Tumor necrosis correlates with angiogenesis and is a predictor of poor prognosis in malignant mesothelioma

Objectives: Malignant mesothelioma (MM) is a fatal tumor of increasing incidence related to asbestos exposure. Microscopic tumor necrosis (TN) is a poor prognostic factor in solid tumors, but it has not been characterized in MM. We wished to evaluate the incidence of TN in MM and its correlations with clinicopathologic factors, angiogenesis, and survival. Methods: TN was graded in 171 routine formalin-fixed, paraffin-embedded hematoxylin-eosinstained tumor sections by two independent observers. Angiogenesis was assessed by the microvessel count (MVC) of CD34 immunostained sections. TN was correlated with survival by Kaplan-Meier and log-rank analysis, and stepwise, multivariate Cox models were used to compare TN with angiogenesis and established prognostic factors and prognostic scoring systems. Results: TN was identified in 39 cases (22.8%) and correlated with low hemoglobin (p = 0.01), thrombocytosis (p = 0.04), and high MVC (p = 0.02). TN was a poor prognostic factor in univariate analysis (p = 0.008). Patients with TN had a median survival of 5.3 months vs 8.3 months in negative cases. Independent indicators of poor prognosis in multivariate analysis were nonepithelioid cell type (p = 0.0001), performance status > 0 (p = 0.007), and increasing MVC (p = 0.004) but not TN. TN contributed independently to the European Organisation for Research and Treatment of Cancer (EORTC) [p = 0.03] and to the Cancer and Leukemia Group B (CALGB) [p = 0.03] prognostic groups in respective multivariate Cox analyses. Conclusions: TN correlates with angiogenesis and is a poor prognostic factor in MM. TN contributes to the EORTC and CALGB prognostic scoring systems.

An evaluation of tumor oxygenation and gene expression in patients with early stage non-small cell lung cancers

Background: To directly assess tumor oxygenation in resectable non - small cell lung cancers (NSCLC) and to correlate tumor pO2 and the selected gene and protein expression to treatment outcomes. Methods: Twenty patients with resectable NSCLC were enrolled. Intraoperative measurements of normal lung and tumor pO2 were done with the Eppendorf polarographic electrode. All patients had plasma osteopontin measurements by ELISA. Carbonic anhydrase-IX (CA IX) staining of tumor sections was done in the majority of patients (n = 16), as was gene expression profiling (n = 12) using cDNA microarrays. Tumor pO2 was correlated with CA IX staining, osteopontin levels, and treatment outcomes. Results: The median tumor pO2 ranged from 0.7 to 46 mm Hg (median, 16.6) and was lower than normal lung pO2 in all but one patient. Because both variables were affected by the completeness of lung deflation during measurement, we used the ratio of tumor/normal lung (T/L) pO2 as a reflection of tumor oxygenation. The median T/L pO 2 was 0.13. T/L pO2 correlated significantly with plasma osteopontin levels (r = 0.53, P = 0.02) and CA IX expression (P = 0.006). Gene expression profiling showed that high CD44 expression was a predictor for relapse, which was confirmed by tissue staining of CD44 variant 6 protein. Other variables associated with the risk of relapse were T stage (P = 0.02), T/L pO2 (P = 0.04), and osteopontin levels (P = 0.001). Conclusions: Tumor hypoxia exists in resectable NSCLC and is associated with elevated expression of osteopontin and CA IX. Tumor hypoxia and elevated osteopontin levels and CD44 expression correlated with poor prognosis. A larger study is needed to confirm the prognostic significance of these factors. © 2006 American Association for Cancer Research.

Galectin-1 : a link between tumor hypoxia and tumor immune privilege

Purpose: To identify a 15-KDa novel hypoxia-induced secreted protein in head and neck squamous cell carcinomas (HNSCC) and to determine its role in malignant progression. Methods: We used surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and tandem MS to identify a novel hypoxia-induced secreted protein in FaDu cells. We used immunoblots, real-time polymerase chain reaction (PCR), and enzyme-linked immunoabsorbent assay to confirm the hypoxic induction of this secreted protein as galectin-1 in cell lines and xenografts. We stained tumor tissues from 101 HNSCC patients for galectin-1, CA IX (carbonic anhydrase IX, a hypoxia marker) and CDS (a T-cell marker). Expression of these markers was correlated to each other and to treatment outcomes. Results: SELDI-TOF studies yielded a hypoxia-induced peak at 15 kDa that proved to be galectin-1 by MS analysis. Immunoblots and PCR studies confirmed increased galectin-1 expression by hypoxia in several cancer cell lines. Plasma levels of galectin-1 were higher in tumor-bearing severe combined immunodeficiency (SCID) mice breathing 10% O 2 compared with mice breathing room air. In HNSCC patients, there was a significant correlation between galectin-1 and CA IX staining (P = .01) and a strong inverse correlation between galectin-1 and CDS staining (P = .01). Expression of galectin-1 and CDS were significant predictors for overall survival on multivariate analysis. Conclusion: Galectin-1 is a novel hypoxia-regulated protein and a prognostic marker in HNSCC. This study presents a new mechanism on how hypoxia can affect the malignant progression and therapeutic response of solid tumors by regulating the secretion of proteins that modulate immune privilege. © 2005 by American Society of Clinical Oncology.

A metastatic neuroendocrine anaplastic small cell tumor in a patient with multiple endocrine neoplasia type 1 syndrome : assessment of disease status and response to doxorubicin, cyclophosphamide, etoposide chemotherapy through scintigraphic imaging with 111In-pentetreotide

Extrapulmonary small cell and small cell neuroendocrine tumors of unknown primary site are, in general, aggressive neoplasms with a short median survival. Like small cell lung cancer (SCLC), they often are responsive to chemotherapy and radiotherapy. Small cell lung cancer and well differentiated neuroendocrine carcinomas of the gastrointestinal tract and pancreas tend to express somatostatin receptors. These tumors may be localized in patients by scintigraphic imaging using radiolabeled somatostatin analogues. A patient with an anaplastic neuroendocrine small cell tumor arising on a background of multiple endocrine neoplasia type 1 syndrome is reported. The patient had a known large pancreatic gastrinoma and previously treated parathyroid adenopathy. At presentation, there was small cell cancer throughout the liver and skeleton. Imaging with a radiolabeled somatostatin analogue, 111In- pentetreotide (Mallinckrodt Medical B. V., Petten, Holland), revealed all sites of disease detected by routine biochemical and radiologic methods. After six cycles of chemotherapy with doxorubicin, cyclophosphamide, and etoposide, there was almost complete clearance of the metastatic disease. 111In-pentetreotide scintigraphy revealed uptake consistent with small areas of residual disease in the liver, the abdomen (in mesenteric lymph nodes), and posterior thorax (in a rib). The primary gastrinoma present before the onset of the anaplastic small cell cancer showed no evidence of response to the treatment. The patient remained well for 1 year and then relapsed with brain, lung, liver, and skeletal metastases. Despite an initial response to salvage radiotherapy and chemotherapy with carboplatin and dacarbazine, the patient died 6 months later.

Lipoxygenase metabolism : roles in tumor progression and survival

The metabolism of arachidonic acid through lipoxygenase pathways leads to the generation of various biologically active eicosanoids. The expression of these enzymes vary throughout the progression of various cancers, and thereby they have been shown to regulate aspects of tumor development. Substantial evidence supports a functional role for lipoxygenase-catalyzed arachidonic and linoleic acid metabolism in cancer development. Pharmacologic and natural inhibitors of lipoxygenases have been shown to suppress carcinogenesis and tumor growth in a number of experimental models. Signaling of hydro[peroxy]fatty acids following arachidonic or linoleic acid metabolism potentially effect diverse biological phenomenon regulating processes such as cell growth, cell survival, angiogenesis, cell invasion, metastatic potential and immunomodulation. However, the effects of distinct LOX isoforms differ considerably with respect to their effects on both the individual mechanisms described and the tumor being examined. 5-LOX and platelet type 12-LOX are generally considered pro-carcinogenic, with the role of 15-LOX-1 remaining controversial, while 15-LOX-2 suppresses carcinogenesis. In this review, we focus on the molecular mechanisms regulated by LOX metabolism in some of the major cancers. We discuss the effects of LOXs on tumor cell proliferation, their roles in cell cycle control and cell death induction, effects on angiogenesis, migration and the immune response, as well as the signal transduction pathways involved in these processes. Understanding the molecular mechanisms underlying the anti-tumor effect of specific, or general, LOX inhibitors may lead to the design of biologically and pharmacologically targeted therapeutic strategies inhibiting LOX isoforms and/or their biologically active metabolites, that may ultimately prove useful in the treatment of cancer, either alone or in combination with conventional therapies. © 2007 Springer Science+Business Media, LLC.

Carbonic anhydrase IX expression, a novel surrogate marker of tumor hypoxia, is associated with a poor prognosis in non-small-cell lung cancer

Purpose To evaluate carbonic anhydrase (CA) IX as a surrogate marker of hypoxia and investigate the prognostic significance of different patterns of expression in non-small-cell lung cancer (NSCLC). Methods Standard immunohistochemical techniques were used to study CA IX expression in 175 resected NSCLC tumors. CA IX expression was determined by Western blotting in A549 cell lines grown under normoxic and hypoxic conditions. Measurements from microvessels to CA IX positivity were obtained. Results CA IX immunostaining was detected in 81.8% of patients. Membranous (m) (P = .005), cytoplasmic (c) (P = .018), and stromal (P < .001) CA IX expression correlated with the extent of tumor necrosis (TN). The mean distance from vascular endothelium to the start of tumor cell positivity was 90 μm, which equates to an oxygen pressure of 5.77 mmHg. The distance to blood vessels from individual tumor cells or tumor cell clusters was greater if they expressed mCA IX than if they did not (P < .001). Hypoxic exposure of A549 cells for 16 hours enhanced CAIX expression in the nuclear and cytosolic extracts. Perinuclear (p) CA IX (P = .035) was associated with a poor prognosis. In multivariate analysis, pCA IX (P = .004), stage (P = .001), platelet count (P = .011), sex (P = .027), and TN (P = .035) were independent poor prognostic factors. Conclusion These results add weight to the contention that mCA IX is a marker of tumor cell hypoxia. The absence of CA IX staining close to microvessels suggests that these vessels are functionally active. pCA IX expression is representative of an aggressive phenotype. © 2003 by American Society of Clinical Oncology.

Macrophage and mast-cell invasion of tumor cell islets confers a marked survival advantage in non-small-cell lung cancer

Purpose The role played by the innate immune system in determining survival from non-small-cell lung cancer (NSCLC) is unclear. The aim of this study was to investigate the prognostic significance of macrophage and mast-cell infiltration in NSCLC. Methods We used immunohistochemistry to identify tryptase+ mast cells and CD68+ macrophages in the tumor stroma and tumor islets in 175 patients with surgically resected NSCLC. Results Macrophages were detected in both the tumor stroma and islets in all patients. Mast cells were detected in the stroma and islets in 99.4% and 68.5% of patients, respectively. Using multivariate Cox proportional hazards analysis, increasing tumor islet macrophage density (P < .001) and tumor islet/stromal macrophage ratio (P < .001) emerged as favorable independent prognostic indicators. In contrast, increasing stromal macrophage density was an independent predictor of reduced survival (P = .001). The presence of tumor islet mast cells (P = .018) and increasing islet/stromal mast-cell ratio (P = .032) were also favorable independent prognostic indicators. Macrophage islet density showed the strongest effect: 5-year survival was 52.9% in patients with an islet macrophage density greater than the median versus 7.7% when less than the median (P < .0001). In the same groups, respectively, median survival was 2,244 versus 334 days (P < .0001). Patients with a high islet macrophage density but incomplete resection survived markedly longer than patients with a low islet macrophage density but complete resection. Conclusion The tumor islet CD68+ macrophage density is a powerful independent predictor of survival from surgically resected NSCLC. The biologic explanation for this and its implications for the use of adjunctive treatment requires further study. © 2005 by American Society of Clinical Oncology.

Targeting amino acid transport in metastatic castration-resistant prostate cancer : effects on cell cycle, cell growth, and tumor development

Background L-type amino acid transporters (LATs) uptake neutral amino acids including L-leucine into cells, stimulating mammalian target of rapamycin complex 1 signaling and protein synthesis. LAT1 and LAT3 are overexpressed at different stages of prostate cancer, and they are responsible for increasing nutrients and stimulating cell growth. Methods We examined LAT3 protein expression in human prostate cancer tissue microarrays. LAT function was inhibited using a leucine analog (BCH) in androgen-dependent and -independent environments, with gene expression analyzed by microarray. A PC-3 xenograft mouse model was used to study the effects of inhibiting LAT1 and LAT3 expression. Results were analyzed with the Mann-Whitney U or Fisher exact tests. All statistical tests were two-sided. Results LAT3 protein was expressed at all stages of prostate cancer, with a statistically significant decrease in expression after 4–7 months of neoadjuvant hormone therapy (4–7 month mean = 1.571; 95% confidence interval = 1.155 to 1.987 vs 0 month = 2.098; 95% confidence interval = 1.962 to 2.235; P = .0187). Inhibition of LAT function led to activating transcription factor 4–mediated upregulation of amino acid transporters including ASCT1, ASCT2, and 4F2hc, all of which were also regulated via the androgen receptor. LAT inhibition suppressed M-phase cell cycle genes regulated by E2F family transcription factors including critical castration-resistant prostate cancer regulatory genes UBE2C, CDC20, and CDK1. In silico analysis of BCH-downregulated genes showed that 90.9% are statistically significantly upregulated in metastatic castration-resistant prostate cancer. Finally, LAT1 or LAT3 knockdown in xenografts inhibited tumor growth, cell cycle progression, and spontaneous metastasis in vivo. Conclusion Inhibition of LAT transporters may provide a novel therapeutic target in metastatic castration-resistant prostate cancer, via suppression of mammalian target of rapamycin complex 1 activity and M-phase cell cycle genes.

Preliminary characterisation of tumor necrosis factor alpha and interleukin-10 responses to chlamydia pecorum infection in the koala (Phascolarctos cinereus)

Debilitating infectious diseases caused by Chlamydia are major contributors to the decline of Australia's iconic native marsupial species, the koala (Phascolarctos cinereus). An understanding of koala chlamydial disease pathogenesis and the development of effective strategies to control infections continue to be hindered by an almost complete lack of species-specific immunological reagents. The cell-mediated immune response has been shown to play an influential role in the response to chlamydial infection in other hosts. The objective of this study, hence, was to provide preliminary data on the role of two key cytokines, pro-inflammatory tumour necrosis factor alpha (TNFα) and anti-inflammatory interleukin 10 (IL10), in the koala Chlamydia pecorum response. Utilising sequence homology between the cytokine sequences obtained from several recently sequenced marsupial genomes, this report describes the first mRNA sequences of any koala cytokine and the development of koala specific TNFα and IL10 real-time PCR assays to measure the expression of these genes from koala samples. In preliminary studies comparing wild koalas with overt chlamydial disease, previous evidence of C. pecorum infection or no signs of C. pecorum infection, we revealed strong but variable expression of TNFα and IL10 in wild koalas with current signs of chlamydiosis. The description of these assays and the preliminary data on the cell-mediated immune response of koalas to chlamydial infection paves the way for future studies characterising the koala immune response to a range of its pathogens while providing reagents to assist with measuring the efficacy of ongoing attempts to develop a koala chlamydial vaccine.

Short-term single treatment of chemotherapy results in the enrichment of ovarian cancer stem cell-like cells leading to an increased tumor burden

Over 80% of women diagnosed with advanced-stage ovarian cancer die as a result of disease recurrence due to failure of chemotherapy treatment. In this study, using two distinct ovarian cancer cell lines (epithelial OVCA 433 and mesenchymal HEY) we demonstrate enrichment in a population of cells with high expression of CSC markers at the protein and mRNA levels in response to cisplatin, paclitaxel and the combination of both. We also demonstrate a significant enhancement in the sphere forming abilities of ovarian cancer cells in response to chemotherapy drugs. The results of these in vitro findings are supported by in vivo mouse xenograft models in which intraperitoneal transplantation of cisplatin or paclitaxel-treated residual HEY cells generated significantly higher tumor burden compared to control untreated cells. Both the treated and untreated cells infiltrated the organs of the abdominal cavity. In addition, immunohistochemical studies on mouse tumors injected with cisplatin or paclitaxel treated residual cells displayed higher staining for the proliferative antigen Ki67, oncogeneic CA125, epithelial E-cadherin as well as cancer stem cell markers such as Oct4 and CD117, compared to mice injected with control untreated cells. These results suggest that a short-term single treatment of chemotherapy leaves residual cells that are enriched in CSC-like traits, resulting in an increased metastatic potential. The novel findings in this study are important in understanding the early molecular mechanisms by which chemoresistance and subsequent relapse may be triggered after the first line of chemotherapy treatment.

Chemotaxis and chemokinesis of human prostate tumor cell lines in response to human prostate stromal cell secretory proteins containing a nerve growth factor-like protein

The migration of three human prostate tumor epithelial cell lines (TSU-pr1, PC-3, DU-145) in response to secreted protein from a human prostate stromal cell line was investigated by using the modified blind-well Boyden chamber assay. Migrated cells were quantified by spectrophotometrically measuring the concentration of crystal violet stain extracted from their nuclei. Cell number was correlated linearly with the concentration of extracted crystal violet stain. All three tumor cell lines showed intrinsic migratory ability in the absence of chemoattractants, such that approximately 1-7% of plated cells migrated across the filter of the Boyden chambers during a 5-h incubation period. Prostate tumor cell migration was significantly enhanced (3-13-fold) in response to stromal cell secretory protein in a dose-dependent manner, whereas bovine serum albumin had no effect on stimulating tumor cell migration. Immunoprecipitation of the stromal cell secreted protein with a nerve growth factor antibody partially and significantly reduced its stimulatory activity for tumor cell migration. A Zigmond-Hirsch matrix assay of tumor cell migration in response to various concentration gradients of stromal cell secreted protein demonstrated both chemotaxis and chemokinesis by all three cell lines. These results are consistent with the stromal cell secretory protein stimulation of chemokinetic tumor cell migration through the capsule of the prostate. Outside of the prostate gland metastasis of tumor cells may occur by chemotaxis to preferential sites containing chemoattractants similar to or related to maintenance factors that can substitute for components of stromal cell secretory protein.

Bimolecular interaction of Insulin-Like Growth Factor (IGF) binding protein-2 with αvβ3 negatively modulates IGF-I-Mediated migration and tumor growth

Both the integrin and insulin-like growth factor binding protein (IGFBP) families independently play important roles in modulating tumor cell growth and progression. We present evidence for a specific cell surface localization and a bimolecular interaction between the αvβ3 integrin and IGFBP-2. The interaction, which could be specifically perturbed using vitronectin and αvβ3 blocking antibodies, was shown to modulate IGF-mediated cellular migration responses. Moreover, this interaction was observed in vivo and correlated with reduced tumor size of the human breast cancer cells, MCF-7β3, which overexpressed the αvβ3 integrin. Collectively, these results indicate that αvβ3 and IGFBP-2 act cooperatively in a negative regulatory manner to reduce tumor growth and the migratory potential of breast cancer cells.

Isolation and characterization of tumor cells from the ascites of ovarian cancer patients : molecular phenotype of chemoresistant ovarian tumors

Tumor cells in ascites are a major source of disease recurrence in ovarian cancer patients. In an attempt to identify and profile the population of ascites cells obtained from ovarian cancer patients, a novel method was developed to separate adherent (AD) and non-adherent (NAD) cells in culture. Twenty-five patients were recruited to this study; 11 chemonaive (CN) and 14 chemoresistant (CR). AD cells from both CN and CR patients exhibited mesenchymal morphology with an antigen profile of mesenchymal stem cells and fibroblasts. Conversely, NAD cells had an epithelial morphology with enhanced expression of cancer antigen 125 (CA125), epithelial cell adhesion molecule (EpCAM) and cytokeratin 7. NAD cells developed infiltrating tumors and ascites within 12-14 weeks after intraperitoneal (i.p.) injections into nude mice, whereas AD cells remained non-tumorigenic for up to 20 weeks. Subsequent comparison of selective epithelial, mesenchymal and cancer stem cell (CSC) markers between AD and NAD populations of CN and CR patients demonstrated an enhanced trend in mRNA expression of E-cadherin, EpCAM, STAT3 and Oct4 in the NAD population of CR patients. A similar trend of enhanced mRNA expression of CD44, MMP9 and Oct4 was observed in the AD population of CR patients. Hence, using a novel purification method we demonstrate for the first time a distinct separation of ascites cells into epithelial tumorigenic and mesenchymal non-tumorigenic populations. We also demonstrate that cells from the ascites of CR patients are predominantly epithelial and show a trend towards increased mRNA expression of genes associated with CSCs, compared to cells isolated from the ascites of CN patients. As the tumor cells in the ascites of ovarian cancer patients play a dominant role in disease recurrence, a thorough understanding of the biology of the ascites microenvironment from CR and CN patients is essential for effective therapeutic interventions.

An MMP13-selective inhibitor delays primary tumor growth and the onset of tumor-associated osteolytic lesions in experimental models of breast cancer

We investigated the effects of the matrix metalloproteinase 13 (MMP13)-selective inhibitor, 5-(4-{4-[4-(4-fluorophenyl)-1,3-oxazol-2-yl]phenoxy}phenoxy)-5-(2-methoxyethyl) pyrimidine-2,4,6(1H,3H,5H)-trione (Cmpd-1), on the primary tumor growth and breast cancer-associated bone remodeling using xenograft and syngeneic mouse models. We used human breast cancer MDA-MB-231 cells inoculated into the mammary fat pad and left ventricle of BALB/c Nu/Nu mice, respectively, and spontaneously metastasizing 4T1.2-Luc mouse mammary cells inoculated into mammary fat pad of BALB/c mice. In a prevention setting, treatment with Cmpd-1 markedly delayed the growth of primary tumors in both models, and reduced the onset and severity of osteolytic lesions in the MDA-MB-231 intracardiac model. Intervention treatment with Cmpd-1 on established MDA-MB-231 primary tumors also significantly inhibited subsequent growth. In contrast, no effects of Cmpd-1 were observed on soft organ metastatic burden following intracardiac or mammary fat pad inoculations of MDA-MB-231 and 4T1.2-Luc cells respectively. MMP13 immunostaining of clinical primary breast tumors and experimental mice tumors revealed intra-tumoral and stromal expression in most tumors, and vasculature expression in all. MMP13 was also detected in osteoblasts in clinical samples of breast-to-bone metastases. The data suggest that MMP13-selective inhibitors, which lack musculoskeletal side effects, may have therapeutic potential both in primary breast cancer and cancer-induced bone osteolysis.

Contribution of fibroblast and mast cell (afferent) and tumor (efferent) IL-6 effects within the tumor microenvironment

Hyperactive inflammatory responses following cancer initiation have led to cancer being described as a 'wound that never heals'. These inflammatory responses elicit signals via NFκB leading to IL-6 production, and IL-6 in turn has been shown to induce epithelial to mesenchymal transition in breast cancer cells in vitro, implicating a role for this cytokine in cancer cell invasion. We previously have shown that conditioned medium derived from cancer-associated fibroblasts induced an Epithelial to Mesenchymal transition (EMT) in PMC42-LA breast cancer cells and we have now identify IL-6 as present in this medium. We further show that IL-6 is expressed approximately 100 fold higher in a cancer-associated fibroblast line compared to normal fibroblasts. Comparison of mouse-specific (stroma) and human-specific (tumor) IL-6 mRNA expression from MCF-7, MDA MB 468 and MDA MB 231 xenografts also indicated the stroma rather than tumor as a significantly higher source of IL-6 expression. Mast cells (MCs) feature in inflammatory cancer-associated stroma, and activated MCs secrete IL-6. We observed a higher MC index (average number of mast cells per xenograft section/average tumor size) in MDA MB 468 compared to MDA MB 231 xenografts, where all MC were observed to be active (degranulating). This higher MC index correlated with greater mouse-specific IL-6 expression in the MDA MB 468 xenografts, implicating MC as an important source of stromal IL-6. Furthermore, immunohistochemistry on these xenografts for pSTAT3, which lies downstream of the IL-6 receptor indicated frequent correlations between pSTAT3 and mast cell positive cells. Analysis of publically available databases for IL-6 expression in patient tissue revealed higher IL-6 in laser capture microdissected stroma compared to adjacent tissue epithelium from patients with inflammatory breast cancer (IBC) and invasive non-inflammatory breast cancer (non-IBC) and we show that IL-6 expression was significantly higher in Basal versus Luminal molecular/phenotypic groupings of breast cancer cell lines. Finally, we discuss how afferent and efferent IL-6 pathways may participate in a positive feedback cycle to dictate tumor progression.