Tumor microenvironment: what can effusions teach us?

Malignant effusions, which are composed of malignant pleural and peritoneal fluid, are an unusual manifestation of cancer and frequently portend a poor prognosis. Neoplastic cells that disseminate into cavities containing effusions are highly metastatic and possess a strong autonomous proliferative drive while concurrently being stimulatory of exudative effusions. Most effusions will respond to transient therapeutic intervention, including the obliteration of potential space via pleurodesis. Cure, however, is rare, thus making effusions a biologically, biochemically, and clinically important topic of study. The local microenvironment that supports malignant growth, invasion, and dissemination of the solid primary tumor into the vasculature is composed of activated stroma that includes scaffolding consisting of materials that promote the tumor function, and vascular structures to provide conduits for travel and nutrient delivery. Less is understood about the tumor-cell microenvironment in malignant effusions. The fluid nature of such a microenvironment when compared with the solid primary tumor may have significant implications for disease dissemination and progression. Dissecting the signaling activity and components of such microenvironments will improve our understanding and ultimately our ability to provide better patient care.

Inhibition of proliferation, migration, and matrix metalloprotease production in malignant mesothelioma cells by tyrosine kinase inhibitors.

The epidermal growth factor receptor (EGFR) is expressed in a variety of human solid tumors, including malignant mesothelioma. EGFR has been implicated in regulation of cell proliferation, survival, angiogenesis, and metastasis, making it an ideal target for drug development. ZD1839 (gefitinib) and OSI-774 (erlotinib) are new, low-molecular-weight, EGFR-selective tyrosine kinase (TK) inhibitors, whereas CI-1033 is a pan-EGFR family TK inhibitor. In the present study, we used ZD1839, OSI-774, and CI-1033 and investigated the effect of these drugs on proliferation, migration, and matrix metalloprotease (MMP) production in three malignant mesothelioma cell lines (M14K, ZL34, and SPC212). Using [3H]thymidine incorporation, DNA synthesis assay, we found that all three drugs inhibited transforming growth factor-alpha (TGF-alpha)-induced cellular proliferation in a dose-dependent manner. In addition, all three drugs induced apoptosis in ZL34 cells as determined by flow cytometry using annexin-V staining. Furthermore, all three drugs inhibited TGF-alpha-induced cell migration (chemotaxis) in a dose-dependent manner as determined by Boyden chamber assay. TGF-alpha-induced MMP-9 production was also inhibited in a dose-dependent manner as determined by gelatin zymography in three cell lines tested. In conclusion, our study demonstrates inhibitory effectiveness of EGFR-TK inhibitors in malignant mesothelioma cells and suggests that these drugs may be an effective treatment strategy for malignant mesothelioma.

Chemotaxis and chemokinesis of malignant mesothelioma cells to multiple growth factors.

BACKGROUND: Chemotaxis is defined as directional cell movement of cells towards concentration gradients of solubilized attractants, whereas chemokinesis is defined as random cell movement in the absence of chemoattractant gradients. Since tumor cell motility plays an important role in the process of tumor invasion and metastasis, we investigated these two distinct motile behaviors in highly invasive tumor, malignant mesothelioma. MATERIALS AND METHODS: Chemotaxis and chemokinesis of mesothelioma cells were assayed using Boyden chambers fitted with filters coated with collagen type IV and different growth factors and cytokines were used as chemoattractants. RESULTS: We found that growth factors such as epidermal growth factor, transforming growth factor-alpha, amphiregulin, heparin-binding epidermal growth factor-like growth factor, beta-cellulin, insulin-like growth factor-I, insulin-like growth factor-II and stem cell factor stimulated directional (chemotactic) and/or random (chemokinetic) motility in all mesothelioma cell lines tested, whereas none of acidic fibroblast growth factor, basic fibroblast growth factor, granulocyte-macrophage colony-stimulating factor or interleukin-6 induced migration in the same mesothelioma cells. CONCLUSION: These findings provide evidence that: (i) multiple growth factors can induce chemotaxis and chemokinesis in malignant mesothelioma cell lines, and (ii) may contribute to our understanding of the highly invasive behavior of malignant mesotheliomas in vivo.

Expression of hyaluronan synthases and hyaluronan in malignant mesothelioma cells.

BACKGROUND: Hyaluronan is one of the main components of the extracellular matrix. It is synthesized at the cell plasma membrane by specific hyaluronan synthases (HAS). Although a large number of studies have described hyaluronan in pleural effusion from malignant mesothelioma, the source of hyaluronan in malignant mesothelioma has been subject to controversy. MATERIALS AND METHODS: The mRNA expression of all three HAS in malignant mesothelioma cells was studied using RT-PCR. The hyaluronan production in culture medium of malignant mesothelioma cells was also examined using high-performance liquid chromatography (HPLC). RESULTS: We found that 9/10 malignant mesothelioma cell lines and one primary culture of malignant mesothelioma cells expressed HAS-1, while 10/10 malignant mesothelioma cell lines and one primary culture of malignant mesothelioma cells expressed HAS-2 and HAS-3. In addition, we demonstrated hyaluronan in the culture medium of 6 out of 10 malignant mesothelioma cell lines and one primary culture of malignant mesothelioma cells. CONCLUSION: Our results show that malignant mesothelioma cells express all three HAS and synthesize hyaluronan. The expression of HAS isoforms and hyaluronan in malignant mesothelioma cells in cultures and previous observations by other investigators indicate that these cells are, at least in part, responsible for hyaluronan synthesis in vivo.

Growth factor-enhanced expression and activity of matrix metalloproteases in human non-small cell lung cancer cell lines.

Growth factors secreted by either host or tumour cells play a major role in tumour cell progression. Besides stimulating cell division, growth factors may also stimulate cell migration and modulate matrix metalloprotease (MMP) production. MMPs are enzymes involved in a variety of physiological and pathological processes including tumour cell invasion and metastasis. We have previously shown that different growth factors regulate the motile behaviour of human lung cancer cell lines. In order to further advance our knowledge of the role the different growth factors play in lung cancer, we investigated their effect on two key enzymes belonging to the MMP family of enzymes, namely MMP-9 and MMP-2. Serum-free cultures of three human non-small cell lung cancer cell lines were exposed to five different growth factors: insulin-like growth factor I (IGF I) and II (IGF II), hepatocyte growth factor (HGF), epidermal growth factor (EGF) and stem cell factor (SCF). The expression of MMP-9 and MMP-2 in growth factor-treated and untreated cell lines was evaluated using gelatine zymography and quantified using computer-assisted image analyses. We found heterogeneous expression and activity of MMP-9 and MMP-2 in all three lung cancer cell lines. The most important finding in our study is that HGF and EGF are capable of stimulating the conversion of MMP-9 from a latent to an active form in human large cell lung cancer cell line U-1810 [corrected]. IGF I, IGF II, HGF and EGF stimulated an enhanced expression and activity of the latent form of MMP-2 and MMP-9. SCF did not enhance MMP activity in any of the cell lines tested. Our previous studies have shown that IGF I, IGF II, HGF, EGF and SCF induce migration of human non-small cell lung cancer cells in the presence of extracellular matrix (ECM) components. In the present study we show that growth factors can also enhance the expression of MMP's in these cells. Taken together these results indicate that certain growth factors may promote invasiveness through their ability to induce not only cell migration, but also by enhancing the expression and activity of matrix degrading MMP-2 and MMP-9.

Accumulation of Tc-99m depreotide (NeoSpect) in axillary sweat glands.

Tc-99m depreotide (NeoSpect) is a radiolabeled somatostatin analog introduced recently for scintigraphic imaging of patients with lung cancer. Eighteen patients were examined 2 to 4 hours after administration of 740 MBq mCi Tc-99m depreotide. In 13 patients (72.2%) bilateral symmetric activity corresponding to the large, deep apocrine sweat glands of the axillae was present. This observation is clinically relevant regardless of its reason or mechanism. It is important to be aware of this reason for activity in the axillae when assessing lymph node involvement not only in patients with lung cancer but also in breast cancer patients using scintigraphy with Tc-99m depreotide.

Effects of neurotrophins on human bronchial smooth muscle cell migration and matrix metalloproteinase-9 secretion.

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) have been found to be upregulated in inflammatory pulmonary diseases, including asthma. The functional role for the neurotrophins in the airways is still not known, but it has been proposed that neurotrophins induce airway hyperreactivity and tissue remodeling. Bronchial smooth muscle cells have been suggested to be involved in the remodeling process through their capacity to proliferate, migrate, and secrete inflammatory mediators and matrix metalloproteinases (MMPs). Therefore, we studied the effect of NGF, BDNF, and NT-3 on human bronchial smooth muscle cell (HBSMC) migration and MMP-2 and MMP-9 secretion. Immunocytochemistry studies showed that HBSMCs expressed the neurotrophin receptors TrkA, TrkB, and TrkC. BDNF, NT-3, and NGF increased MMP-9, but not MMP-2, secretion as shown by zymography. BDNF and NT-3, but not NGF, stimulated HBSMC migration as evaluated by Boyden chamber. Taken together, our data indicate that the neurotrophins may stimulate events important for airway remodeling.

Decreasing mortality rate in early pneumonia following hematopoietic stem cell transplantation.

Pulmonary complications after allogeneic hematopoietic stem-cell transplantation (HSCT) remain 1 of the most important causes of morbidity and mortality. This study evaluates the change over time of incidence, aetiology and risk factors for death related to pneumonia within 3 months after HSCT. 997 patients who underwent HSCT were studied retrospectively. Most patients (83%) had a haematological malignancy. The majority (89%) had an HLA-A, -B, and -DR matched related or unrelated donor. Conditioning consisted of cyclophosphamide and total-body irradiation or busulfan and graft-versus-host disease prophylaxis of cyclosporin and methotrexate in most cases. Death related to pneumonia occurred in 56 (5.6%) patients. Cytomegalovirus (37%) was the main pathogen involved, especially during the first 2 decades studied. In the multivariate risk factor analysis, we found that death from pneumonia was significantly associated with receiving a T-cell depleted graft (p<0.001), bacteraemia (p=0.001), and y of transplantation (p<0.001). In patients receiving a transplant during the last decade, the incidence of death related to pneumonia was 2.8% compared to 8.9% during the first decade. We conclude that the rate of mortality related to pneumonia has decreased over time, possibly as a result of improved diagnostic, prophylactic and therapeutic methods and treatment.

Anti-Gal IgG potentiates natural killer cell migration across porcine endothelium via endothelial cell activation and increased natural killer cell motility triggered by CD16 cross-linking.

Xenoreactive antibodies (Ab) are important for the development of acute vascular rejection (AVR) of xenografts characterized by monocytes, natural killer (NK) cells and neutrophils infiltrating the graft. The mechanisms by which anti-galactose alpha 1,3galactose (alpha-Gal) IgG influence NK cell migration across porcine aortic endothelium (PAEC) were investigated. NK cell migration across PAEC increased in the presence of anti-alpha-Gal IgG. Anti-alpha-Gal IgG exposure activated PAEC as shown by an increased expression of CD62E and CD106. NK cells adhered, spread and showed motile forms on plastic surfaces coated with human IgG, IgG Fc and on mAb against CD16, but not on mouse IgG or BSA, suggesting that CD16 cross-linking can mediate increased adhesiveness. Increased NK cell motility was observed on Boyden filters coated with human IgG, IgG Fc, and mAb against CD16 and the alpha 4, alpha 5, alpha L, beta 1 and beta 2 integrin chains. No motile response was seen on mouse IgGor CD7, CD56 and alpha 6 integrin mAb. NK cell migration on human IgG and anti-CD16 Ab was blocked by anti-CD16 or anti-beta 2, but not anti-beta 1 Ab, implying that the motile response triggered by CD16 cross-linking is mediated via beta 2 integrins. Preformed or induced anti-alpha-Gal IgG may therefore contribute to AVR by stimulating innate immune cell infiltration of the graft.