Kathy J. Carter

Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in vivo

Expression of lipoprotein lipase (LPL) by the macrophage has been proposed to promote foam cell formation and atherosclerosis, primarily on the basis of in vitro studies. LPL-deficient mice might provide a model for testing the role of LPL secretion by the macrophage in an in vivo system. Unfortunately, homozygous deficiency of LPL in the mouse is lethal shortly after birth. Because the fetal liver is the major site of hematopoiesis in the developing fetus, transplantation of C57BL/6 mice with LPL-/- fetal liver cells (FLCs) was used to investigate the physiologic role of macrophage LPL expression in vivo. Thirty-four female C57BL/6 mice were lethally irradiated and reconstituted with FLCs from day 14 LPL+/+, LPL+/-, and LPL-/- donors. No significant differences were detected in plasma levels of post-heparin LPL activity or in serum cholesterol or triglyceride levels between the 3 groups on either a chow diet or an atherogenic diet. After 19 weeks on the atherogenic diet, aortae were collected for quantitative analysis of the extent of aortic atherosclerosis. LPL expression was detected by immunocytochemistry and in situ hybridization in macrophages of aortic atherosclerotic lesions of LPL+/+-->C57BL/6 and LPL+/--->C57BL/6 mice, but not in LPL-/--->C57BL/6 mice, whereas myocardial cells expressed LPL in all groups. The mean aortic lesion area was reduced by 55% in LPL-/--->C57BL/6 mice compared with LPL+/+-->C57BL/6 mice and by 45% compared with LPL+/--->C57BL/6 mice, respectively. These data demonstrate in vivo that LPL expression by macrophages in the artery wall promotes foam cell formation and atherosclerosis. off

Reduced Atherosclerotic Lesions in Mice Deficient for Total or Macrophage-Specific Expression of Scavenger Receptor-A

Abstract—The absence of the scavenger receptor A (SR-A)-I/II has produced variable effects on atherosclerosis in different murine models. Therefore, we examined whether SR-AI/II deficiency affected atherogenesis in C57BL/6 mice, an inbred strain known to be susceptible to diet-induced atherosclerotic lesion formation, and whether the deletion of macrophage SR-AI/II expression would modulate lesion growth in C57BL/6 mice and LDL receptor (LDLR)−/− mice. SR-AI/II–deficient (SR-AI/II−/−) female and male mice on the C57BL/6 background were challenged with a butterfat diet for 30 weeks. No differences were detected in plasma lipids between SR-AI/II−/− and SR-AI/II+/+ mice, whereas both female and male SR-AI/II−/− mice had a tremendous reduction (81% to 86%) in lesion area of the proximal aorta compared with SR-AI/II+/+ mice. Next, to analyze the effect of macrophage-specific SR-AI/II deficiency in atherogenesis, female C57BL/6 mice were lethally irradiated, transplanted with SR-AI/II−/− or SR-AI/II+/+ fetal liver cells, and challenged with the butterfat diet for 16 weeks. In a separate experiment, male LDLR−/− mice were reconstituted with SR-AI/II−/− or SR-AI/II+/+ fetal liver cells and challenged with a Western diet for 10 weeks. No significant differences in plasma lipids and lipoprotein profiles were noted between the control and experimental groups in either experiment. SR-AI/II−/−→C57BL/6 mice, however, had a 60% reduction in lesion area of the proximal aorta compared with SR-AI/II+/+→C57BL/6 mice. A similar level of reduction (60%) in lesion area was noted in the proximal aorta and the entire aorta en face of SR-AI/II−/−→LDLR−/− mice compared with SR-AI/II+/+→LDLR−/− mice. These results demonstrate in vivo that SR-AI/II expression has no impact on plasma lipid levels and that macrophage SR-AI/II contributes significantly to atherosclerotic lesion formation.

Matrix Metalloproteinase-9 from Bone Marrow–Derived Cells Contributes to Survival but not Growth of Tumor Cells in the Lung Microenvironment

The role of specific stromal-derived matrix metalloproteinases (MMPs) was analyzed in experimental metastasis assays in wild-type and either MMP-9, MMP-7, or MMP-2 null mice. MMP-9 null mice showed an 81% reduction in Lewis lung carcinoma tumor number, whereas MMP-7 null mice showed a 42% increase in tumor number, and there was no difference in tumor number in MMP-2 null mice compared with wild-type controls. Similarly, in an orthotopic model of lung cancer, 50% fewer MMP-9 null mice were able to establish tumors in the lung compared with control mice, although the size of the tumors was not different. The effect of MMP-9 on lung tumor colonization was dependent on the expression of MMP-9 from bone marrow-derived cells and is most likely contributed by neutrophils. To examine temporal effects of stromal MMP-9, bioluminescence imaging from luciferase-expressing human lung cancer-derived A549 cells revealed that there were fewer tumor cells in the lungs of MMP-9 null mice as early as 19 hours after injection compared with control mice, with no difference in subsequent growth rates. Six hours after injection of tumor cells, MMP-9 null mice showed a 4-fold increase in the percent of tumor cells undergoing apoptosis compared with control mice. We conclude that MMP-9 from the bone marrow contributes to the early survival and establishment of tumors in the lung and has no effect on subsequent growth. These results provide insights into the failure of MMP inhibitors in clinical trials in patients with late-stage lung cancer.

Effect of Ablation or Inhibition of Stromal Matrix Metalloproteinase-9 on Lung Metastasis in a Breast Cancer Model Is Dependent on Genetic Background

Matrix metalloproteinases (MMP) are a family of enzymes with a myriad of functions. Lately, we have come to realize that broad-spectrum inhibition of these enzymes, as was tried unsuccessfully in multiple phase III trials in cancer patients, is likely unwise given the protumorigenic and antitumorigenic functions of various family members. Here, we used the multistage mammary tumor model MMTV-PyVT to investigate roles for either MMP7 or MMP9 in tumor progression. We found no effect of genetic ablation of MMP7 or MMP9 on the multifocal tumors that developed in the mammary glands. Lack of MMP7 also had no effect on the development of lung metastases, suggesting that MMP7 is irrelevant in this model. In contrast, MMP9 deficiency was associated with an 80% decrease in lung tumor burden. The predominant cellular source of MMP9 was myeloid cells, with neutrophils being the largest contributor in tumor-bearing lungs. Experimental metastasis assays corroborated the role of host-derived MMP9 in lung metastasis and also facilitated determination of a time frame most relevant for the MMP9-mediated effect. The lung tumors from MMP9-deficient mice showed decreased angiogenesis. Surprisingly, the antimetastatic outcome of MMP9 ablation seemed to be dependent on strain. Only mice that had genetic background derived from C57BL/6 showed reduced metastasis, whereas mice fully of the FVB/N background showed no significant effect. These strain-specific responses were also observed in a study using a highly selective pharmacologic inhibitor of MMP9 and thus suggest that responses to MMP inhibition are controlled by genetic differences.

A protective role of mast cells in intestinal tumorigenesis

Mast cells have been observed in numerous types of tumors; however, their role in carcinogenesis remains poorly understood. The majority of epidemiological evidence suggests a negative association between the presence of mast cells and tumor progression in breast, lung and colonic neoplasms. Intestinal adenomas in the multiple intestinal neoplasia (Min, APC(Min/+)) mouse displayed increased numbers of mast cells and increased abundance of mast cell-associated proteinases as determined by transcriptional profiling with the Hu/Mu ProtIn microarray. To examine the role of mast cells in intestinal tumorigenesis, a mutant mouse line deficient in mast cells, Sash mice (c-kit(W-sh/W-sh)), was crossed with the Min mouse, a genetic model of intestinal neoplasia. The resulting mast cell-deficient Min-Sash mice developed 50% more adenomas than littermate controls and the tumors were 33% larger in Min-Sash mice. Mast cell deficiency did not affect tumor cell proliferation; however, apoptosis was significantly inhibited in mast cell-deficient mice. Mast cells have been shown to act as critical upstream regulators of numerous inflammatory cells. Neutrophil, macrophage and T cell populations were similar between Min and Min-Sash mice; however, eosinophils were significantly less abundant in tumors obtained from Min-Sash animals. These results indicate a protective, antitumor role of mast cells in a genetic model of early-stage intestinal tumorigenesis.

Matrix metalloproteinase-9 contributes to intestinal tumourigenesis in the adenomatous polyposis coli multiple intestinal neoplasia mouse

Matrix metalloproteinases (MMPs) are a family of 23 extracellular proteases that are best known for their collective ability to degrade all components of the extracellular matrix. We previously demonstrated that genetic ablation of MMP‐7 reduced tumour multiplicity in multiple intestinal neoplasia (Min) mice possessing a genetic alteration in the adenomatous polyposis coli gene (APC). These mice, commonly referred to as APC‐Min mice, are a frequently used model of early intestinal tumourigenesis. To examine further the role of MMPs in intestinal tumour development, we generated APC‐Min mice genetically deficient in MMP‐2, ‐9, ‐12 or ‐19. Genetic ablation of MMP‐2, ‐12 or ‐19 did not affect multiplicity or size of intestinal tumours when crossed into the APC‐Min system. However, MMP‐9 deficient animals developed 40% fewer tumours than littermate controls, although tumour size distribution remained unaffected. Intestinal adenomas from MMP‐9 deficient mice demonstrated a 50% decrease in proliferating cells compared with control tissues, with no difference in apoptosis. To determine the cellular origin of MMP‐9 in these tumours, immunofluorescent co‐staining with markers for different leucocyte lineages was used to demonstrate that intratumoural MMP‐9 is largely a product of neutrophils. These studies extend the potential targets for chemoprevention of intestinal adenomas to MMP‐9 in addition to MMP‐7 and exclude MMP‐2,‐12,‐19 as attractive targets for intervention.

Proteinase Activity in Human and Murine Saliva as a Biomarker for Proteinase Inhibitor Efficacy

As molecularly targeted agents reach the clinic, there is a need for assays to detect their presence and effectiveness against target molecules in vivo. Proteinase inhibitors are one example of a class of therapeutic agent for which satisfactory methods of identifying successful target modulation in vivo are lacking. This is of particular importance while these drugs are in clinical trials because standard maximum-tolerated dose-finding studies often are not suitable due to lack of toxicity. Saliva represents a readily accessible bodily fluid that can be repeatedly sampled and used for assaying in vivo effects of systemic drugs. Here we show the development of a simple assay that can be used to measure proteinase activity in saliva and proteinase inhibition after systemic treatment with three different proteinase inhibitors. A variety of gelatinolytic activities present in human and murine saliva have been assayed with a fluorescent dye-labeled substrate and assigned to different proteinase categories by inclusion of specific classes of inhibitors. Treatment of mice with either matrix metalloproteinase inhibitors or a urokinase inhibitor for a period as short as 48 hours results in levels of the drugs that can be detected in saliva by mass spectrometry and concomitant decreases in salivary proteinase activity, thus demonstrating that these inhibitors successfully modulate their targets in vivo.

New tools for the quantitative assessment of prodrug delivery and neurotoxicity

Systemic off-target toxicities, including neurotoxicity, are prevalent side effects in cancer patients treated with a number of otherwise highly efficacious anticancer drugs. In the current study, we have: (1) developed a new analytical metric for the in vivo preclinical assessment of systemic toxicities/neurotoxicity of new drugs and delivery systems; and (2) evaluated, in mice, the in vivo efficacy and toxicity of a versatile and modular NanoDendron (ND) drug delivery and imaging platform that we recently developed. Our paclitaxel-carrying ND prodrug, ND(PXL), is activated following proteolytic cleavage by MMP9, resulting in localized cytotoxic chemotherapy. Using click chemistry, we combined ND(PXL) with a traceable beacon, ND(PB), yielding ND(PXL)-ND(PB) that functions as a theranostic compound. In vivo fluorescence FRET imaging of this theranostic platform was used to confirm localized delivery to tumors and to assess the efficiency of drug delivery to tumors, achieving 25-30% activation in the tumors of an immunocompetent mouse model of breast cancer. In this model, ND-drug exhibited anti-tumor efficacy comparable to nab-paclitaxel, a clinical formulation. In addition, we combined neurobehavioral metrics of nociception and sensorimotor performance of individual mice to develop a novel composite toxicity score that reveals and quantifies peripheral neurotoxicity, a debilitating long-term systemic toxicity of paclitaxel therapy. Importantly, mice treated with nab-paclitaxel developed changes in behavioral metrics with significantly higher toxicity scores indicative of peripheral neuropathy, while mice treated with ND(PXL) showed no significant changes in behavioral responses or toxicity score. Our ND formulation was designed to be readily adaptable to incorporate different drugs, imaging modalities and/or targeting motifs. This formulation has significant potential for preclinical and clinical tools across multiple disease states. The studies presented here report a novel toxicity score for assessing peripheral neuropathy and demonstrate that our targeted, theranostic NDs are safe and effective, providing localized tumor delivery of a chemotherapeutic and with reduced common neurotoxic side-effects.

Abstract PR13: Interleukin-4 receptor α chain regulates the growth of primary and metastatic mammary tumors

Interleukin-4 (IL-4), a cytokine produced mainly by immune cells, may promote the growth of epithelial tumors by mediating increased proliferation and survival. The IL-4 Receptor (IL-4R) is overexpressed in human and murine epithelial mammary cancers, and may be a promising target for anti-tumor therapy. Here, we show that loss of IL-4Rα, a component of the IL-4 receptor, in two murine mammary tumor cell lines (R221A and 4T1), reduces tumor cell survival and outgrowth ability, and results in attenuated growth at primary and metastatic sites. RNA interference was used to knock down the expression of IL-4Rα in both cell lines. In support of our hypothesis, reduced IL-4Rα expression in R221A cells nearly abolished tumor take after orthotopic mammary injection in vivo, decreased the median latency of 4T1 primary tumors, and resulted in a significant reduction in tumor size at endpoint. In addition, reduced IL-4Rα expression in both cell lines resulted in a decrease [>2.4 fold] in lung and [>2 fold] liver metastatic tumor burden following experimental metastasis assays in vivo. The decrease in metastatic ability of IL-4Rα knockdown cells was attributed to 1) reduced tumor cell survival as determined by seeding ability (number of tumors) and an increase in apoptosis by caspase-3 positivity, and 2) a reduction in proliferation as determined by outgrowth ability (tumor size) and a decrease in Ki67 positivity. In addition, an overall increase in immune infiltrates (macrophages, neutrophils, and T lymphocytes) within IL-4Rα knockdown tumors was observed, indicating that enhanced clearance of knock down tumor cells could also contribute to the reduction in knock down tumor size visualized at endpoint. Finally, it was determined by western blot analysis that IL-4 ligand stimulates the phosphorylation of AKT (s373) and ERK in R221a and 4T1 cells in vitro. Immunohistochemical analysis of R221a and 4T1 lung metastases showed significantly reduced levels of pAKT (s472) and pERK in IL-4Rα knock down tumors compared to control tumors, implicating a possible role for AKT and ERK in mediating pro-tumor effects IL-4Rα signaling. Collectively, our results demonstrate that the IL-4 receptor promotes the growth of mammary cancer cells at primary and metastatic sites, possibly through activated AKT and ERK signaling, and suggests that available drugs targeting IL4-Rα signaling may have potential for treating primary breast cancers and limiting metastatic disease. This abstract is also presented as poster C51. Citation Format: Katherine T. Venmar, Daniel Hwang, Ashley Dozier, Kathy Carter, Sareena Gillani, Barbara Fingleton. Interleukin-4 receptor α chain regulates the growth of primary and metastatic mammary tumors. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR13.

Abstract 5328: Interleukin-4 receptor regulates metastasis of mammary epithelial cancer cells

Interleukin-4 (IL-4), a cytokine produced mainly by immune cells, may promote cancer cell survival by mediating apoptosis resistance in epithelial tumors. The IL-4 Receptor (IL-4R) is overexpressed in a variety of human solid cancer cell lines and primary cell lines, and may be a promising target for anti-tumor therapy. Here, we show that loss of IL-4Rα, a component of the IL-4 receptor, in two murine mammary tumor cell lines (R221A and 4T1), reduces tumor cell survival and results in attenuated metastatic growth in secondary sites. Proliferation and direct cell counting analyses, following knockdown of IL-4Rα with RNA interference, show that R221A clones with reduced IL-4Rα expression proliferate at a comparable rate to control clones, but the number of cells is significantly reduced. This data correlates with the finding that reduced IL-4Rα expression in R221A cells almost completely abolishes tumor take after orthotopic mammary injection in vivo. Furthermore, reduced IL-4Rα expression in R221A and 4T1 cell lines results in a decrease [2.4 fold] in lung metastatic growth in vivo. Reduced expression of IL-4Rα in 4T1 cells results in a similar decrease of liver metastasis. In support of this data, proliferation assays following serum starvation and a modified clonogenic survival assay show that both 4T1 and R221A knockdown clones have a decrease [2 fold] in survival ability in comparison to control clones. Taken together, our results indicate that the IL-4 receptor promotes survival of breast cancer cells at metastatic sites, and suggests that drugs targeting this pathway, currently in clinical trials for respiratory ailments, may have potential for limiting metastatic disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5328. doi:1538-7445.AM2012-5328