We recently reported that SHIP restrains LPS-induced classical (M1) activation of in vitro differentiated, bone marrow-derived macrophages (BMMPhis) and that SHIP upregulation is essential for endotoxin tolerance. Herein, we show that in vivo differentiated SHIP-/- peritoneal (PMPhis) and alveolar (AMPhis) 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, BMMPhis from SHIP-/- mice do not display this M2 phenotype unless exposed to TGFbeta 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 TGFbeta if progenitors have elevated PIP3.

Purpose: Limited drug penetration in solid tumors is a potential mechanism of resistance for many anticancer drugs. Taxanes represent a class of drugs that are currently undergoing a new round of development, but with little known of their ability to penetrate and distribute relative to blood vessels within solid tumors. Experimental Design: We assessed the tissue penetration of paclitaxel and docetaxel in HCT-116 tumor xenografts and in multilayered cell culture (MCC), a three-dimensional cell culture model of the tumor extravascular compartment. In xenografts, taxanes were mapped relative to blood vessels to obtain drug profiles as a function of distance from vasculature. For MCC, cultures were exposed to stirred drug reservoirs and taxanes measured as a function of depth into tissue. Results: Both taxanes exhibited limited penetration, with little drug reaching further than 100 Am into the tissue. Of the two, paclitaxel exhibited up to 2-fold greater penetration than docetaxel. Mapping tumor cell proliferation following treatment allowed the consequences of limited drug penetration to be assessed. In tumor xenografts where reduced drug exposure to cells far from vasculature is one of several factors influencing response to treatment, up to a 75% reduction in S-phase cells was achieved in cells nearest the vessels, but only 50% reduction was observed in the tissue 150 Am away. In MCC-based data, where the influence of reduced cell proliferation with depth into tissue was circumvented, a 5-fold (paclitaxel) and 10-fold (docetaxel) increase in reservoir drug concentration was required to produce a response in cells 150 Am into the tissue equivalent to that seen in cells directly exposed to the drug. Conclusion: These results indicate that limited distribution is an important mechanism of tumor resistance to taxanes.

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/LCC6 HER2 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, DioC 7 (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 Am 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.

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 mol/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.

Integrin-linked kinase (ILK) was assesed as a therapeutic target in glioblastoma xenograft models through multiple endpoints including treatment related changes in the tumor microenvironment. Glioblastoma cell lines were tested in vitro for sensitivity toward the small-molecule inhibitors QLT0254 and QLT0267. Cell viability, cell cycle, and apoptosis were evaluated using MTT assay, flow cytometry, caspase activation, and DAPI staining. Western blotting and ELISA were used for protein analysis (ILK, PKB/Akt, VEGF, and HIF-1A). In vivo assessment of growth rate, cell proliferation, BrdUrd, blood vessel mass (CD31 labeling), vessel perfusion (Hoechst 33342), and hypoxia (EF-5) was done using U87MG glioblastoma xenografts in RAG2-M mice treated orally with QLT0267 (200 mg/kg q.d.). ILK inhibition in vitro with QLT0254 and QLT0267 resulted in decreased levels of phospho-PKB/Akt (Ser 473 ), secreted VEGF, G 2 -M block, and apoptosis induction. Mice treated with QLT0267 exhibited significant delays in tumor growth (treated 213 mm 3 versus control 549 mm 3 ). In situ analysis of U87MG tumor cell proliferation from QLT0267-treated mice was significantly lower relative to untreated mice. Importantly, VEGF and HIF-1A expression decreased in QLT0267-treated tumors as did the percentage of blood vessel mass and numbers of Hoechst 33342 perfused tumor vessels compared with control tumors (35% versus 83%). ILK inhibition with novel small-molecule inhibitors leads to treatment-associated delays in tumor growth, decreased tumor angiogenesis, and functionality of tumor vasculature. The therapeutic effects of a selected ILK inhibitor (QLT0267) should be determined in the clinic in cancers that exhibit dysregulated ILK, such as PTEN-null glioblastomas.