RhoA knockout fibroblasts lose tumor-inhibitory capacity in vitro and promote tumor growth in vivo.

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 tumor-inhibitory 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.

Confrontation of fibroblasts with cancer cells in vitro: gene network analysis of transcriptome changes and differential capacity to inhibit tumor growth.

BACKGROUND: There is growing evidence that emerging malignancies in solid tissues might be kept under control by physical intercellular contacts with normal fibroblasts. METHODS: Here we characterize transcriptional landscapes of fibroblasts that confronted cancer cells. We studied four pairs of in vitro and ex vivo fibroblast lines which, within each pair, differed in their capacity to inhibit cancer cells. The natural process was modeled in vitro by confronting the fibroblasts with PC-3 cancer cells. Fibroblast transcriptomes were recorded by Affymetrix microarrays and then investigated using network analysis. RESULTS: The network enrichment analysis allowed us to separate confrontation- and inhibition-specific components of the fibroblast transcriptional response. Confrontation-specific differences were stronger and were characterized by changes in a number of pathways, including Rho, the YAP/TAZ cascade, NF-kB, and TGF-beta signaling, as well as the transcription factor RELA. Inhibition-specific differences were more subtle and characterized by involvement of Rho signaling at the pathway level and by potential individual regulators such as IL6, MAPK8, MAP2K4, PRKCA, JUN, STAT3, and STAT5A. CONCLUSIONS: We investigated the interaction between cancer cells and fibroblasts in order to shed light on the potential mechanisms and explain the differential inhibitory capacity of the latter, which enabled both a holistic view on the process and details at the gene/protein level. The combination of our methods pointed to proteins, such as members of the Rho pathway, pro-inflammatory signature and the YAP1/TAZ cascade, that warrant further investigation via tools of experimental perturbation. We also demonstrated functional congruence between the in vitro and ex vivo models. The microarray data are made available via the Gene Expression Omnibus as GSE57199.

Inhibition of tumor cell proliferation and motility by fibroblasts is both contact and soluble factor dependent.

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 up-regulation 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-monocyte-activating polypeptide II, ectodysplasin A2, Galectin-3, chemokine (C-X-C motif) ligand 2, Nidogen1, urokinase, and matrix metalloproteinase 3.

Decreased decorin expression in the tumor microenvironment.

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.

Control of cyclooxygenase-2 expression and tumorigenesis by endogenous 5-methoxytryptophan.

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.

The architecture of fibroblast monolayers of different origin differentially influences tumor cell growth.

Normal human and murine fibroblasts can inhibit proliferation of tumor cells when co-cultured in vitro. The inhibitory capacity varies depending on the donor and the site of origin of the fibroblast. It requires direct cell-to-cell contact and is not transferable with supernatant. Here, we show that effective inhibition also requires the formation of a morphologically intact fibroblast monolayer before the seeding of the tumor cells. Interference with the formation of the monolayer impairs the inhibition. Subclones of TERT-immortalized fibroblasts were selected on the basis of differences in the growth pattern and related inhibitory activity. Whereas the well-organized "whirly" (WH) growth pattern was associated with strong inhibition, the disorganized "crossy" (CR) growth pattern was linked to reduced inhibition. Time lapse imaging of tumor-fibroblast co-cultures using extended field live cell microscopy revealed that fibroblast monolayers with growth inhibitory capacity also reduced the motility of the tumor cells whereas noninhibitory monolayers had no effect on tumor cell motility. Gene expression pattern of two isogenic pairs of fibroblasts, WH and CR subclones of the TERT immortalized line (inhibitory, and less inhibitory subsequently) and freshly explanted skin (inhibitory) and hernia (noninhibitory) fibroblasts derived from the same patient, identified a set of genes that co-segregated with the inhibitory phenotype. This suggests that our model system may reveal molecular mechanisms involved in contact-mediated microenvironmental surveillance that may protect the organism from the outgrowth of disseminated tumor cells.

Human CD80/IL2 lentivirus-transduced acute myeloid leukaemia (AML) cells promote natural killer (NK) cell activation and cytolytic activity: implications for a phase I clinical study.

Immunotherapeutic strategies may promote T and/or natural killer (NK) cell cytotoxicity. NK cells have the potential to exert a powerful anti-leukaemia effect, as demonstrated by studies of allogeneic transplantation. We have previously shown that CD80/interleukin 2 (IL2) lentivirus (LV)-transduced AML cells stimulate in-vitro T cell activation. The present study demonstrated that allogeneic and autologous culture of peripheral blood mononuclear cells with CD80/IL2-expressing AML cells also promoted NK cell cytotoxicity. Expression of the activation receptors NKp30, NKp44, CD244, CD25, CD69 and HLA-DR significantly increased following allogeneic culture and a consistent increased expression of NKp30, NKp44, NKp46, NKG2D, NKG2C and CD69, and up-regulation of the cytolytic marker CD107a was detected following autologous culture with LV-CD80/IL2 AML cells. Furthermore, increased NK cell lysis of K562 and primary AML blasts was detected. The lytic activity increased by twofold against K562 (from 46.6% to 90.4%) and allogeneic AML cells (from 11.8% to 20.1%) following in-vitro stimulation by CD80/IL2-expressing AML cells. More importantly for potential therapeutic applications, lysis of primary AML cells by autologous NK cells increased by more than 40-fold (from 0.4% to 22.5%). These studies demonstrated that vaccination of patients with CD80/IL2-transduced AML cells could provide a powerful strategy for T/NK cell-mediated stimulation of anti-leukaemic immunological responses.

Safety analysis of ex vivo-expanded NK and NK-like T cells administered to cancer patients: a phase I clinical study.

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.

Efficient gene transfer into primary human natural killer cells by retroviral transduction.

OBJECTIVE: To optimize retroviral gene transfer into primary human natural killer (NK) cells. MATERIALS AND METHODS: NK cells from healthy donors were expanded ex vivo for a period of 21 days. Retroviral transductions were carried out by replacing culture media with retrovirus-containing supernatant during 2-hour incubations on days 3, 4, 5, 6, 10, 15, or 20. In some experiments, NK cells were transduced on 2 consecutive days (days 5 and 6). Green fluorescent protein served as a marker for detection of transduced cells. RESULTS: NK cells showed a median of 27.2% transduction efficiency after a single transduction round (transduction on day 5) and a median of 47.1% transduction efficiency after two rounds of transduction (transduction on days 5 and 6), 24 hours after exposure to retrovirus-containing supernatants. On day 21 after initial culture, 51.9% of NK cells were transduced after a single transduction round (transduction on day 5) and 75.4% after two rounds of transduction (transduction on days 5 and 6). Gene transfer did not change the function or phenotype of NK cells as determined by phenotypical analysis, nor did the proliferative ability or cytotoxic function change. CONCLUSION: The results show that NK cells can successfully be transduced with retroviral vectors, without any detectable changes in phenotype or function. This may open up new possibilities in the studies of NK cell biology and the development of NK cells for immunotherapy regimens.

Enhanced ouabain resistance gene as a eukaryotic selection marker.

Current selection markers allow selection by antibiotics or fluorescent/magnetic sorting by green fluorescent protein or membrane antigens. Antibiotic selection proceeds on a time scale of weeks, and flourescence-activated cell sorting requires complex equipment and may generate false-positive results when selection is performed too early after transduction with membrane markers. We have characterized an endogenous eukaryotic selection marker, the ouabain resistance gene (Oua(r)), which has the potential for quick and efficient in vitro selection of target cells. The Oua(r) used by us is derived from the rat alpha(1) isoform of Na(+),K(+)-ATPase, where leucine at position 799 is substituted for cysteine by targeted mutagenesis. This mutation confers resistance to more than 1 mM ouabain in vitro. We show that cells transfected with plasmid or transduced with a retrovirus vector encoding Oua(r) can be selected efficiently with ouabain in 48 hr and that a pure population of cells can be obtained. The ouabain resistance gene may be useful as a selection marker in general molecular biology, preclinical, and clinical applications because of its short selection time and also because of the safety of ouabain for human use.