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PDF file - 153K, LY294002 attenuates mast cell mediated CD11b+ cell migration. Representative images of migration of CD11b cells in response to (A) Stempro +SCF (control), (B) LAD-2 CM (LAD-2 conditioned medium), (C) LAD-2 CM + 10 �M LY294002 (LAD-2 CM with 10 �M LY294002) and (D) CM obtained after pretreatment of LAD-2 cells with 10 �M LY294002. (E) Purity of CD11b+ cells obtained after using either biotinylated Rat ant-human CD11b or biotinylated Rat IgG2bK. Black arrows indicate migrated CD11b+ cells. Black scale bar = 20 �m.

PDF file - 173K, Tumor/Margin frequency of CD4 T cells is inversely related to Macrophages and Mast cells. (A) Graphical Representation and (B) Representative images of the CD11b, Mac387, Tryptase, CAE and CD4 cells in tumor and tumor margin area.

PDF file - 54K, Toluidine Blue staining protocol.

PDF file - 55K, Mast cells promote in vitro and in vivo Gr1+ neutrophil and Foxp3+ Tregs migration in PI3K dependent manner. (A) GMMCs promote Gr1+ neutrophil in vitro migration and pretreatment of GMMCs with LY294002 attenuates migration of Gr1+ cells (B) LY294002 in vivo treatment attenuates migration of Gr1+ neutrophils (C) LY294002 in vivo treatment attenuates migration of Foxp3+ Tregs, * P < 0.05 represents the result of ANOVA.

Purpose: To understand signaling pathways that shape inflamed tissue and predispose to cancer is critical for effective prevention and therapy for chronic inflammatory diseases. We have explored phosphoinositide 3-kinase (PI3K) activity in human inflammatory bowel diseases and mouse colitis models.Experimental Design: We conducted immunostaining of phosphorylated AKT (pAKT) and unbiased high-throughput image acquisition and quantitative analysis of samples of noninflamed normal colon, colitis, dysplasia, and colorectal cancer. Mechanistic insights were gained from ex vivo studies of cell interactions, the piroxicam/IL-10−/− mouse model of progressive colitis, and use of the PI3K inhibitor LY294002.Results: Progressive increase in densities of pAKT-positive tumor-associated macrophages (TAM) and increase in densities of mast cells in the colonic submucosa were noted with colitis and progression to dysplasia and cancer. Mast cells recruited macrophages in ex vivo migration assays, and both mast cells and TAMs promoted invasion of cancer cells. Pretreatment of mast cells with LY294002 blocked recruitment of TAMs. LY294002 inhibited mast cell and TAM-mediated tumor invasion, and in mice, blocked stromal PI3K, colitis, and cancer.Conclusion: The PI3K/AKT pathway is active in cells infiltrating inflamed human colon tissue. This pathway sustains the recruitment of inflammatory cells through a positive feedback loop. The PI3K/AKT pathway is essential for tumor invasion and the malignant features of the piroxicam/IL-10−/− mouse model. LY294002 targets the PI3K pathway and hinders progressive colitis. These findings indicate that colitis and progression to cancer are dependent on stromal PI3K and sensitive to treatment with LY294002. Clin Cancer Res; 19(9); 2342–54. ©2013 AACR.

PDF file - 97K, Multiplex ELISA data for human inflammatory chemoattractants.

Purpose: To assess the clinical and pathologic significance of mast cell infiltration in human pancreatic cancer and evaluate crosstalk between mast cells and cancer cells in vitro.Experimental Design: Immunohistochemistry for tryptase was done on 53 pancreatic cancer specimens. Mast cell counts were correlated with clinical variables and survival. Serum tryptase activity from patients with cancer was compared with patients with benign pancreatic disease. In vitro, the effect of pancreatic cancer–conditioned medium on mast cell migration was assessed. The effect of conditioned medium from the human mast cell line, LAD-2, on cancer and normal ductal cell proliferation was assessed by thymidine incorporation. Matrigel invasion assays were used to evaluate the effect of mast cell–conditioned medium on cancer cell invasion in the presence and absence of a matrix metalloproteinase inhibitor, GM6001.Results: Mast cell infiltration was significantly increased in pancreatic cancer compared with normal pancreatic tissue (11.4 ± 6.7 versus 2.0 ± 1.4, P < 0.001). Increased infiltrating mast cells correlated with higher grade tumors (P < 0.0001) and worse survival. Patients with pancreatic cancer had elevated serum tryptase activity (P < 0.05). In vitro, AsPC1 and PANC-1 cells induced mast cell migration. Mast cell–conditioned medium induced pancreatic cancer cell migration, proliferation, and invasion but had no effect on normal ductal cells. Furthermore, the effect of mast cells on cancer cell invasion was, in large part, matrix metalloproteinase–dependent.Conclusions: Tumor-infiltrating mast cells are associated with worse prognosis in pancreatic cancer. In vitro, the interaction between mast cells and pancreatic cancer cells promotes tumor growth and invasion. Clin Cancer Res; 16(8); 2257–65. ©2010 AACR.

PDF file - 125K, (A) Representative images of HT-29 invasion study in response to Stempro + SCF (internal control for LAD-2 CM), LAD-2 CM, CM + 10 �M LY294002 (LAD-2 CM with 10 �M LY294002) and 10 �M LY pretreated CM (CM obtained after treatment of LAD-2 cells with 10 �M LY294002). (B) Representative images of CFSE labeled HT-29 invasion study in response to tumor infiltrating leukocytes (TILs) isolated from UC associated cancer patients. Red arrows show invaded HT-29. Red scale bar = 50 �m, * P < 0.05 represents the result of Student t test.

PDF file - 40K, Multiplex ELISA data for human inflammatory chemoattractants.

PDF file - 74K

Supplementary Data from Crosstalk between Mast Cells and Pancreatic Cancer Cells Contributes to Pancreatic Tumor Progression

PDF file - 61K, Table.1 Patient Cohorts. Table 2. Ulcerative Colitis Patients Absent Colonic Dysplasia. 3.1.3 Ulcerative Colitis patients with invasive cancer used in this study. Table 4. Patients diagnosed with ulcerative colitis and associated colorectal cancers.

Supplementary Figure 4 from Mast Cell 5-Lipoxygenase Activity Promotes Intestinal Polyposis in APCΔ468 Mice

Commentary on this Article from T-Regulatory Cells Shift from a Protective Anti-Inflammatory to a Cancer-Promoting Proinflammatory Phenotype in Polyposis

Supplementary Figure 1 from Mast Cell 5-Lipoxygenase Activity Promotes Intestinal Polyposis in APCΔ468 Mice

Supplementary Figure 2 from Mast Cell 5-Lipoxygenase Activity Promotes Intestinal Polyposis in APCΔ468 Mice

Supplementary Figure 3 from Mast Cell 5-Lipoxygenase Activity Promotes Intestinal Polyposis in APCΔ468 Mice

TNF plays an integral role in inflammatory bowel disease (IBD), as evidenced by the dramatic therapeutic responses in Crohn’s disease (CD) patients induced by chimeric anti-TNF mAbs. However, treatment of CD patients with etanercept, a decoy receptor that binds soluble TNF, fails to improve disease. To explore this discrepancy, we investigated the role of TNF signaling in Wnt/β-catenin–mediated intestinal stem cell and progenitor cell expansion in CD patients, human cells, and preclinical mouse models. We hypothesized that TNF exerts beneficial effects on intestinal epithelial cell (IEC) responses to injury. In CD patients, intestinal stem cell and progenitor cell Wnt/β-catenin signaling correlates with inflammation status. TNF-deficient (Tnf−/−) mice exhibited increased apoptosis, less IEC proliferation, and less Wnt signaling when stimulated with anti-CD3 mAb. Bone marrow (BM) chimera mice revealed that mucosal repair depended on TNF production by BM–derived cells and TNFR expression by radioresistant IECs. Wild-type→Tnfr1/2−/− BM chimera mice with chronic dextran sodium sulfate colitis exhibited delayed ulcer healing, more mucosal inflammation, and impaired Wnt/β-catenin signaling, consistent with the hypothesis that epithelial TNFR signaling participates in mucosal healing. The direct effect of TNF on stem cells was demonstrated by studies of TNF-induced Wnt/β-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced β-catenin activation in nontransformed human NCM460 cells (TOPFlash) and mice (TOP-GAL). Together, these data support the hypothesis that TNF plays a beneficial role in enhancing Wnt/β-catenin signaling during ulcer healing in IBD. These novel findings will inform clinicians and therapeutic chemists alike as they strive to develop novel therapies for IBD patients.

Purpose The development of NIRF cathepsin activity probes offered the ability to visualize tumor associated tumor reaction and act as a surrogate marker to delineate the dysplastic lesions. One major type is a NIRF substrate of cathepsins (SBP), which is involved in catalytic way to produce high levels of fluorescence emission. The other major type (ABP) reacts with active cathepsins in stoichiometric manner since they bind covalently with their active center. Little is known about the sensitivity and the specificity of the NIRF probes to detect autochthonous developed dysplastic lesions. Dual laser NIRF endoscope provides a good tool to determine the efficiency of various NIRF probes in vivo in the same lesions. Experimental design In the current study, we validated both types of NIRF probes by using the dual laser NIRF endoscope to detect lesions colon cancer mouse model (TS4Cre/cAPC +/lox). Results The dual laser NIRF endoscope is emitting equal power with both lasers. It can detect with the same efficiency in 680 mode, as well as, 750 mode when NIFR probes of the same scaffold in vivo. When SBP and ABP were used, our results showed both probes are efficient enough to detect large polyps but small dysplastic lesions could not efficiently imaged with the ABP. Conclusions The dual laser NIRF endoscope is a powerful tool to validate probes. The probes that react catalytically with the active center of cathepsins are more efficient than the ones that react stoichiometrically in detecting small lesions.

[This corrects the article DOI: 10.1371/journal.pone.0206568.].

Near-infrared fluorescence (NIRF) endoscopy has a great potential for efficient early detection of dysplastic lesions in the colon. For preclinical studies, we developed a small animal NIRF endoscope and successfully used this device to identify dysplastic lesions in a murine model of chronic colitis. In this chapter, we present a step-by-step protocol for using NIRF endoscopy to examine the location, the size, and the borders of the dysplastic lesions developed in murine colitis. Our studies suggest that NIRF endoscopy is a specific and sensitive technique that provides a unique opportunity to analyze early stages of tumorigenesis in animal models of colon cancer and to perform surveillance colonoscopy in patients with colitis-associated colon cancer.

Near-infrared fluorescence (NIRF) endoscopy has a great potential for efficient early detection of dysplastic lesions in the colon. For preclinical studies, we developed a small animal NIRF endoscope and successfully used this device to identify dysplastic lesions in a murine model of chronic colitis. In this chapter, we present a step-by-step protocol for using NIRF endoscopy to examine the location, the size, and the borders of the dysplastic lesions developed in murine colitis. Our studies suggest that NIRF endoscopy is a specific and sensitive technique that provides a unique opportunity to analyze early stages of tumorigenesis in animal models of colon cancer and to perform surveillance colonoscopy in patients with colitis-associated colon cancer.

Inflammatory bowel disease (IBD) is known for causing disturbed homeostatic balance among the intestinal immune compartment, epithelium and microbiota. Owing to the emergence of IBD as a major cause of morbidity and mortality, great efforts have been put into understanding the sequence of intestinal inflammatory events. Intestinal macrophages and dendritic cells act in a synergistic fashion with intestinal epithelial cells and microbiota to initiate the triad that governs the intestinal immune responses (whether inflammatory or regulatory). In this review, we will discuss the interplay of intestinal epithelial cells, bacteria and the innate immune component. Moreover, whether or not genetic intervention of probiotic bacteria is a valid approach for attenuating/mitigating exaggerated inflammation and IBD will also be discussed.

T-regulatory (Treg) cells play a major role in cancer by suppressing protective antitumor immune responses. A series of observations (from a single laboratory) suggest that Treg cells are protective in cancer by virtue of their ability to control cancer-associated inflammation in an interleukin (IL)-10–dependent manner. Here, we report that the ability of Treg cells to produce IL-10 and control inflammation is lost in the course of progressive disease in a mouse model of hereditary colon cancer. Treg cells that expand in adenomatous polyps no longer produce IL-10 and instead switch to production of IL-17. Aberrant Treg cells from polyp-ridden mice promote rather than suppress focal mastocytosis, a critical tumor-promoting inflammatory response. The cells, however, maintain other Treg characteristics, including their inability to produce IL-2 and ability to suppress proliferation of stimulated CD4 T cells. By promoting inflammation and suppressing T-helper functions, these cells act as a double-edged knife propagating tumor growth. [Cancer Res 2009;69(13):5490–7]

2-Phenylethylamine is an endogenous constituent of the human brain and is implicated in cerebral transmission. This bioactive amine is also present in certain foodstuffs such as chocolate, cheese and wine and may cause undesirable side effects in susceptible individuals. Metabolism of 2-phenylethylamine to phenylacetaldehyde is catalysed by monoamine oxidase B but the oxidation to its acid is usually ascribed to aldehyde dehydrogenase and the contribution of aldehyde oxidase and xanthine oxidase, if any, is ignored. The objective of this study was to elucidate the role of the molybdenum hydroxylases, aldehyde oxidase and xanthine oxidase, in the metabolism of phenylacetaldehyde derived from its parent biogenic amine. Treatments of 2-phenylethylamine with monoamine oxidase were carried out for the production of phenylacetaldehyde, as well as treatments of synthetic or enzymatic-generated phenylacetaldehyde with aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase. The results indicated that phenylacetaldehyde is metabolised mainly to phenylacetic acid with lower concentrations of 2-phenylethanol by all three oxidising enzymes. Aldehyde dehydrogenase was the predominant enzyme involved in phenylacetaldehyde oxidation and thus it has a major role in 2-phenylethylamine metabolism with aldehyde oxidase playing a less prominent role. Xanthine oxidase does not contribute to the oxidation of phenylacetaldehyde due to low amounts being present in guinea pig. Thus aldehyde dehydrogenase is not the only enzyme oxidising xenobiotic and endobiotic aldehydes and the role of aldehyde oxidase in such reactions should not be ignored.

2-Phenylethylamine is an endogenous constituent of human brain and is implicated in cerebral transmission. It is also found in certain foodstuffs and may cause toxic side-effects in susceptible individuals. Metabolism of 2-phenylethylamine to phenylacetaldehyde is catalyzed by monoamine oxidase and the oxidation of the reactive aldehyde to its acid derivative is catalyzed mainly by aldehyde dehydrogenase and perhaps aldehyde oxidase, with xanthine oxidase having minimal transformation. The present investigation examines the metabolism of 2-phenylethylamine to phenylacetaldehyde in liver slices and compares the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activity in the oxidation of phenylacetaldehyde with precision-cut fresh liver slices in the presence/absence of specific inhibitors of each enzyme. In liver slices, phenylacetaldehyde was rapidly converted to phenylacetic acid. Phenylacetic acid was the main metabolite of 2-phenylethylamine, via the intermediate phenylacetaldehyde. Phenylacetic acid formation was completely inhibited by disulfiram (specific inhibitor of aldehyde dehydrogenase), whereas isovanillin (specific inhibitor of aldehyde oxidase) inhibited acid formation to a lesser extent and allopurinol (specific inhibitor of xanthine oxidase) had little or no effect. Therefore, in liver slices, phenylacetaldehyde is rapidly oxidized by aldehyde dehydrogenase and aldehyde oxidase with little or no contribution from xanthine oxidase.

Purpose Leukotrienes and prostaglandins, products of arachidonic acid metabolism, sustain both systemic and lesion-localized inflammation. Tumor-associated Inflammation can also contribute to the pathogenesis of colon cancer. Patients with inflammatory bowel disease (IBD) have increased risk of developing colon cancer. The levels of 5-lipoxygenase (5-LO), the key enzyme for leukotrienes production, are increased in colon cancer specimens and colonic dysplastic lesions. Here we report that Zileuton, a specific 5-LO inhibitor, can prevent polyp formation by efficiently reducing the tumor-associated and systemic inflammation in APCΔ468 mice. Experimental Design In the current study, we inhibited 5-LO by dietary administration of Zileuton in the APCΔ468 mouse model of polyposis and analyzed the effect of in vivo 5-LO inhibition on tumor-associated and systemic inflammation. Results Zileuton-fed mice developed fewer polyps and displayed marked reduction in systemic and polyp-associated inflammation. Pro-inflammatory cytokines and pro-inflammatory innate and adaptive immunity cells were reduced both in the lesions and systemically. As part of tumor-associated inflammation Leukotriene B4 (LTB4), product of 5-LO activity, is increased focally in human dysplastic lesions. The 5-LO enzymatic activity was reduced in the serum of Zileuton treated polyposis mice. Conclusions This study demonstrates that dietary administration of 5-LO specific inhibitor in the polyposis mouse model decreases polyp burden, and suggests that Zileuton may be a potential chemo-preventive agent in patients that are high-risk of developing colon cancer.

Purpose: To examine whether induction of autophagy is a mechanism of leukemic cell resistance to dual mTORC1/mTORC2 inhibitors in acute myelogenous leukemia (AML) leukemic progenitors. Experimental Design: Combinations of different experimental approaches were used to assess induction of autophagy, including immunoblotting to detect effects on LC3II and p62/SQTM1 expression and on ULK1 phosphorylation, immunofluorescence, and electron microscopy. Functional responses were assessed using cell viability and apoptosis assays, and clonogenic leukemic progenitor assays in methylcellulose. Results: We provide evidence that treatment of AML cells with catalytic mTOR inhibitors results in induction of autophagy, which acts as a regulatory mechanism to promote leukemic cell survival. Such induction of autophagy by dual mTORC1/mTORC2 inhibitors partially protects primitive leukemic precursors from the inhibitory effects of such agents and limits their activities. Simultaneous blockade of the autophagic process using chloroquine or by knockdown of ULK1 results in enhanced antileukemic responses. Conclusions: Dual targeting of mTORC2 and mTORC1 results in induction of autophagy in AML cells. Combinations of catalytic mTOR targeting agents and autophagy inhibitors may provide a unique approach to target primitive leukemic precursors in AML. Clin Cancer Res; 20(9); 2400–9. ©2014 AACR.

BACKGROUND Surveillance colonoscopy using random biopsies to detect colitis-associated cancer (CAC) suffers from poor sensitivity. Although chromoendoscopy improves detection, acceptance in the community has been slow. Here, we examine the usefulness of near infrared fluorescence (NIRF) endoscopy to image molecular probes for cathepsin activity in colitis-induced dysplasia. METHODS In patient samples, cathepsin activity was correlated with colitis and dysplasia. In mice, cathepsin activity was detected as fluorescent hydrolysis product of substrate-based probes after injection into Il10(-/-) colitic mice. Fluorescence colonoscopy and colonic whole-mount imaging were performed before complete sectioning and pathology review of resected colons. RESULTS Cathepsin activity was 5-fold and 8-fold higher in dysplasia and CAC, respectively, compared with areas of mild colitis in patient tissue sections. The signal-to-noise ratios for dysplastic lesions seen by endoscopy in Il10(-/-) mice were 5.2 ± 1.3 (P = 0.0001). Lesions with increased NIRF emissions were classified as raised or flat dysplasia, lymphoid tissue, and ulcers. Using images collected by endoscopy, a receiver operating characteristic curve for correctly diagnosing dysplasia was calculated. The area under the curve was 0.927. At a cutoff of 1000 mean fluorescence intensity, the sensitivity and specificity for detecting dysplasia were 100% and 83%, respectively. Analysis revealed that focally enhanced NIRF emissions derived from increased numbers of infiltrating myeloid-derived suppressor cells and macrophages with equivalent cathepsin activity. CONCLUSIONS These studies indicate that cathepsin substrate-based probe imaging correctly identifies dysplastic foci within chronically inflamed colons. Combined white light and NIRF endoscopy presents unique advantages that may increase sensitivity and specificity of surveillance colonoscopy in patients with CAC.

We have recently shown that arsenic trioxide (As 2O 3) is a potent inducer of autophagic degradation of the BCR-ABL1 oncoprotein, which is the cause of chronic myeloid leukemia (CML) and Ph+ acute lymphoid leukemia (Ph+ ALL). Our recently published work has shown that pharmacological inhibition of autophagy or molecularly targeting of elements of the autophagic machinery partially reverses the suppressive effects of As 2O 3 on primitive leukemic precursors from CML patients. Altogether, our studies have provided direct evidence that arsenic-induced, autophagy-mediated, degradation of BCR-ABL1 is an important mechanism for the generation of the effects of As 2O 3 on BCR-ABL1 transformed leukemic progenitors. These studies raise the potential of future clinical-translational efforts employing combinations of arsenic trioxide with autophagy-modulating agents to promote elimination of early leukemic progenitors and, possibly, leukemia-initiating stem cells.

We provide evidence that arsenic trioxide (As2O3) targets the BCR-ABL oncoprotein via a novel mechanism involving p62/SQSTM1-mediated localization of the oncoprotein to the autolysosomes and subsequent degradation mediated by the protease cathepsin B. Our studies demonstrate that inhibitors of autophagy or cathepsin B activity and/or molecular targeting of p62/SQSTM1, Atg7, or cathepsin B result in partial reversal of the suppressive effects of AS2O3 on BCR-ABL expressing leukemic progenitors, including primitive leukemic precursors from chronic myelogenous leukemia (CML) patients. Altogether, these findings indicate that autophagic degradation of BCR-ABL is critical for the induction of the antileukemic effects of As2O3 and raise the potential for future therapeutic approaches to target BCR-ABL expressing cells by modulating elements of the autophagic machinery to promote BCR-ABL degradation.

To address the role of Tpl2, a MAP3K8 that regulates innate/adaptive immunity and inflammation, in intestinal tumorigenesis, we crossed a Tpl2 KO allele into the Apc min/+ genetic background. Here, we show that Apc min/+ /Tpl2 −/− mice exhibit a fivefold increase in the number of intestinal adenomas. Bone marrow transplantation experiments revealed that the enhancement of polyposis was partially hematopoietic cell-driven. Consistent with this observation, Tpl2 ablation promoted intestinal inflammation. IL-10 levels and regulatory T-cell numbers were lower in the intestines of Tpl2 −/− mice, independent of Apc and polyp status, suggesting that they were responsible for the initiation of the enhancement of tumorigenesis caused by the ablation of Tpl2 . The low IL-10 levels correlated with defects in mTOR activation and Stat3 phosphorylation in Toll-like receptor-stimulated macrophages and with a defect in inducible regulatory T-cell generation and function. Both polyp numbers and inflammation increased progressively with time. The rate of increase of both, however, was more rapid in Apc min/+ /Tpl2 −/− mice, suggesting that the positive feedback initiated by inflammatory signals originating in developing polyps is more robust in these mice. This may be because these mice have a higher intestinal polyp burden as a result of the enhancement of tumor initiation.

Aim: To investigate the mechanism(s) by which the intestinal commensal microbe Lactobacillus acidophilus can affect host immunity, we studied the role of a component of the cell wall, lipoteichoic acid, in colitis. Materials & methods: Colitis was induced by the intraperitoneal injection of pathogenic CD4+CD25-CD45RBhi T cells into Rag1-/- mice. The parental strain, NCK56, or the lipoteichoic acid-deficient strain, NCK2025, was then administered orally. Fluorescent microscopy was employed to examine resulting cell populations and their cytokine production in the colon. Results: NCK2025 enhanced IL-10 production by dendritic cells and macrophages. Increased numbers of regulatory dendritic cells coincided with the induction of activated FoxP3+ Tregs. Conclusion: These results suggest that the oral administration of the genetically modified strain NCK2025 may be an effective immunotherapeutic approach that reprograms the immune response in colonic inflammatory conditions.

Mast cells (MC) are a bone marrow-derived, long-lived, heterogeneous cellular population that function both as positive and negative regulators of immune responses. They are arguably the most productive chemical factory in the body and influence other cells through both soluble mediators and cell-to-cell interaction. MC are commonly seen in various tumors and have been attributed alternatively with tumor rejection or tumor promotion. Tumor-infiltrating MC are derived both from sentinel and recruited progenitor cells. MC can directly influence tumor cell proliferation and invasion but also help tumors indirectly by organizing its microenvironment and modulating immune responses to tumor cells. Best known for orchestrating inflammation and angiogenesis, the role of MC in shaping adaptive immune responses has become a focus of recent investigations. MC mobilize T cells and antigen-presenting dendritic cells. They function as intermediaries in regulatory T cells (Treg)-induced tolerance but can also modify or reverse Treg-suppressive properties. The central role of MC in the control of innate and adaptive immunity endows them with the ability to tune the nature of host responses to cancer and ultimately influence the outcome of disease and fate of the cancer patient.

Arachidonic acid metabolism has been implicated in colon carcinogenesis, but the role of hematopoietic 5-lipoxygenase (5LO) that may impact tumor immunity in development of colon cancer has not been explored. Here we show that tissue-specific deletion of the 5LO gene in hematopoietic cells profoundly attenuates polyp development in the APCΔ468 murine model of colon polyposis. In vitro analyses indicated that mast cells in particular utilized 5LO to limit proliferation of intestinal epithelial cells and to mobilize myeloid-derived suppressor cells (MDSCs). Mice lacking hemapoietic expression of 5LO exhibited reduced recruitment of MDSCs to the spleen, mesenteric lymph nodes, and primary tumor site. 5LO deficiency also reduced the activity in MDSCs of arginase-1, which is thought to be critical for MDSC function. Together, our results establish a pro-tumorigenic role of hematopoietic 5LO in the immune microenvironment and suggest 5LO inhibition as an avenue for future investigation in treatment of colorectal polyposis and cancer. Cancer Res; 71(5); 1627–36. ©2011 AACR.

Purpose: To assess the clinical and pathologic significance of mast cell infiltration in human pancreatic cancer and evaluate crosstalk between mast cells and cancer cells in vitro. Experimental Design: Immunohistochemistry for tryptase was done on 53 pancreatic cancer specimens. Mast cell counts were correlated with clinical variables and survival. Serum tryptase activity from patients with cancer was compared with patients with benign pancreatic disease. In vitro, the effect of pancreatic cancer–conditioned medium on mast cell migration was assessed. The effect of conditioned medium from the human mast cell line, LAD-2, on cancer and normal ductal cell proliferation was assessed by thymidine incorporation. Matrigel invasion assays were used to evaluate the effect of mast cell–conditioned medium on cancer cell invasion in the presence and absence of a matrix metalloproteinase inhibitor, GM6001. Results: Mast cell infiltration was significantly increased in pancreatic cancer compared with normal pancreatic tissue (11.4 ± 6.7 versus 2.0 ± 1.4, P < 0.001). Increased infiltrating mast cells correlated with higher grade tumors (P < 0.0001) and worse survival. Patients with pancreatic cancer had elevated serum tryptase activity (P < 0.05). In vitro, AsPC1 and PANC-1 cells induced mast cell migration. Mast cell–conditioned medium induced pancreatic cancer cell migration, proliferation, and invasion but had no effect on normal ductal cells. Furthermore, the effect of mast cells on cancer cell invasion was, in large part, matrix metalloproteinase–dependent. Conclusions: Tumor-infiltrating mast cells are associated with worse prognosis in pancreatic cancer. In vitro, the interaction between mast cells and pancreatic cancer cells promotes tumor growth and invasion. Clin Cancer Res; 16(8); 2257–65. ©2010 AACR.

Autophagy is increasingly an area of high potential therapeutic interest in hematopoietic malignancies, but its precise involvement in the control of leukemic cell growth and survival remain to be defined. We provide evidence that arsenic trioxide (As2O3) targets the BCR-ABL oncoprotein for degradation via autophagy, involving the p62/SQSTM1 and the protease cathepsin B. Utilizing either florescence probes or gold-conjugate antibodies we show co-localization of BCR-ABL and p62/SQSTM1 in autolysosomes. Also, by drug inhibition of either autophagy or cathepsin B, or by molecularly targeting p62/SQSMT1, Atg7 or cathepsin B, we documented reversal of the suppressive effect of As2O3 on BCR-ABL expressing cells, including primitive leukemic precursors from chronic myelogenous leukemia (CML) patients. Altogether, our data indicate that autophagy-induced degradation of BCR-ABL is critical for the generation of As2O3 antileukemic effects. These results raise the potential of targeting the autophagic machinery to enhance the antileukemic properties of arsenic trioxide on leukemia initiating stem cells. 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 LB-81. doi:1538-7445.AM2012-LB-81

Phosphofructokinase (PFK) from Rana ridibunda erythrocytes was purified about 570-fold by column chromatography on Cibacron Blue Sepharose. The resulting enzyme preparation had a specific activity of 1.94 U/mg protein and a pH maximum of 7.6. The molecular weight as determined by HPLC chromatography was 330,000 Da. The S 0.5 value for fructose-6-phosphate (F6P) was 5.6 mM and the K m for ATP 0.87 mM. The enzyme was sensitive to inhibition by ATP which was increased with lower F6P concentrations. At physiological levels of 2,3-diphosphoglycerate (0.35 μmol/ml RBC), 20% of PFK activity was inhibited. Significant activations under cellular conditions were exercised by AMP and, to a lesser extent, by P 1 . Micromolar concentrations of fructose-2,6-bisphosphate and glucose-1,6-bisphosphate were also potent activators of the erythrocyte enzyme. Fructose-1,6-bisphosphate (10–50) μM activated the enzyme to a limited extent. With respect to these effects, it is suggested that PFK is a significant enzyme in regulating the glycolytic flux of Rana ridibunda red blood cells. The existence of a regulatory mechanism controlled by the energy status of the red cell, as well as the state of oxygenation of haemoglobin, is discussed, in which PFK occupies a central role.

Normal mammalian cells have a limited lifespan in culture and hypotheses explaining cellular senescence usually fall into one of two categories. One of these postulates that random errors or damage accumulate in essential macromolecules and eventually outstrip the cell's capacity for resynthesis and repair. The second considers the changes when immortal clones are produced from normal cells and in particular the lifespans of hybrids when cells of differing growth potentials are fused. These data can be explained by postulating that the mortal phenotype is dominant and that trans-acting growth inhibitors are involved in limiting lifespan. But the results do not indicate if the inhibitors are the primary cause of senescence or a secondary effect induced by quite different initial events. We suggest that normal cells possess proof-reading mechanisms which monitor the accuracy of chromosome segregation and replication and which can induce the synthesis of growth inhibitors when they detect major errors in chromosome metabolism. It is further postulated that random damage accumulates during the growth of normal cells and eventually leads to detectable chromosome changes and the synthesis of inhibitors. Our hypothesis predicts that the emergence of immortal clones will be linked to the absence of active inhibitors and therefore to a loss in the fidelity of chromosome metabolism. Data are quoted which show that in contrast to normal cells, immortal clones have highly irregular karyotypes, amplify segments of their chromosomes, integrate exogenous DNA efficiently, maintain a constant level of 5-methylcytosine residues and have high frequencies of chromosomal aberrations. The mechanism of the proof-reading is unknown, but it may monitor changes in the patterns by which chromosome domains are attached to the nuclear matrix. J Theor Biol

It has been estimated that up to 30% of detectable polyps in patients regress spontaneously. One major challenge in the evaluation of effective therapy of cancer is the readout for tumor regression and favorable biological response to therapy. Inducible near infra-red (NIR) fluorescent probes were utilized to visualize intestinal polyps of mice hemizygous for a novel truncation of the Adenomatous Polyposis coli (APC) gene. Laser Scanning Confocal Microscopy in live mice allowed visualization of cathepsin activity in richly vascularized benign dysplastic lesions. Using biotinylated suicide inhibitors we quantified increased activities of the Cathepsin B & Z in the polyps. More than (3/4) of the probe signal was localized in CD11b(+)Gr1(+) myeloid derived suppressor cells (MDSC) and CD11b(+)F4/80(+) macrophages infiltrating the lesions. Polyposis was attenuated through genetic ablation of cathepsin B, and suppressed by neutralization of TNFalpha in mice. In both cases, diminished probe signal was accounted for by loss of MDSC. Thus, in vivo NIR imaging of focal cathepsin activity reveals inflammatory reactions etiologically linked with cancer progression and is a suitable approach for monitoring response to therapy.