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ABSTRACT: The role of paracrine tumor-stroma regulation in the progression of cancer is under intense investigation. Activated fibroblasts are key components of the tumor microenvironment providing the soluble factors mediating the regulation. Nemosis is an experimental model to study these parameters: formation of a multicellular spheroid activates fibroblasts and leads to increased production of soluble factors involved in the promotion of growth and motility. Role of nemosis was investigated in the tumorigenesis of HaCaT derivatives representing skin carcinoma progression. Conditioned medium from fibroblast spheroids increased proliferation rate of HaCaT derivatives. Expression of proliferation marker Ki-67 increased significantly in benign A5 and low-grade malignant II-4 cells, but did not further increase in the metastatic RT3 cells. Expression of p63, keratinocyte stem cell marker linked to cancer progression, was augmented by medium from nemotic fibroblasts; this increase was also seen in RT3 cells. Scratch-wound healing of the keratinocytes was enhanced in response to fibroblast nemosis. Neutralizing antibodies against growth factors inhibited wound healing to some extent; the response varied between benign and malignant keratinocytes. Migration and invasion were enhanced by conditioned medium from nemotic fibroblasts in benign and low-grade malignant cells. RT3 keratinocyte migration was further augmented, but invasion was not, indicating their intrinsic capacity to invade. Our data demonstrate that fibroblast nemosis increases proliferation and motility of HaCaT keratinocyte derivatives, and thus nemosis can be used as a model to study the role of soluble factors secreted by fibroblasts in tumor progression.
Experimental Cell Research 06/2010; 316(10):1739-47. · 3.58 Impact Factor
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ABSTRACT: Transforming growth factor beta (TGF-beta) acts as a tumor promoter by inducing epithelial-mesenchymal transition (EMT), which leads to a motile phenotype, enabling invasion and metastasis of cancer cells. Cancer-related inflammation, mediated by prostaglandins, has been proposed as a critical mechanism in conversion of benign cells to malignant.
Induction of cyclooxygenase 2 (COX-2), producer of prostaglandins, is thought to be a prerequisite for TGF-beta-induced EMT in benign cells. We used HaCaT derivatives, representative of skin cancer progression, to investigate TGF-beta1 mediated EMT response, and the role of COX-2 in it.
Effect of TGF-beta1 was investigated by analyzing cell proliferation, morphology and protein expression. Chemotaxis and scratch-wound assays were used to study migration.
TGF-beta1 caused proliferation arrest of benign and malignant HaCaT cells, and changed the epithelial morphology of benign and low-grade malignant cells, but not metastatic cells, to mesenchymal spindle-shape. Epithelial junction proteins ZO-1 and E-cadherin were downregulated in all cell lines in response to TGF-beta1, but mesenchymal markers were not induced, suggesting a partial EMT response. COX-2 and migration were induced only in benign HaCaT derivatives. Malignant derivatives did not induce COX-2 in response to TGF-beta 1 treatment, thus emphasizing the role of inflammation in EMT response of benign cells.
TGF-beta1 operates via distinct mechanisms in inducing EMT and metastasis, and supporting this we show that TGF-beta1 induces COX-2 and promotes the migration of benign cells, but does not further augment the migration of malignant cells, indicating their resistance to TGF-beta1 in the context of motility.
Journal of dermatological science 05/2010; 58(2):97-104. · 3.71 Impact Factor
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ABSTRACT: Tumor microenvironment has emerged as an important target for cancer therapy. In particular, cancer-associated fibroblasts (CAF) seem to regulate many aspects of tumorigenesis. CAFs secrete a variety of soluble factors that act in a paracrine manner and thus affect not only cancer cells, but also other cell types present in the tumor stroma. Acting on cancer cells, CAFs promote tumor growth and invasion. They also enhance angiogenesis by secreting factors that activate endothelial cells and pericytes. Tumor immunity is mediated via cytokines secreted by immune cells and CAFs. Both immune cells and CAFs can exert tumor-suppressing and -promoting effects. CAFs, and the factors they produce, are attractive targets for cancer therapy, and they have proven to be useful as prognostic markers. In this review we focus mainly on carcinomas and discuss the recent findings regarding the role of activated fibroblasts in driving tumor progression.
Experimental Cell Research 05/2010; 316(17):2713-22. · 3.58 Impact Factor
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ABSTRACT: Malignant cells when grown in suspension, as a rule, proliferate and can form spheroids that have been used as a model of tumor nodules, micrometastases and avascular tumors. In contrast, normal adherent cells cannot be stimulated to grow as multicellular aggregates. Now, recent results show that normal fibroblasts if forced to cluster (spheroid formation) do not grow but undergo a new pathway of cell activation (nemosis) leading to a massive proinflammatory, proteolytic and growth factor response. The clustering and activation are initiated by fibronectin-integrin interaction. The activated fibroblasts are able to modulate the behavior of cancer cells and, furthermore malignant cells boost this activation even further. In this model, the activation of fibroblasts terminates in programmed necrosis-like cell death. Activation of the tumor stroma, especially of fibroblasts, is of critical importance for tumor progression, although mechanisms leading to their activation are still largely uncharacterized. In summary, our results suggest that this kind of fibroblast activation (nemosis) may be involved in pathological conditions such as inflammation and cancer.
Experimental Cell Research 04/2009; 315(10):1633-8. · 3.58 Impact Factor
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ABSTRACT: Tumor-stroma reaction is associated with activation of fibroblasts. Nemosis is a novel type of fibroblast activation. It leads to an increased production of growth factors and proinflammatory and proteolytic proteins, while at the same time cytoskeletal proteins are degraded. Here we used paired normal skin fibroblasts and cancer-associated fibroblasts (CAF) and primary and recurrent oral squamous cell carcinoma (SCC) cells to study the nemosis response.
Fibroblast nemosis was analyzed by protein and gene expression and the paracrine regulation with colony formation assay. One of the normal fibroblast strains, FB-43, upregulated COX-2 in nemosis, but FB-74 cells did not. In contrast, CAF-74 spheroids expressed COX-2 but CAF-43 cells did not. Alpha-SMA protein was expressed in both CAF strains and in FB-74 cells, but not in FB-43 fibroblasts. Its mRNA levels were downregulated in nemosis, but the CAFs started to regain the expression. FSP1 mRNA was downregulated in normal fibroblasts and CAF-74 cells, but not in CAF-43 fibroblasts. Serine protease FAP was upregulated in all fibroblasts, more so in nemotic CAFs. VEGF, HGF/SF and FGF7 mRNA levels were upregulated to variable degree in nemosis. CAFs increased the colony formation of primary tumor cell lines UT-SCC-43A and UT-SCC-74A, but normal fibroblasts inhibited the anchorage-independent growth of recurrent UT-SCC-43B and UT-SCC-74B cells.
Nemosis response, as observed by COX-2 and growth factor induction, and expression of CAF markers alpha-SMA, FSP1 and FAP, varies between fibroblast populations. The expression of CAF markers differs between normal fibroblasts and CAFs in nemosis. These results emphasize the heterogeneity of fibroblasts and the evolving tumor-promoting properties of CAFs.
PLoS ONE 02/2009; 4(9):e6879. · 4.09 Impact Factor
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ABSTRACT: Cell-cell clustering of fibroblasts, called nemosis, leads to a massive growth factor, proteolytic and proinflammatory response. Culturing fibroblasts in conditioned medium collected from HaCaT keratinocyte cell panel representing different stages of skin carcinogenesis had a differential effect on fibroblast nemosis. Non-malignant keratinocytes had a nemosis-inhibiting effect on fibroblasts as seen by inhibition of COX-2 protein expression. Conditioned medium from malignant cells promoted fibroblast nemosis by inducing higher levels of COX-2, HGF/SF and VEGF. Even a small amount of malignant medium converted the inhibitory effect of benign medium, whereas non-malignant medium neutralized the nemosis-promoting effect of malignant medium. In collagen co-cultures benign keratinocytes caused a nemosis-inhibiting effect on fibroblast spheroids by inhibiting COX-2 induction, while with malignant keratinocytes myofibroblastic differentiation of fibroblasts was seen.
Molecular oncology 01/2009; 2(4):340-8. · 4.10 Impact Factor
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Kati Räsänen
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ABSTRACT: Paracrine regulation between the components of the tumour microenvironment cancer cells, activated fibroblasts, immune and endothelial cells is under intense investigation. The signals between the different cell types are mediated by soluble factors, such as growth factors, proinflammatory cytokines and proteolytic enzymes. Nemosis is an experimental in vitro model of fibroblast activation, leading to increased production of such mediators. Nemotic activation of fibroblasts occurs as they are forced to cluster thereby forming a multicellular spheroid. The aim of the present studies was to elucidate the mechanisms underlying the nemotic response of cancer-associated fibroblasts (CAF) and the role of nemosis in paracrine regulation between activated fibroblasts and benign and malignant epithelial cells. The results presented in this thesis demonstrate that the nemotic response of CAFs and normal fibroblasts differs, and inter-individual variations exist between fibroblast populations. In co-culture experiments, fibroblasts increased colony formation of squamous cell carcinoma (SCC) cells, and CAFs further augmented this, highlighting the tumour-evolving properties of CAFs. Furthermore, fibroblast monolayers in those co-cultures started to cluster spontaneously. This kind of spontaneous nemosis response might take place also in vivo, although more direct evidence of this still needs to be obtained. The HaCaT skin carcinoma progression model was used to study the effects of benign and malignant keratinocytes on fibroblast nemosis. Benign HaCaT cells inhibited fibroblast nemosis, observed as inhibition of cyclooxygenase 2 (COX-2) induction in nemotic spheroids. In contrast, malignant HaCaTs further augmented the nemotic response by increasing expression of COX-2 and the growth factors hepatocyte growth factor / scatter factor (HGF/SF) and vascular endothelial growth factor (VEGF), as well as causing a myofibroblastic differentiation of nemotic fibroblasts into fibroblasts resembling CAFs. On the other side of this reciprocal signalling, factors secreted into conditioned medium by the nemotic fibroblasts promoted proliferation and motility of the HaCaT cell lines. Notably, the nemotic fibroblast medium increased the expression of p63, a transcription factor linked to carcinogenesis, also in the highly metastatic HaCaT cells. These results emphasize the paracrine role of factors secreted by activated fibroblasts in driving tumour progression. We also investigated the epithelial-mesenchymal transition (EMT) of the HaCaT clones in response to transforming growth factor β (TGF-β), which is a well-characterized inducer of EMT. TGF-β caused growth arrest and loss of epithelial cell junctions in the HaCaT derivatives, but mesenchymal markers were not induced, suggesting a partial, but not complete EMT response. Inflammation induced by COX-2 has been proposed to be a key mechanism in EMT of benign cells. Corroborating this notion, COX-2 was induced only in benign, not in malignant HaCaT derivatives. Furthermore, in cells in which TGF-β caused COX-2 induction, migration was clearly augmented. The concept of treating cancer is changing from targeting solely the cancer cells to targeting the whole microenvironment. The results of this work emphasise the role of activated fibroblasts in cancer progression and that CAFs should also be taken into consideration in the treatment of cancer. The results from these studies suggests that nemosis could be used as a diagnostic tool to distinguish in vitro activated fibroblasts from tumour stroma and also in studying the paracrine signalling that is mediated to other cell types via soluble factors. Syöpäkasvaimet eivät ole eristäytyneet muusta elimistöstä, vaan syöpäsolut ovat tiiviissä vuorovaikutuksessa ympäröivien solujen, kuten aktivoituneiden sidekudos-, puolustus- ja verisuonisolujen kanssa. Valtaosa syövistä on lähtöisin epiteelistä, joka on kudosten pintoja päällystävä kerros; näistä syövistä käytetään nimitystä karsinooma. Sidekudoksen pääasiallinen solutyyppi on mesenkymaalinen fibroblasti. Liukoiset tekijät välittävät signaaleja näiden eri solutyyppien välillä, edistäen täten syövän kasvua ja leviämistä. Kasvainten mikroympäristön tutkimuksen avulla pyritään löytämään uusia hoitomuotoja syöpätauteihin. Nemoosi on tutkimusryhmämme kehittämä kokeellinen fibroblastien aktivoitumisen malli. Väitöskirjatutkimuksen tarkoituksena oli selvittää nemoosi-mallia hyödyntäen, miten aktivoituneiden fibroblastien tuottamat liukoiset tekijät vaikuttavat epiteelisolujen muuttumiseen hyvänlaatuisesta pahanlaatuisiksi. Väitöskirjan ensimmäisessä osatyössä tutkittiin suusyöpäpotilaista eristettyjen normaalien ja kasvaimen fibroblastien nemoosi-vastetta ja vuorovaikutusta levyepiteelistä lähtöisin olevien karsinoomasolujen kanssa. Kasvaimen fibroblastit aktivoituivat eri tavalla normaaleihin verrattuna ja ne edistivät karsinoomasolujen kasvua. Toisessa työssä käytettiin ihon pintakerroksen keratinosyyteistä koostuvaa HaCaT-solupaneelia, joka sisältää kaksi hyvän- ja kaksi pahanlaatuista solulinjaa. Hyvänlaatuiset HaCaT-solut ehkäisivät fibroblastien aktivoitumista. Vastaavasti pahanlaatuiset HaCaT-solut edistivät nemoosi-vastetta, lisäten fibroblastien tuottaman syklo-oksigenaasi 2 (COX-2) tulehdustekijän, hepatosyyttikasvutekijän (HGF/SF) ja verisuonikasvutekijän (VEGF) määrää. Kolmannessa osajulkaisussa tutkittiin, kuinka nämä aktivoituneiden fibroblastien tuottamat liukoiset tekijät vaikuttavat HaCaT-solupaneelin eriasteisten solujen käyttäytymiseen. Nemoottisten fibroblastien kasvatusliuokseen erittämät tekijät lisäsivät HaCaT-solulinjojen jakautumis- ja liikkumiskapasiteettia. Nemoosi ei kuitenkaan aikaansaanut näiden epiteelisolujen muuttumista mesenkymaalisiksi. HaCaT-solupaneelin epiteeli-mesenkyymitransformaatiota (EMT) tutkittiin väitöskirjan neljännessä osassa. Transformoiva kasvutekijä beta (TGF-beta) on tunnettu EMT-aiheuttaja, ja sen todettiin pysäyttävän kaikkien HaCaT-solulinjojen jakautumisen ja johtavat epiteelisolujen välisten solu-solu liitoksien heikkenemiseen. Soluliitoksien katoaminen mahdollistaa epiteelisolujen yksittäisen liikkumisen. TGF-beta sai aikaan COX-2 entsyymin tuotannon hyvän- mutta ei pahanlaatuisissa HaCaT-soluissa ja samalla lisäsi näiden solujen liikkumiskapasiteettia. Tämä väitöskirjatyö osoittaa mikroympäristön, ja erityisesti aktivoituneiden fibroblastien, olevan olennainen osa kasvaimen kehittymisessä pahanlaatuiseksi. Näiden solujen rooli tulisi huomioida kehitettäessä syöpähoitoja. Työn tulokset osoittavat myös, että nemoosi-mallia voidaan mahdollisesti jatkossa hyödyntää karsinoomien patogeneesi-, diagnostiikka- ja hoitotutkimuksissa.
