against human xenografts. Citation Format: Alastair H. Kyle, Jennifer H.E. Baker, Karen H. Fryer, Judit Bana áth, Taixiang Wang, Soraya S. Porres, Sam E. Mann, George Hynd, Keith Menear, Andrew I. Minchinton. Hypoxia-selective DNA-PK inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A110.

Notch signaling is important for tumor angiogenesis induced by vascular endothelial growth factor A. Blockade of the Notch ligand Dll4 inhibits tumor growth in a paradoxical way. Dll4 inhibition increases endothelial cell sprouting, but vessels show reduced perfusion. The reason for this lack of perfusion is not currently understood. Here we report that inhibition of Notch signaling in endothelial cell using an inducible binary transgenic system limits VEGFA-driven tumor growth and causes endothelial dysfunction. Neither excessive endothelial cell sprouting nor defects of pericyte abundance accompanied the inhibition of tumor growth and functional vasculature. However, biochemical and functional analysis revealed that endothelial nitric oxide production is decreased by Notch inhibition. Treatment with the soluble guanylate cyclase activator BAY41-2272, a vasorelaxing agent that acts downstream of endothelial nitric oxide synthase (eNOS) by directly activating its soluble guanylyl cyclase receptor, rescued blood vessel function and tumor growth. We show that reduction in nitric oxide signaling is an early alteration induced by Notch inhibition and suggest that lack of functional vessels observed with Notch inhibition is secondary to inhibition of nitric oxide signaling. Coculture and tumor growth assays reveal that Notch-mediated nitric oxide production in endothelial cell requires VEGFA signaling. Together, our data support that eNOS inhibition is responsible for the tumor growth and vascular function defects induced by endothelial Notch inhibition. This study uncovers a novel mechanism of nitric oxide production in endothelial cells in tumors, with implications for understanding the peculiar character of tumor blood vessels. Cancer Res; 74(9); 2402–11. ©2014 AACR.

Many cancer research efforts focus on exploiting genetic-level features that may be targeted for therapy. Tissue-level features of the tumour microenvironment also represent useful therapeutic targets. Here we investigate the presence of low oxygen tension and sensitivity to NOS inhibition of tumour vasculature as potential tumour-specific features that may be targeted by hypoxic cytotoxins, a class of therapeutics currently under investigation. We have previously demonstrated that tirapazamine (TPZ) mediates central vascular dysfunction in tumours. TPZ is a hypoxic cytotoxin that is also a competitive inhibitor of NOS. Here we further investigated the vascular-targeting activity of TPZ by combining it with NOS inhibitor L-NNA, or with low oxygen content gas breathing. Tumours were analyzed via multiplex immunohistochemical staining that revealed irreversible loss of perfusion and enhanced tumour cell death when TPZ was combined with either low oxygen or a NOS inhibitor. Tumour growth rate was reduced by TPZ + NOS inhibition, and tumours previously resistant to TPZ-mediated vascular dysfunction were sensitized by low oxygen breathing. Additional mapping analysis suggests that tumours with reduced vascular-associated stroma may have greater sensitivity to these effects. These results indicate that poorly oxygenated tumour vessels, also being abnormally organized and with inadequate smooth muscle, may be successfully targeted for significant anti-cancer effects by inhibition of NOS and hypoxia-activated prodrug toxicity. This strategy illustrates a novel use of hypoxia-activated cytotoxic prodrugs as vascular targeting agents, and also represents a novel mechanism for targeting tumour vessels.

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.

Background HER2-positive breast cancers exhibit high rates of innate and acquired resistance to trastuzumab (TZ), a HER2-directed antibody used as a first line treatment for this disease. TZ resistance may in part be mediated by frequent co-expression of EGFR and by sustained activation of the mammalian target of rapamycin (mTOR) pathway. Here, we assessed feasibility of combining the EGFR inhibitor gefitinib and the mTOR inhibitor everolimus (RAD001) for treating HER2 overexpressing breast cancers with different sensitivity to TZ. Methods The gefitinib and RAD001 combination was broadly evaluated in TZ sensitive (SKBR3 and MCF7-HER2) and TZ resistant (JIMT-1) breast cancer models. The effects on cell growth were measured in cell based assays using the fixed molar ratio design and the median effect principle. In vivo studies were performed in Rag2M mice bearing established tumors. Analysis of cell cycle, changes in targeted signaling pathways and tumor characteristics were conducted to assess gefitinib and RAD001 interactions. Results The gefitinib and RAD001 combination inhibited cell growth in vitro in a synergistic fashion as defined by the Chou and Talalay median effect principle and increased tumor xenograft growth delay. The improvement in therapeutic efficacy by the combination was associated in vitro with cell line dependent increases in cytotoxicity and cytostasis while treatment in vivo promoted cytostasis. The most striking and consistent therapeutic effect of the combination was increased inhibition of the mTOR pathway (in vitro and in vivo) and EGFR signaling in vivo relative to the single drugs. Conclusions The gefitinib and RAD001 combination provides effective control over growth of HER2 overexpressing cells and tumors irrespective of the TZ sensitivity status.

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.

Purpose: To determine whether vascular-targeting effects can be detected in vivo using magnetic resonance imaging (MRI). Methods and Materials: MR images of HCT-116 xenograft-bearing mice were acquired at 7 Tesla before and 24 hours after intraperitoneal injections of tirapazamine. Quantitative dynamic contrast-enhanced MRI analyses were performed to evaluate changes in tumor perfusion using two biomarkers: the volume transfer constant (Ktrans) and the initial area under the concentration-time curve (IAUC). We used novel implanted fiducial markers to obtain cryosections that corresponded to MR image planes from excised tumors; quantitative immunohistochemical mapping of tumor vasculature, perfusion, and necrosis enabled correlative analysis between these and MR images. Results: Conventional histological analysis showed lower vascular perfusion or greater amounts of necrosis in the central regions of five of eight tirapazamine-treated tumors, with three treated tumors showing no vascular dysfunction response. MRI data reflected this result, and a striking decrease in both Ktrans and IAUC values was seen with the responsive tumors. Retrospective evaluation of pretreatment MRI parameters revealed that those tumors that did not respond to the vascular-targeting effects of tirapazamine had significantly higher pretreatment Ktrans and IAUC values. Conclusions: MRI-derived parameter maps showed good agreement with histological tumor mapping. MRI was found to be an effective tool for noninvasively monitoring and predicting tirapazamine-mediated central vascular dysfunction. © 2009 Elsevier Inc. All rights reserved.

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.

Despite significant success, the response of Her2+ patients to trastuzumab (TzMAb, Herceptin ®) is varied, with many still experiencing tumor progression. We have used 3D tissue, tumor xenograft and metastatic models to examine the microregional distribution of TzMAb when administered alone or in combination with bevacizumab (BvMAb). Methods: Her2 positive (Her2+) SKOV-3 ovarian and MDA361, JIMT-1, BT474 mammary cancer cells were grown as 3D tissue discs, spheroids and as xenografts. Her2 expression was ranked as SKOV3 > BT474 > JIMT-1 > MDA361. Variable concentrations (25-100 µg/mL for in vitro; 2.5-10 mg/kg ip q3d for in vivo) of TzMAb, BvMAb or isotype IgG control antibodies were administered and tissues collected. Multiplexed immunohistochemistry generated maps of whole tissue sections for quantitative and qualitative analysis. In addition to direct visualization of fluorescent-tagged antibody therapeutics, features including Her2 expression (Her2), hypoxia (pimonidazole), blood vessels (CD31), vascular perfusion (carbocyanine fluorescent dye), pericytes (SMA, desmin), basal lamina (CIV) and tight junctions (ZO-1) were mapped relative to each other. Findings: In vitro: The rate of TzMAb distribution through 3D Her2+ tissue models was not significantly different to that of BvMAb or an isotype control (IgG). Inter-model variability was not correlated with the degree of Her2 expression. In vivo: All the xenograft models exhibited highly heterogeneous distribution of TzMAb, with variation at inter-vessel, inter-tumor and intra-tumor levels. There was no discernible pattern in the deposition of TzMAb; it was not limited to tumor margins or the central core, and was often bound up to 200 µm away from the nearest blood vessels. Other areas containing perfused vessels had little to no binding. Areas of limited TzMAb binding persisted after repeat dosing. SKOV-3 and BT474 metastatic lesions collected from liver and lung tissues had on average greater TzMAb staining intensity than was seen in subcutaneous tumors. However, there was a significant range in the ability of TzMAb to access and bind Her2+ve cells. Lesions ranged from less than 150 µm to several mm in diameter, with some metastases showing bound TzMAb on every cell and others showing no observable bound drug despite containing perfused vessels or being surrounded by highly perfused normal lung or liver tissue. For both subcutaneous tumors and metastatic lesions, no consistent, quantifiable difference or correlation was found between the amount of bound TzMAb and the presence of tight junctions, the density of nearest CD31 vessels, the location of hypoxic cells or the fraction of perfused, mature or immature vessels. Pretreatment with BvMAb for 24-72h resulted in significant reductions (up to 90% reduced) in tumor accumulation of TzMAb in all tumor models examined, including lung and liver metastases, despite the continued presence of perfused vessels. Summary: The extravascular distribution of TzMAb in Her2-overexpressing xenografts and metastases is heterogeneous and is not explained simply by the presence or absence of functioning vasculature. The relatively good distribution of TzMAb through 3D tissue models suggest that tight or irreversible binding to Her2 may not adequately explain the limited distribution of TzMAb seen in vivo. Pre-treatment with BvMAb dramatically decreases the access of TzMAb to the tumor microenvironment. Persistence of metastases and tissues with poor access to TzMAb suggests the possibility of inadequate drug exposure as a mechanism for resistance to TzMAb activity that could apply to additional targeted monoclonal antibody therapeutics. This resistance may be exacerbated when TzMAb or other antibodies are administered in combination with anti VEGF therapy. Citation Format: Jennifer H.E. Baker, Alastair H. Kyle, Jordan Cran, Haley Patrick, Maria-Jose Gandolfo, Andrew I. Minchinton. Microenvironmental distribution of trastuzumab in metastases and xenograft models is highly heterogeneous and decreases sharply when administered in combination with bevacizumab. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B32.

s: AACR Special Conference: Advances in Breast Cancer; October 17-20, 2015; Bellevue, WA The gene and protein expression profile of a metastatic lesion can vary from the primary tumor. As a result, behavior of a secondary lesion can differ in the context of drug uptake and sensitivity as compared to the parent tumor. A better understanding of phenotypic variations that arise as a result of location of disease burden may be able to shed light on why widely used subcutaneous and orthotopic preclinical models fail to predict clinical success of drug candidates evaluated in the metastatic setting. These data can be used as a guide for preclinical study design in drug discovery. Our objective was to examine metastatic lesions from a wide range of organs in order to establish patterns of microarchitecture that may influence drug biodistribution and therefore drug efficacy in vivo. We hypothesize that metastatic lesions are phenotypically unique tumors with heterogeneous microenvironments which results in variable drug delivery and therefore drug efficacy. The investigations described compare tumor and vascular architecture of an orthotopic tumor with metastatic lesions. To study this, female NCr nude mice were inoculated with Her2/neu positive human breast cancer cells (JIMT-1) transfected with a fluorescence protein (mkate). Animals were inoculated either in the mammary fat pad (o.t.) to replicate a primary tumor, or directly into the left ventricle (i.c.) to establish systemic disease. Tumor development was monitored using in vivo fluorescence imaging (IVFI). Once metastases were established, animals were dosed with a custom formulated fluorescent labelled liposome (LipoDiR). The distribution of LipoDiR was imaged using IVFI. Subsequently, animals were sacrificed, mammary fat pad tumors and organs with metastatic lesions were excised and imaged ex vivo for mkate and LipoDiR. Tumors within organ tissues were further analyzed via multiplex immunohistochemical staining. Tumor sections were stained with antibodies against Her2/neu, smooth muscle actin (SMA), Collagen IV (CIV), a blood vessel marker (CD31), and a marker of hypoxia (CAIX) in order to examine the variability of tumor and vascular architecture. JIMT-1mKate cells proved to be a valuable Her2/neu positive cell line with the ability to form consistent systemic disease in the lung, liver, brain, kidney, ovaries, and adrenal glands after i.c. inoculation. LipoDiR, was successfully used to image distribution of nanoparticles in vivo, ex vivo and in tissue sections at the microscopic level. Although LipoDiR pharmacokinetics were comparable between the two models (o.t. and i.c.), orthotopic tumors showed a significant enhanced permeability and retention (EPR) effect while most organ tumors were unable to accumulate drug over a 24 hours. When comparing the tumors collected from the mammary fat pad to disseminated tumors, vessel density (CD31), vessel maturity (CIV), vessel perfusion (DiR), hypoxia (CAIX) and tumor marker expression (Her2/neu) were highly variable. Interestingly, tumors seeded within an organ did not take on the organ architecture, nor did they appear similar to the primary tumor. Rather, metastatic lesions showed considerable variability suggesting that each secondary tumor is a distinct disease entity. Our data suggests that more heterogeneity in tumor architecture and protein expression exists in metastatic lesions than has been previously appreciated. This variability is fairly profound even between tumors found in the same organ and leads to a significant impact on nanoparticle distribution. These studies highlight a need to investigate candidate drugs in multiple disease models that recapitulate the most aggressive disease. Not only will this improve the ability of preclinical studies to predict clinical success of experimental drugs, it will also help to concentrate research efforts on those drugs with the ability to engender better outcomes for patients with the most refractory disease. Citation Format: Jessica Kalra, Jennifer Baker, Alastair Kyle, Andrew Minchinton, Marcel Bally. Location of tumor burden influences tumor and vascular architecture, necrosis, and nanoparticle delivery. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B54.

Background and purpose Tirapazamine is a hypoxic cytotoxin currently undergoing Phase II/III clinical evaluation in combination with radiation and chemotherapeutics for the treatment of non-hematological cancers. Tissue penetration studies using multicellular models have suggested that tirapazamine exposure may be limited to cells close to blood vessels. However, animal studies show tirapazamine enhances the anti-tumour activity of radiation and chemotherapy and clinical studies with tirapazamine, so far, are promising. To investigate this apparent paradox we examined the microregional effects of tirapazamine in vivo by mapping drug effects with respect to the position of blood vessels in tumour cryosections. Patients and methods Tirapazamine was administered i.p. to mice bearing HCT-116 tumours, which were excised at various times after treatment. Images of multiple-stained cryosections were overlaid to provide microregional information on the relative position of proliferating cells, hypoxia, perfusion and vasculature. Results We observed extensive and permanent vascular dysfunction in a large proportion of tumours from mice treated with tirapazamine. In the affected tumours, blood flow ceased in the centrally located tumour vessels, leaving a rim of functional vessels around the periphery of the tumour. This vascular dysfunction commenced within 24 h after tirapazamine administration and the areas affected appeared to be replaced by necrosis over the following 24–48 h. Conclusions Because the majority of hypoxic cells are located in the center of tumours we propose that the activity of tirapazamine in vivo may be related to its effects on tumour vasculature and that its activity against hypoxic cells located distal to functional blood vessels may not be as important as previously believed.

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

The blood supply of solid tumours affects the outcome of treatment via its influence on the microenvironment of tumour cells and drug delivery. In addition, tumour blood vessels are an important target for cancer therapy. Intravital microscopy of tumours growing in 'window chambers' in animal models provides a means of directly investigating tumour angiogenesis and vascular response to treatment, in terms of both the morphology of blood vessel networks and the function of individual vessels. These techniques allow repeated measurements of the same tumour. Recently, multi-photon fluorescence microscopy techniques have been applied to these model systems to obtain 3D images of the tumour vasculature, whilst simultaneously avoiding some of the problems associated with the use of conventional fluorescence microscopy in living tissues. Here, we review the current status of this work and provide some examples of its use for studying the dynamics of tumour angiogenesis and vascular function.

Despite significant success, the response of Her2+ patients to trastuzumab (Herceptin ®) is varied, with many still experiencing tumour progression. In previous studies we have shown that trastuzumab has limited distribution in MDA-435-LCC6-Her2 over-expressing breast cancer xenografts. We have used biomarkers derived from MRI and multiplexed immunohistochemistry to examine the limited distribution of trastuzumab in the context of the tumour microenvironment. Methods: Endogenously over-expressing SKOV-3 ovarian carcinomas, MDA361, BT474 and vector-overexpressed Her2+ve MCF7 mammary (MCF7Her2) tumours were grown as xenografts in NOD/SCID mice in the sacral region, or were injected ip or iv to generate metastases in the peritoneal or lung compartments. Animals were administered 10 mg/kg trastuzumab with tissues harvested & frozen at 20-24h. MRI: Tumours were imaged for vascular function using DCE-MRI (Gadovist, 60mM), then treated with trastuzumab. Implanted fiducial markers enabled cryosections that closely approximated MR imaged slices to be obtained for comparison to tumour maps. Area Under the Curve (AUC) was calculated for the first 60 seconds post contrast agent injection. Tumour mapping: Multiplexed immunohistochemistry generated maps of whole tumour sections for quantitative and qualitative analysis. In addition to direct visualization of trastuzumab, features including Her2 expression (Her2), blood vessels (CD31), vascular perfusion (Dil18) and pericytes (SMA, desmin), basal lamina (CIV) and tight junctions (ZO-1) were stained and mapped relative to each other. Findings: All models exhibit highly heterogeneous distribution of trastuzumab, with variation at the inter-vessel, inter-tumour and intra-tumoural levels. Accumulation of trastuzumab is not limited to the tumour margins, though sections obtained on the distal ends of tumours have greater average amounts of bound drug. Trastuzumab is bound to tumour cells up to 200 µm away from the nearest perfused blood vessels in some areas, while other areas containing perfused vessels have little to no binding despite Her2 expression. The average amount of Her2+ve tissue positive for bound trastuzumab at 20-24h post administration is: MCF7-Her2 (68.6±7.2%), SKOV-3 (31.5±1.9%), BT474 (33.2%±1.6), MDA361 (21.5%+3.2). Small SKOV-3 metastatic lesions collected from the peritoneal cavity and lung tissues have an average of 68.5±3.5% of Her2+ve tissue bound for trastuzumab, although many have as little as 30%, and some very small lesions have no bound trastuzumab at all. For both the solid tumours and the metastatic lesions, no consistent, quantifiable difference or correlation was found between the amount of bound trastuzumab and the presence of greater ZO-1 labeled tight junctions, the density of nearest CD31 vessels, or the fraction of perfused, desmin +ve or SMA +ve vessels. Maps derived from DCE-MRI data showed that in some cases a high AUC value matched with high trastuzumab saturation, however these patterns were not consistent; in every tumour significant areas of mismatched high and low regions were observed. Summary: The highly heterogeneous microregional distribution of trastuzumab in these models appears to be independent of conventionally considered barriers to drug delivery, including ZO-1 tight junctions, microvessel density, blood flow and vascular maturity. The common occurrence of perfused vessels with no trastuzumab on the perivacular Her2+ve cells suggests that limited distribution is not a consequence of the binding site barrier hypothesis. The absence of a consistent pattern of greater accumulation of trastuzumab at the tumour margins suggests that interstitial fluid pressure (IFP) is not primarily responsible for the observed heterogeneity and limited distribution. These data suggest an as yet unidentified barrier may be responsible for inadequate access of trastuzumab to all Her2+ve cells. Citation Format: Jennifer Baker, Alastair H. Kyle, Firas Moosvi, Andrew I. Minchinton. Heterogeneous accumulation of trastuzumab in Her2+ve tumor and metastases models. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A12. doi:10.1158/1538-7445.CHTME14-A12

We have evaluated distribution and micro-regional activity of members of the camptothecin class of topoisomerase I targeting agents including topotecan (TPT), irinotecan (IRN), Irinophore C (IrC), a liposomal formulation of IRN and SN38, the active metabolite of IRN. Despite having a similar mechanism of action these agents are used against different cancers and possess significantly different pharmacological and physicochemical properties. The native fluorescence of each agent was used to visualize its distribution in HCT116 & HT29 tissue cryosections that were subsequently processed via multiplexed staining to relate drug exposure to its effect on tumor cell status (proliferation, apoptosis) and distance from vasculature. TPT exhibited the most uniform tumor distribution but it also experienced a significantly faster tumor wash-out while IRN was retained longer in tumors but exhibited a larger concentration gradient in relation to tumor blood vessels. IrC, the high molecular weight liposomal formulation, exhibited the poorest tissue distribution but it also displayed longer tumor retention due to its extended plasma half-life. At a depth of 100 µm away from vessels drug levels reached 94 ± 5 % (TPT), 41 ± 10 % (IRN) and 4.8 ± 2 % (IrC) of maximal levels seen near vessels. In terms of vessel permeability, IRN and IrC did not extravasate equally from all vessels, ∼50% of vessels showed little permeation by the two drugs and, because of this, some microregions of the tumors received much higher exposure than others. However, despite the significant differences in their tissue distribution, all 3 agents were able to exert a complete suppression of cycling tumor cells at 4 hours following treatment. At 24 hours following TPT or IRN tumor proliferation status returned to normal levels indicating that neither drug was retained long enough to exert activity on out of cycle cells. In contrast IrC benefited from an extended plasma exposure and was able to inhibit proliferation over a period of several days. The time to 50% recovery in proliferation was ∼3 days for TPT and IRN versus 14 days for IrC. Tumor growth delay studies showed that both TPT and IRN benefited somewhat from a dose splitting schedule designed to specifically target proliferation at peak levels two days in row. However, this strategy was still outperformed by IrC. Tumor growth delay following 50 mg/kg drug qwx2 was 9.6 ± 3 days (IRN) versus 27.8 ± 3.8 days (IrC). To determine the root cause of the discrepancy between the poor distribution but good microregional activity of IRN and IrC an in vitro 3D tissue-disc model was employed. It was found that IRN tissue penetration was limited by its high degree of cellular accumulation. SN38, the active metabolite of IRN, did not accumulate to the same extent and did penetrate tissue effectively. Hence it was concluded that the poor tissue distribution observed for both IRN and IrC was offset by systemic conversion to SN38, which was able to exert a more uniform activity within tumors. Citation Format: Alastair H. Kyle, Maria Jose Gandolfo, Jennifer H.E. Baker, Andrew I. Minchinton. Camptothecins: Tissue penetration and implications for therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 827. doi:10.1158/1538-7445.AM2014-827

We have previously shown that trastuzumab distributes heterogeneously within MDA-435-Her2 xenografts. Though peak accumulation occurs 24h after administration & trastuzumab was detected greater than 150 µm from many vessels, there remained substantial Her2+ve areas that were negative for bound drug. Methods: Her2+ve human carcinoma xenografts were grown in NOD/SCID mice as solid tumors or as metastatic models following ip or iv injection. Tumors were MR-imaged (T1-weighted) with a 500kDa Gd-labeled hyperbranched polyglycerol (HPG) contrast agent (CA). Treatments included bevacizumab (BvMAb, 10 mg/kg), trastuzumab (TsMab, 10 mg/kg) and human IgG (10 mg/kg). Multiplexed staining and imaging of whole tumor sections was used to evaluate the distribution of TsMAb in the context of the tumor microenvironment. Findings: BT474, MDA361 and SKOV3 primary tumor xenografts all show highly heterogeneous distribution of TsMAb at 24h, where some areas are highly saturated with TsMAb and others contain unbound Her2+ tissue. Small metastases in the peritoneal cavity and lung also exhibit substantial proportions of tissue that are negative for TsMAb. CD31-stained vessels dual-labeled for a perfusion marker are often seen to have little or no bound TsMAb on perivascular tumor cells. Pretreatment with BvMAb for 24-72h results in significant reductions in tumor accumulation of TsMAb, with tumors showing 60-90% of bound TsMAb relative to IgG-treated controls (p Summary: The extravascular distribution of TsMAb in Her2-overexpressing xenografts, including micrometastases, is heterogeneous and is not explained simply by the presence or absence of vascular function. Pre-treatment with BvMAb for even short exposures of 24-72h worsens the distribution of TsMAb within the tumor microenvironment. The persistence of tissues that are unbound for trastuzumab suggests the possibility of inadequate drug exposure as a mechanism for drug resistance that may be exacerbated by pre-treatment with a VEGF blockade. Citation Format: Jennifer H. E. Baker, Alastair H. Kyle, Stefan A. Reinsberg, Firas Moosvi, Jordan Cran, Urs Hafeli, Katayoun Saatchi, Andrew I. Minchinton. Microenvironmental distribution of trastuzumab is heterogeneous and decreases sharply when administered following a single dose of bevacizumab in Her2+ve xenografts and metastases models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2988. doi:10.1158/1538-7445.AM2014-2988

Background: The tumor microenvironment plays an important role in determining the activity of many anticancer drugs and one critically important factor that has recently garnered considerable interest is the micro-regional distribution of anticancer drugs within tumors. One of the key issues being whether chemotherapy drugs reach all the cells within a tumor at concentrations high and long enough to exert a therapeutic effect. Irinotecan and topotecan are two camptothecin analogues with differing pharmacological and physicochemical properties, both exhibit clinical activity though are not curative. In this study the micro-regional distribution of the two analogues was compared to their pattern of activity with tumor cryosections. Methods: Using whole tissue section mapping and multiplexed immunostaining techniques irinotecan and topotecan fluorescence was detected in relation to tumor vasculature in cryosections from HCT116 xenografts 1, 2, 4, 8 & 24 hours following their administration. The tissue sections were subsequently stained to map drug activity via apoptosis and proliferation status at early (1–24 hours) and late (1–14 days) times after treatment. Results: Topotecan exhibited a faster and more uniform tissue distribution than irinotecan but experienced significant wash-out within the first 2 hours following treatment. Irinotecan exhibited a larger concentration gradient in relation to tumor blood vessels but appeared to benefit from a longer retention within the tissue, with significant exposure occurring out to 8–24 hours following treatment. Interestingly, irinotecan did not appear to permeate equally from all vessels within the tumors. Some areas of the tumors received much higher exposure than others. At 50 mg/kg irinotecan and 10 mg/kg topotecan, both agents were able to exert a uniform block of cycling tumor cells within 4 hours of treatment but by 24 hours tumor proliferation status had returned to near normal levels indicating neither drug was retained long enough to exert activity on cells that were out of cycle at time of treatment. Conclusions: Development of new camptothecin analogues might benefit from design strategies to increase tumor retention and uniformity in tissue distribution. While both irinotecan and topotecan were able to exert a significant and uniform cell cycle block within 4 hours of treatment in the HCT116 xenografts, due to their short retention proliferation was seen to recover by 24 hours. Irinotecan appeared to distribute less uniformly within the tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C210.

Overview: Quiescent tumor cells in solid cancers contribute significantly to treatment failure of conventional anti-proliferative based therapies. The hypothesis of this study is that transiently increasing the proliferative fraction of a tumor will increase response to chemotherapy and result in a net benefit to overall survival. Initial work was carried out using an in vitro engineered-tissue assay to develop and test strategies to induce proliferation. Using this assay a combination of growth factor stimulation and O2 supplementation was found to override tumor-derived quiescence and enhance response to a panel of chemotherapy drugs. Background: The microenvironment in solid tumors limits the efficacy of anti-proliferative therapies by generating a quiescent tumor cell subpopulation through diffusion-limited supply of nutrients supplied by the blood. Recent findings in our laboratory have shown that the distribution of proliferating cells in solid tumors can be modified if the limiting factors controlling it are understood. Quiescence in tissues grown from two human colorectal cancer cell lines was shown to be driven by oxygen and growth factor deprivation. The combined supplementation of these two factors was found to reverse tumor microenvironment derived quiescence. Methods: An in vitro engineered-tissue model grown from HCT116 & HT29 cells was used to study the interrelation of key factors involved in limiting tumor cell proliferation. The model was able to replicate gradients in diffusible factors and reproduce quiescence seen in solid tumors. Findings were validated in tumor xenografts using a technique in which proliferation was mapped in relation to tumor vasculature. Strategies to stimulate proliferation in combination with treatment were then tested against a panel of drugs with cell survival as the endpoint (doxorubicin, paclitaxel, vinorelbine, gemcitabine & SN38). Results: Stimulation of the IGF-1 receptor in combination with O2 supplementation in the engineered-tissue model was able to initiate proliferation in quiescent areas with maximal induction occurring after 16–24 hours, tissue proliferation increased 3±0.4-fold (HCT116) and 4±1-fold (HT29) as measured using a BrdUrd endpoint. Chemotherapy given at the time of maximal induction induced a 3 to 6-fold increase in cell kill when compared to tissue grown under normal physiological conditions. Findings showed that combined stimulation always yielded a greater effect than individual stimulation via IGF-1R or oxygen alone. Cell kill was also greater when chemotherapy was given 16 hours following initiation of stimulation compared to when given simultaneously. Typical results: 1µM gemcitabine with no induction SF=0.71±0.08, with IGF-1R stimulation SF=0.25±0.03, with O2 supplementation SF=0.47±0.04, with combined induction 12 hours prior SF=0.10±0.03, with combined induction at time SF=0.43±0.03 (mean ± SD, n=8). Conclusions: Findings from this study indicate that transient stimulation of quiescent tumor cells represents a promising target for improving the activity of most standard chemotherapy regimes. The in vitro engineered-tissue model was found to be a powerful tool to study diffusion limited supply of molecules in solid tumors. Initial results using the engineered-tissue model have been validated in tumor xenografts and further work to translate these findings is underway. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):PR-7.

Background: Tirapazamine (TPZ; SR 4233; 3-amino-1,2,4-benzotriazine-1,4-di-N-oxide) is a Phase II/III bioreductive anti-cancer agent with greater toxicity to hypoxic vs oxygenated cells in vitro. We have previously reported that TPZ is able to mediate dose-dependent, irreversible central vascular dysfunction in vivo, leaving a hallmark viable rim of surviving peripheral vessels as seen with other vascular targeting agents (VTAs). Purpose: To explore how this agent, which is a bioreductive cytotoxin in vitro, is able to mediate catastrophic damage to tumor endothelium, likely the most oxygenated cell population in solid tumors, we have investigated the role of the tumor microenvironment including tumor hypoxia and vascular function. Methods: Vascular dysfunction may be qualitatively and quantitatively assessed using multiplex immunohistochemistry-based staining of frozen tumor sections for vasculature, perfusion, hypoxia, apoptosis and proliferation. Additional features of the microenvironment such as vascular permeability, architecture and support cells were also assessed using this tumor-mapping approach. Modification of tumor hypoxia was achieved via induction of mild anemia or low oxygen content gas breathing of tumor-bearing mice, and vascular function was increased by over-expression of VEGF in tumor models or systemic administration of nitric oxide (NO), or decreased via inhibition of nitric oxide synthase (NOS). Results: DCE-MRI studies correlated with tumor mapping data have shown that greater pre-treatment perfusion in HCT116 colorectal xenografts predicts for decreased sensitivity to the anti-vascular effects of TPZ. In contrast to observations using DCE-MRI, increasing tumor perfusion and vascular function caused an increase in tumor vessel sensitivity to TPZ, increasing both the magnitude and frequency of response in tumors. Both NOS inhibition and excess NO availability were able to potentiate anti-cancer effects of TPZ through enhancement of its vascular damage. Increasing tumor hypoxia by decreasing blood oxygenation was also found to potentiate the anti-vascular effects of TPZ, with otherwise resistant HT29 colorectal xenograft tumors showing a strong vascular dysfunction effect. Vascular architecture and tumor microenvironmental features were mapped in sensitive HCT116 and resistant HT29 colorectal xenograft models, with vascular maturity, permeability, microregional location of proliferating cells and hypoxia all identified as distinct in the two models. Summary: This work demonstrates that the vascular targeting effects of TPZ are potentiated by both hypoxia and vascular destabilization, and emphasizes the importance of specifically investigating the activity of anti-cancer agents in the context of the tumor microenvironment in vivo. This work was funded by a grant from CIHR & Jennifer Baker is a recipient of a graduate studentship from Michael Smith Foundation for Health Research. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C248.