Cyclooxygenase-2 (COX-2) expression is induced by mitogenic and proinflammatory factors. Its overexpression plays a causal role in inflammation and tumorigenesis. COX-2 expression is tightly regulated, but the mechanisms are largely unclear. Here we show the control of COX-2 expression by an endogenous tryptophan metabolite, 5-methoxytryptophan (5-MTP). By using comparative metabolomic analysis and enzyme-immunoassay, our results reveal that normal fibroblasts produce and release 5-MTP into the extracellular milieu whereas A549 and other cancer cells were defective in 5-MTP production. 5-MTP was synthesized from L-tryptophan via tryptophan hydroxylase-1 and hydroxyindole O-methyltransferase. 5-MTP blocked cancer cell COX-2 overexpression and suppressed A549 migration and invasion. Furthermore, i.p. infusion of 5-MTP reduced tumor growth and cancer metastasis in a murine xenograft tumor model. We conclude that 5-MTP synthesis represents a mechanism for endogenous control of COX-2 overexpression and is a valuable lead for new anti-cancer and anti-inflammatory drug development.tumor suppression | tryptophan metabolism | inflammation control C yclooxygenase-2 (COX-2) is a rate-limiting enzyme in the production of diverse prostanoids with potent biological activities. It is involved in multiple physiological functions and triggers key pathological processes, such as tumorigenesis and inflammation (1, 2). COX-2 is constitutively overexpressed in a wide variety of human cancers and is enhanced by proinflammatory stimuli (3, 4). There is convincing evidence for a causal role of COX-2 in tumorigenesis. Inhibition of COX-2 activities was reported to control human colorectal cancer (5-8). COX-2 induces tumorigenesis by promoting important cellular functions including cell proliferation, migration, and resistance to apoptosis (9-11). The induced COX-2 expression by proinflammatory and mitogenic factors in normal cells is tightly controlled (12) whereas its overexpression in cancer cells is attributed to dysregulated transcription (13). The endogenous control mechanisms for COX-2 expression in normal cells and the mechanisms underlying the dysregulation in cancer cells are poorly understood. We previously identified in the conditioned medium of human fibroblasts small molecules (named cytoguardins) that suppress COX-2 expression induced by proinflammatory mediators (14). NMR analysis of a semipurified fraction revealed compounds with indole moieties (14). However, the exact chemical structures remain elusive. In this study, we elucidated the structure of cytoguardins by comparing the metabolomic profiles between normal and cancer cells. ResultsCytoguardins Inhibit Cancer Cell COX-2. To determine that fibroblast factors are capable of suppressing cancer cell COX-2 expression, we cocultured human Hs68 foreskin fibroblasts (HsFb) with A549 lung cancer cells in a Boyden chamber for 24 h. A549 cells were removed and treated with phorbol 12-myristate 13-acetate (PMA) for 4 h, and COX-2 proteins were analyzed. HsFb suppressed A549 ...

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The chimeric state after allogeneic hematopoietic stem cell transplantation provides a platform for adoptive immunotherapy using donor-derived immune cells. The major risk with donor lymphocyte infusions (DLIs) is the development of graft-versus-host disease (GvHD). Development of new DLI products with antitumor reactivity and reduced GvHD risk represents a challenging task in cancer immunotherapy. Although natural killer (NK) and NK-like T cells are promising owing to their antitumor activity, their low concentrations in peripheral blood mononuclear cells reduces their utility in DLIs. We have recently developed a system that allows expansion of clinical-grade NK and NK-like T cells in large numbers. In this study, the safety of donor-derived long-term ex vivo-expanded human NK and NK-like T cells given as DLIs was investigated as immunotherapy for cancer in five patients following allogeneic stem cell infusion. Infusion of the cells was safe whether administered alone or with IL-2 subcutaneously. No signs of acute GvHD were observed. One patient with hepatocellular carcinoma showed markedly decreased serum alpha-fetoprotein levels following cell infusions. These findings suggest that the use of ex vivo-expanded NK and NK-like T cells is safe and appears an attractive approach for further clinical evaluation in cancer patients.

Normal human and murine fibroblasts can inhibit proliferation of tumor cells when cocultured in vitro. The inhibitory capacity varies depending on the donor and the site of origin of the fibroblast. We showed previously that effective inhibition requires formation of a morphologically intact fibroblast monolayer before seeding of the tumor cells. Here we show that inhibition is extended to motility of tumor cells and we dissect the factors responsible for these inhibitory functions. We find that inhibition is due to two different sets of molecules: (i) the extracellular matrix (ECM) and other surface proteins of the fibroblasts, which are responsible for contact-dependent inhibition of tumor cell proliferation; and (ii) soluble factors secreted by fibroblasts when confronted with tumor cells (confronted conditioned media, CCM) contribute to inhibition of tumor cell proliferation and motility. However, conditioned media (CM) obtained from fibroblasts alone (nonconfronted conditioned media, NCM) did not inhibit tumor cell proliferation and motility. In addition, quantitative PCR (Q-PCR) data show upregulation of proinflammatory genes. Moreover, comparison of CCM and NCM with an antibody array for 507 different soluble human proteins revealed differential expression of growth differentiation factor 15, dickkopf-related protein 1, endothelial-monocyteactivating polypeptide II, ectodysplasin A2, Galectin-3, chemokine (C-X-C motif) ligand 2, Nidogen1, urokinase, and matrix metalloproteinase 3.tumor microenvironment | cancer-associated fibroblast | motility | extracellular matrix | soluble factors T he normal balance between epithelial cells and the surrounding stroma is disrupted during tumor development. Developing preneoplastic cells in the process of escaping from their intrinsic checkpoints that prevent illegitimate cell proliferation also have to overcome the microenvironmental forces that maintain the integrity of the normal tissue architecture. It is becoming increasingly clear that the normal microenvironment can restrict cancer development and progression (1-3). Inhibition of tumor cell growth by normal fibroblasts is one measurable manifestation of this multicomponential control. Part of this process is reflected by the ability of the tumor cell to corrupt the surrounding stroma and turn it from restrictive to supportive. The generation of cancer-associated fibroblasts (CAFs) that enhance angiogenesis and support tumor growth and spreading through the release of growth factors and cytokines is a case in point (1-5).We have departed from the observation of Stoker et al. that normal fibroblasts can inhibit the growth of admixed tumor cells upon contact (6). Having confirmed their findings, we have extended such findings into a high throughput microwell system and showed that the strength of the inhibition differs depending on the source of the fibroblasts. Moreover, such inhibition is contact dependent as well as requires an intact fibroblast monolayer (7, 8).Here we report the surprising finding that the inhi...

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the Western world. It is currently an incurable disease, making new treatment options such as immunotherapy desirable. Monoclonal antibodies (Mabs) to surface antigens of the tumor cell is one option. Administration of cytotoxic cells such as natural killer (NK) and natural killerlike T (NKT) cells expanded in vitro might be a useful treatment modality alone or in combination with MAbs. A limiting step in the development of successful cellular immunotherapy has been the availability of appropriate cytotoxic cells. Here, we report the feasibility of expanding populations of the human killer cells, CD3ÀCD56 þ NK and CD3 þ CD56 þ NKT cells, from peripheral blood mononuclear cells (PBMCs) of B-CLL patients. The influence of tumor B cells on the in vitro expansion of killer cells was assessed by depleting B cells from PBMCs by microbead separation before culture. The 21-day cultures from both B-cell-and non-B-cell-depleted PBMC showed a marked expansion of NK cells, and also of T cells, among which almost half had the NKT phenotype. Depletion of B cells before culture did not change the expansion rates of NK and NKT cells significantly. In patients with progressive B-CLL, NK cell expansion capacity was improved after fludarabine treatment when compared to samples obtained before treatment. Repeated samples of PBMCs from individual untreated patients with both indolent and progressive disease cultured under identical conditions gave similar NK cell expansion rates. Expanded killer cell populations had cytotoxic function against the NK-sensitive target K562 cell line and expressed high levels of Granzyme B. From our studies, we conclude that NK cells as well as NKT cells from the peripheral blood of B-CLL patients can be expanded, and that these cells have cytotoxic capacity.

Fibroblasts are a main player in the tumor-inhibitory microenvironment. Upon tumor initiation and progression, fibroblasts can lose their tumor-inhibitory capacity and promote tumor growth. The molecular mechanisms that underlie this switch have not been defined completely. Previously, we identified four proteins overexpressed in cancer-associated fibroblasts and linked to Rho GTPase signaling. Here, we show that knocking out the Ras homolog family member A (RhoA) gene in normal fibroblasts decreased their tumorinhibitory capacity, as judged by neighbor suppression in vitro and accompanied by promotion of tumor growth in vivo. This also induced PC3 cancer cell motility and increased colony size in 2D cultures. RhoA knockout in fibroblasts induced vimentin intermediate filament reorganization, accompanied by reduced contractile force and increased stiffness of cells. There was also loss of wide F-actin stress fibers and large focal adhesions. In addition, we observed a significant loss of α-smooth muscle actin, which indicates a difference between RhoA knockout fibroblasts and classic cancer-associated fibroblasts. In 3D collagen matrix, RhoA knockout reduced fibroblast branching and meshwork formation and resulted in more compactly clustered tumor-cell colonies in coculture with PC3 cells, which might boost tumor stem-like properties. Coculturing RhoA knockout fibroblasts and PC3 cells induced expression of proinflammatory genes in both. Inflammatory mediators may induce tumor cell stemness. Network enrichment analysis of transcriptomic changes, however, revealed that the Rho signaling pathway per se was significantly triggered only after coculturing with tumor cells. Taken together, our findings in vivo and in vitro indicate that Rho signaling governs the inhibitory effects by fibroblasts on tumor-cell growth.Rho GTPases | RhoA | cancer-associated fibroblasts | tumor-inhibitory capacity | cytoskeleton

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Decorin is a small leucine-rich proteoglycan, synthesized and deposited by fibroblasts in the stroma where it binds to collagen I. It sequesters several growth factors and antagonizes numerous members of the receptor tyrosine kinase family. In experimental murine systems, it acted as a potent tumor suppressor. Examining the Human Protein Atlas online database of immunostained tissue samples we have surveyed decorin expression in silico in several different tumor types, comparing them with corresponding normal tissues. We found that decorin is abundantly secreted and deposited in normal connective tissue but its expression is consistently decreased in the tumor microenvironment. We developed a software to quantitate the difference in expression. The presence of two closely related proteoglycans in the newly formed tumor stroma indicated that the decreased decorin expression was not caused by the delay in proteoglycan deposition in the newly formed connective tissue surrounding the tumor.