Vascularization predicts overall survival and risk of transformation in follicular lymphoma

STP-53912) and by the Fundacao para a Ciencia e Tecnologia (FCT BD13230/2003), Portugal.Follicular lymphoma patients display heterogeneous overall survival and variable risk of transformation. Recent studies have highlighted the role of the microenvironment. The contribution of microvessel density to follicular lymphoma survival remains controversial. We used a quantitative tumor mapping approach to determine whether the degree of vascularization correlated with outcome in a uniformly treated cohort. Whole-tissue sections of diagnostic biopsies from 84 cases were stained for CD34 and tumor-to-vessel-distance that encompassed 90% of the tumor (TVD90) was determined using image analysis. Twenty-one cases with lower TVD90 showed inferior overall survival (P=0.0001) and high risk of transformation (P=0.01). These cases significantly correlated with increased Lymphoma-Associated Macrophages (x(2)=0.025). In multivariate analysis macrophages content, IPI and TVD90 were independent predictors of overall survival (P=0.05, P=0.001 and P=0.01, respectively) and IPI and TVD90 predicted risk of transformation (P=0.008 and P=0.08, respectively). Increased angiogenesis is an independent marker of inferior survival and may promote transformation.publishersversionpublishe

SHIP Represses the Generation of Alternatively Activated Macrophages

SummaryWe recently reported that SHIP restrains LPS-induced classical (M1) activation of in vitro differentiated, bone marrow-derived macrophages (BMMΦs) and that SHIP upregulation is essential for endotoxin tolerance. Herein, we show that in vivo differentiated SHIP−/− peritoneal (PMΦs) and alveolar (AMΦs) macrophages, unlike their wild-type counterparts, are profoundly M2 skewed (alternatively activated), possessing constitutively high arginase I (ArgI) and Ym1 levels and impaired LPS-induced NO production. Consistent with this, SHIP−/− mice display M2-mediated lung pathology and enhanced tumor implant growth. Interestingly, BMMΦs from SHIP−/− mice do not display this M2 phenotype unless exposed to TGFβ within normal mouse plasma (MP) during in vitro differentiation. Our results suggest that SHIP functions in vivo to repress M2 skewing and that macrophage polarization can occur during differentiation in response to TGFβ if progenitors have elevated PIP3

The combination of gefitinib and RAD001 inhibits growth of HER2 overexpressing breast cancer cells and tumors irrespective of trastuzumab sensitivity

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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. <it>In vivo </it>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.</p> <p>Results</p> <p>The gefitinib and RAD001 combination inhibited cell growth <it>in vitro </it>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 <it>in vitro </it>with cell line dependent increases in cytotoxicity and cytostasis while treatment <it>in vivo </it>promoted cytostasis. The most striking and consistent therapeutic effect of the combination was increased inhibition of the mTOR pathway (<it>in vitro </it>and <it>in vivo</it>) and EGFR signaling <it>in vivo </it>relative to the single drugs.</p> <p>Conclusions</p> <p>The gefitinib and RAD001 combination provides effective control over growth of HER2 overexpressing cells and tumors irrespective of the TZ sensitivity status.</p

A Systems Approach for Tumor Pharmacokinetics

Recent advances in genome inspired target discovery, small molecule screens, development of biological and nanotechnology have led to the introduction of a myriad of new differently sized agents into the clinic. The differences in small and large molecule delivery are becoming increasingly important in combination therapies as well as the use of drugs that modify the physiology of tumors such as anti-angiogenic treatment. The complexity of targeting has led to the development of mathematical models to facilitate understanding, but unfortunately, these studies are often only applicable to a particular molecule, making pharmacokinetic comparisons difficult. Here we develop and describe a framework for categorizing primary pharmacokinetics of drugs in tumors. For modeling purposes, we define drugs not by their mechanism of action but rather their rate-limiting step of delivery. Our simulations account for variations in perfusion, vascularization, interstitial transport, and non-linear local binding and metabolism. Based on a comparison of the fundamental rates determining uptake, drugs were classified into four categories depending on whether uptake is limited by blood flow, extravasation, interstitial diffusion, or local binding and metabolism. Simulations comparing small molecule versus macromolecular drugs show a sharp difference in distribution, which has implications for multi-drug therapies. The tissue-level distribution differs widely in tumors for small molecules versus macromolecular biologic drugs, and this should be considered in the design of agents and treatments. An example using antibodies in mouse xenografts illustrates the different in vivo behavior. This type of transport analysis can be used to aid in model development, experimental data analysis, and imaging and therapeutic agent design.National Institutes of Health (U.S.) (grant T32 CA079443

Characterization of Three-Dimensional Tissue Cultures Using Electrical Impedance Spectroscopy

AbstractElectrical impedance spectroscopy was used to characterize the cell environment of multilayered cell cultures (MCCs), a culture system in which cells are grown on a permeable support membrane to form a thick disc of cells with tumor-like properties. Cultures were grown using SiHa tumor cells as well as V79 wild-type cells and V79/DOX cells cultivated to exhibit multidrug resistance. Electrical impedance measurements were made on MCCs over a frequency range of 0.1kHz to 1MHz. Data analysis using a simple electrical model for the cell environment yielded estimates for parameters related to the intra- and extracellular resistance and net membrane capacitance, which were then related to MCC thickness. The extracellular fraction and tortuosity of the MCCs were determined in separate experiments where the rate of diffusion and the equilibrium level of C14-inulin, which does not penetrate the cell membrane, was measured within MCCs. Impedance measurements predicted the barrier to diffusion posed by the extracellular space of MCCs to be roughly two times greater than that inferred from the C14-inulin experiments. However, the relative ranking of the three cell types used to grow MCCs was similar for the two methods. Results indicate that impedance spectroscopy is well suited for use in characterizing the MCC cell environment, offering a fast, nondestructive method for monitoring cell culture growth and integrity

Targeting the tumour vasculature: exploitation of low oxygenation and sensitivity to NOS inhibition by treatment with a hypoxic cytotoxin.

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

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