Aneuploidy, an incorrect chromosome number, is the leading cause of miscarriages and mental retardation in humans and is a hallmark of cancer. We examined the effects of aneuploidy on primary mouse cells by generating a series of cell lines that carry an extra copy of one of four mouse chromosomes. In all four trisomic lines proliferation was impaired and metabolic properties were altered. Immortalization, the acquisition of the ability to proliferate indefinitely, was also affected by the presence of an additional copy of certain chromosomes. Our data indicate that aneuploidy decreases not only organismal but also cellular fitness and elicits traits that are shared between different aneuploid cells.

Innate immune cells can constitute a substantial proportion of the cells within the tumor microenvironment and have been associated with tumor malignancy in patients and animal models of cancer; however, the mechanisms by which they modulate cancer progression are incompletely understood. Here, we show that high levels of cathepsin protease activity are induced in the majority of macrophages in the microenvironment of pancreatic islet cancers, mammary tumors, and lung metastases during malignant progression. We further show that tumor-associated macrophage (TAM)-supplied cathepsins B and S are critical for promoting pancreatic tumor growth, angiogenesis, and invasion in vivo, and markedly enhance the invasiveness of cancer cells in culture. Finally, we demonstrate that interleukin-4 (IL-4) is responsible for inducing cathepsin activity in macrophages in vitro and in vivo. Together, these data establish IL-4 as an important regulator, and cathepsin proteases as critical mediators, of the cancer-promoting functions of TAMs.[Keywords: Tumor-associated macrophage; tumor microenvironment; cysteine cathepsin; protease; interleukin-4; invasion] Supplemental material is available at http://www.genesdev.org.

During the process of tumor progression, cancer cells can produce the requisite growth-and invasion-promoting factors and can also rely on noncancerous cells in the tumor microenvironment as an alternative, cell-extrinsic source. However, whether the cellular source influences the function of such tumor-promoting factors remains an open question. Here, we examined the roles of the cathepsin Z (CtsZ) protease, which is provided by both cancer cells and macrophages in pancreatic neuroendocrine tumors in humans and mice. We found that tumor proliferation was exclusively regulated by cancer cell-intrinsic functions of CtsZ, whereas tumor invasion required contributions from both macrophages and cancer cells. Interestingly, several of the tumor-promoting functions of CtsZ were not dependent on its described catalytic activity but instead were mediated via the Arg-Gly-Asp (RGD) motif in the enzyme prodomain, which regulated interactions with integrins and the extracellular matrix. Together, these results underscore the complexity of interactions within the tumor microenvironment and indicate that cellular source can indeed impact molecular function.[Keywords: cell invasion; cell migration; tumor microenvironment; protease] Supplemental material is available for this article. Received July 26, 2014; revised version accepted August 29, 2014. Tumors arise in complex tissue microenvironments in which a multitude of different noncancerous cell types can potently regulate disease initiation and progression (Hanahan and Coussens 2012;Quail and Joyce 2013). In addition, interactions with the extracellular matrix (ECM) are critical for modulating cell behavior, including enhancing cell survival and promoting invasion via ECM turnover and proteolysis (Lu et al. 2012;Sevenich and Joyce 2014). The ECM is a heterogeneous mix of proteins and polysaccharides, including different collagens, laminins, fibronectin, and heparan sulfate proteoglycans, which form an intricate network that confers tissue structure and regulates growth factor availability (Hynes and Naba 2012;Lu et al. 2012). Integrins are central in mediating interactions between cells and the surrounding ECM, and integrin engagement at the cell surface results in activation of downstream signaling nodes, including focal adhesion kinase (FAK) and Src kinase, to promote cancer cell proliferation, survival, migration, and invasion (Desgrosellier and Cheresh 2010;Huttenlocher and Horwitz 2011;Moreno-Layseca and Streuli 2014).Among the noncancerous cell types that modulate tumorigenesis, tumor-associated macrophages (TAMs) have emerged as critical regulators of tumor progression (Biswas et al. 2013;Noy and Pollard 2014). This is particularly evident in tumor invasion, as TAMs provide a major source of proteases that modulate the ECM (Joyce and Pollard 2009). In determining the mechanisms by which TAMs promote different tumorigenic processes, the focus to date has been predominantly centered on identifying factors that are TAM-specific or TAM-enriched, which are not necessa...

Tumor-associated macrophages have recently emerged as a key regulatory cell type during cancer progression, and have been found to promote tumor malignancy in the majority of studies performed to date. We show in this study that CD68þ macrophages positively correlate with tumor grade and liver metastasis in human pancreatic neuroendocrine tumors (PNETs). To investigate the potential mechanisms whereby macrophages can promote PNET progression, we crossed the RIP1-Tag2 (RT2) mouse model of pancreatic islet cancer to colonystimulating factor-1 (CSF-1)-deficient Csf1 op/op mice, which have reduced numbers of tissue macrophages. Csf1 op/op RT2 mice had a substantial reduction in cumulative tumor burden, which interestingly resulted from a significant decrease in angiogenic switching and tumor number, rather than an evident effect on tumor growth. In the tumors that did develop in CSF-1-deficient animals, however, there were no significant differences in tumor cell proliferation, apoptosis, angiogenesis or invasion. CSF-1 deficiency decreased macrophage infiltration by approximately 50% during all stages of RT2 tumor progression. Interestingly, several cytokines were upregulated in CSF-1-deficient RT2 tumors, and neutrophil infiltration was increased. These results show that macrophages are important for promoting PNET development and suggest that additional factors contribute to the recruitment and survival of myeloid cells in RT2 tumors in the absence of CSF-1.

Heparan sulfate proteoglycans are an important and abundant component of the extracellular matrix, which undergo substantial remodeling throughout tumorigenesis via the enzymatic activity of heparanase. Heparanase has been shown to be upregulated in many human cancers; however, its specific functions in human pancreatic neuroendocrine tumors (PanNETs) and spontaneous mouse models of cancer have not been evaluated. Here, we investigated the role of heparanase in PanNETs using patient samples and the RIP1-Tag2 (RT2) PanNET-transgenic mouse model. High heparanase expression significantly correlated with more advanced tumor stage, higher tumor grade and the presence of distant metastasis in PanNET patients. We genetically manipulated heparanase levels in the RT2 model using heparanase-transgenic mice, which constitutively overexpress heparanase, and heparanase-knockout mice. Heparanase was found to have a critical role in promoting tumor invasion, through both macrophage and cancer cell sources in the tumor microenvironment. In addition, elevated heparanase levels significantly increased peritumoral lymphangiogenesis in vivo and promoted the trans-differentiation of macrophages into lymphatic endothelial cell-like structures in culture. Conversely, we found that heparanase deletion led to increased angiogenesis and pericyte coverage. Together, these data identify important roles for heparanase in regulating several critical aspects of tumorigenesis, demonstrating that heparanase represents a potential therapeutic target for PanNET patients.

Pancreatic neuroendocrine tumors (PanNETs) are a relatively rare but clinically challenging tumor type. In particular, high grade, poorly-differentiated PanNETs have the worst patient prognosis, and the underlying mechanisms of disease are poorly understood. In this study we have identified and characterized a previously undescribed class of poorly differentiated PanNETs in the RIP1-Tag2 mouse model. We found that while the majority of tumors in the RIP1-Tag2 model are well-differentiated insulinomas, a subset of tumors had lost multiple markers of beta-cell differentiation and were highly invasive, leading us to term them poorly differentiated invasive carcinomas (PDICs). In addition, we found that these tumors exhibited a high mitotic index, resembling poorly differentiated (PD)-PanNETs in human patients. Interestingly, we identified expression of Id1, an inhibitor of DNA binding gene, and a regulator of differentiation, specifically in PDIC tumor cells by histological analysis. The identification of PDICs in this mouse model provides a unique opportunity to study the pathology and molecular characteristics of PD-PanNETs.

Innate immune cells can constitute a substantial proportion of the cells within the tumor microenvironment and have been associated with tumor malignancy in patients and animal models of cancer; however, the mechanisms by which they modulate cancer progression are incompletely understood. Here, we show that high levels of cathepsin protease activity are induced in the majority of macrophages in the microenvironment of pancreatic islet cancers, mammary tumors and lung metastases during malignant progression. We further show that tumor-associated macrophage (TAM)-supplied cathepsins B and S are critical for promoting pancreatic tumor growth, angiogenesis and invasion in vivo, and markedly enhance the invasiveness of cancer cells in culture. Finally, we demonstrate that interleukin (IL)-4 is responsible for inducing cathepsin activity in macrophages in vitro and in vivo. Together, these data establish IL-4 as an important regulator, and cathepsin proteases as critical mediators, of the cancer-promoting functions of TAMs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-379.

Circulating concentrations of hCG free alpha-subunit (alpha hCG) increase throughout pregnancy. To address the hypothesis that maternal plasma alpha hCG may reflect placental dysfunction and/or adverse perinatal outcome during insulin-dependent diabetic pregnancy, alpha hCG was measured serially throughout gestation, beginning before week 12, with a specific RIA using a monoclonal antibody in 54 insulin-dependent diabetic (randomly assigned to strict and customary glycemic control) and 25 nondiabetic pregnancies. alpha hCG was significantly lower in pregnant insulin-dependent diabetic subjects than in nondiabetics subjects until 24 weeks gestation, after which it was higher until delivery. Plasma alpha hCG stabilized in nondiabetics at 32 weeks, whereas it continued to increase in diabetics until delivery, at which time it was 37% greater than that in nondiabetics (mean +/- SE, 1441 +/- 90 vs. 1052 +/- 78 micrograms/L; P less than 0.002). Values in diabetic subjects assigned to strict control were intermediate between those in diabetic subjects assigned to customary control and nondiabetic subjects. alpha hCG was greater in diabetic subjects with pregestational hypertension or microvascular disease, but not in those with pregnancy-induced hypertension. These findings were independent of the assigned goals of glycemic control. alpha hCG was not correlated with the duration of diabetes or related to premature delivery, fetal distress, birth asphyxia, or macrosomia. Thus, alpha hCG is increased during the third trimester of the type I diabetic pregnancy and is associated with preexisting hypertension and maternal microangiopathy, but is not a predictor of adverse perinatal outcome. Excessive alpha hCG secretion in diabetes may share pathophysiological mechanisms in common with those underlying diabetic microangiopathy.