Cell line authentication: a necessity for reproducible biomedical research.

Immortalized or continuous cell lines are invaluable tools in basic and preclinical research. However, the widespread use of misidentified cell lines is a serious threat to scientific reproducibility. Based on the experiences of mandatory cell line authentication at the International Journal of Cancer (IJC), we provide an overview of the issues pertinent to misidentified cell lines and discuss available solutions. We also summarize the lessons learned, revealing that at least 5% of the human cell lines used in manuscripts considered for peer review are misidentified. About 4% of the considered manuscripts are rejected for severe cell line problems, and most are subsequently published in other journals. In order to diminish such malpractice and its consequences for the scientific record, we postulate that strict multi-layered quality control is essential. Besides journals and publishers, we encourage scientists, research institutions, and funders to take action on the matter and revise their respective policies. Hence, we provide concrete recommendations on introducing regular authentication schemes and staff training, and discuss future steps for enhancing good cell culture practices.

The need for a worldwide consensus for cell line authentication: Experience implementing a mandatory requirement at the International Journal of Cancer.

Cell lines are used in life science research worldwide as biological surrogates. All cell lines are subject to major limitations when used as research tools, including (i) cross-contamination with other cells cultured in the same laboratory environment and (ii) evolution in vitro that renders a given cell line inappropriate as a surrogate for a specific biological hypothesis. There is ample evidence that cross-contamination or phenotypic drift of cells in culture can generate irreproducible or misleading data. A small number of scientific journals-the International Journal of Cancer being at the forefront-and funding agencies have recently moved forward to ask for obligatory cell line authentication data. The history of implementing such rules by the International Journal of Cancer exemplifies the difficulties encountered when installing mandatory quality measures in life sciences.

IL-6 promotes malignant growth of skin SCCs by regulating a network of autocrine and paracrine cytokines.

Cytokines play a crucial role in tumor initiation and progression. Here, we demonstrate that interleukin (IL)-6 is a key factor by driving tumor progression from benign to malignant, invasive tumors in the HaCaT-model of human skin carcinoma. IL-6 activates STAT3 and directly stimulates proliferation and migration of the benign noninvasive HaCaT-ras A-5 cells in vitro. Furthermore, IL-6 induces a complex, reciprocally regulated cytokine network in the tumor cells that includes inflammatory and angiogenic factors such as IL-8, GM-CSF, VEGF and MCP-1. These IL-6 effects lead to tumor cell invasion in organotypic cultures in vitro and to the formation of malignant and invasive s.c. tumors in vivo. Tumor invasion is supported by the IL-6 induced overexpression of MMP-1 in vitro and in vivo. These data demonstrate a key function of IL-6 in the progression of skin SCCs by regulating a complex cytokine and protease network and suggest new therapeutic approaches to target this central player in skin carcinogenesis.

Platelet-derived growth factor-B normalizes micromorphology and vessel function in vascular endothelial growth factor-A-induced squamous cell carcinomas.

Vascular endothelial growth factor (VEGF), which is a key regulator of angiogenesis, often induces formation of immature vessels with increased permeability and reduced vessel functionality. Here, we demonstrate that de novo expression of murine (m)VEGF-164 induces malignant and invasive tumor growth of HaCaT keratinocytes. However, the mVEGF-164-induced tumors are ulcerated with a disorganized epithelium that is interrupted by lacunae with limited basement membrane and endothelial cell coverage. Vessel maturation is strongly impaired. Tumor and vessel micromorphology are markedly improved by the combined expression of human platelet-derived growth factor (hPDGF)-B and mVEGF-164. Although tumor size and malignancy are comparable with either mVEGF-164 alone or combined human PDGF-B and mVEGF-164 expression, combined hPDGF-B and mVEGF-164 expression leads to a more solid and compact tumor tissue with a mature functional tumor vasculature and a higher microvessel density, as demonstrated histologically and by dynamic contrast-enhanced magnetic resonance imaging. Treatment of the hPDGF-B- and mVEGF-164-expressing tumors with imatinib mesylate to block PDGF-B signaling reverses this effect. In addition, tumor cell invasion of mVEGF-164 transfectants and mVEGF-164 plus hPDGF-B transfectants in vivo is associated with a marked induction of tumor-derived matrix metalloproteinase-1 and stromal matrix metalloproteinase-9 and -13, as was confirmed in three-dimensional organotypic co-cultures with fibroblasts in vitro. These data clearly demonstrate the need for a concerted action of different growth factors in the establishment of solid tumors with functional vasculature and emphasize the need for a multifactorial therapy.

Distinct progression-associated expression of tumor and stromal MMPs in HaCaT skin SCCs correlates with onset of invasion.

Matrix metalloproteinases (MMPs) are critically involved in tumor invasion and metastasis. However, failure of broad spectrum MMP inhibitors in clinical trials emphasizes the need for detailed analyses of the specific role of different MMPs in tumor malignancy. Using HaCaT-keratinocyte clones representing distinct stages in skin squamous cell carcinoma (SCC) progression, we demonstrate the expression of specific tumor and stroma-derived MMPs with the onset and maintenance of tumor invasion. Although MMP-9-positive leukocytes are present in benign and malignant tumor transplants at the onset of stromal activation and angiogenesis, mRNA expression of stroma-derived MMP-9 as well as MMP-2, -13 and -14 is exclusively found in enhanced malignant tumor transplants. Their expression initiates with the onset of invasion, whereas being absent in early noninvasive stages of malignant transplants. In addition, a high expression of tumor-derived MMP-1, -2 and -14 contributes to malignant and invasive tumor growth. However, stroma-derived MMP-3 is exclusively restricted to very late-stage invasive and malignant transplants. The functional contribution of these proteases to invasive growth is supported by the gelatinolytic activity in the tumor transplants that again initiates with the onset of invasive growth suggesting a crucial role of MMP-2, -9, -13 and -14 for the establishment of a reactive stroma that promotes tumor invasion. These data demonstrate a complex cooperation of distinct tumor and stroma-derived MMPs in the establishment of malignant tumors and provide the basis for a more specific use of highly selective MMP inhibitors during distinct stages of tumor progression.

E-cadherin suppression directs cytoskeletal rearrangement and intraepithelial tumor cell migration in 3D human skin equivalents.

The link between loss of cell-cell adhesion, the activation of cell migration, and the behavior of intraepithelial (IE) tumor cells during the early stages of skin cancer progression is not well understood. The current study characterized the migratory behavior of a squamous cell carcinoma cell line (HaCaT-II-4) upon E-cadherin suppression in both 2D, monolayer cultures and within human skin equivalents that mimic premalignant disease. The migratory behavior of tumor cells was first analyzed in 3D tissue context by developing a model that mimics transepithelial tumor cell migration. We show that loss of cell adhesion enabled migration of single, IE tumor cells between normal keratinocytes as a prerequisite for stromal invasion. To further understand this migratory behavior, E-cadherin-deficient cells were analyzed in 2D, monolayer cultures and displayed altered cytoarchitecture and enhanced membrane protrusive activity that was associated with circumferential actin organization and induction of the nonmuscle, beta actin isoform. These features were associated with increased motility and random, individual cell migration in response to scrape-wounding. Thus, loss of E-cadherin-mediated adhesion led to the acquisition of phenotypic properties that augmented cell motility and directed the transition from the precancer to cancer in skin-like tissues.

Effects of fibroblasts and microenvironment on epidermal regeneration and tissue function in long-term skin equivalents.

In vitro generated skin models find growing interest as promising tools in basic research and clinical application in regenerative medicine. Here, we present further details of an improved long-term skin equivalent (SE) enabling mechanistic studies on skin reconstruction and epidermal function. Growth conditions of fibroblasts in a 3D scaffold were analysed to optimise the dermal microenvironment by providing an authentic dermal matrix for regular tissue reconstruction and function of cocultured keratinocytes. These SEs demonstrate sustained epidermal viability - over 12 weeks - with regular differentiation as substantiated by in vivo-like patterns of all differentiation products, exemplified here by the cornified envelope components loricrin and repetin. The continuous expression of all major tight junction components in the granular layer, shown here for ZO-1 in coherence with the presence of epidermal barrier lipids, and ultrastructural accumulation of lamellar bodies, collectively indicate proper epidermal barrier structures. Remarkably, cocultured keratinocytes exerted an ongoing proliferation-stimulating effect on fibroblasts colonising the scaffold comparable to a cocktail of fibroblast growth factors. Consequently, precultivation of dermal equivalents (DEs) in basal or growth factor-enriched media had only minor effects on the quality of epidermal regeneration in cocultures. As to the role of fibroblast numbers, complete absence of dermal cells resulted in atrophic epithelia but the effect of cell numbers as low as 5 x 10(4)cells/cm(2) on epidermal tissue quality equalled that of the standard density (2 x 10(5)cells/cm(2)). Surprisingly, precultivation of fibroblasts in the DEs for 7 days (standard) showed no better effect on epidermal tissue reformation as compared to 2 days whereas a precultivation period of 14 days resulted in atrophic epidermal and dermal tissue development. These data demonstrate, (i) the strict dependence of epidermal tissue regeneration on the presence of fibroblasts, (ii) the mutual keratinocyte-fibroblast interactions for cell proliferation and organogenesis, and (iii) the importance of the proper microenvironment for epidermal tissue function and supposedly for establishment of a stem cell niche in vitro.

Critical role for NF-kappaB-induced JunB in VEGF regulation and tumor angiogenesis.

Regulation of vascular endothelial growth factor (VEGF) expression is a complex process involving a plethora of transcriptional regulators. The AP-1 transcription factor is considered as facilitator of hypoxia-induced VEGF expression through interaction with hypoxia-inducible factor (HIF) which plays a major role in mediating the cellular hypoxia response. As yet, both the decisive AP-1 subunit leading to VEGF induction and the molecular mechanism by which this subunit is activated have not been deciphered. Here, we demonstrate that the AP-1 subunit junB is a target gene of hypoxia-induced signaling via NF-kappaB. Loss of JunB in various cell types results in severely impaired hypoxia-induced VEGF expression, although HIF is present and becomes stabilized. Thus, we identify JunB as a critical independent regulator of VEGF transcription and provide a mechanistic explanation for the inherent vascular phenotypes seen in JunB-deficient embryos, ex vivo allantois explants and in vitro differentiated embryoid bodies. In support of these findings, tumor angiogenesis was impaired in junB(-/-) teratocarcinomas because of severely impaired paracrine-acting VEGF and the subsequent inability to efficiently recruit host-derived vessels.

Reversion of tumor phenotype in surface transplants of skin SCC cells by scaffold-induced stroma modulation.

Interactions between cancer cells and the tissue microenvironment play an essential role in controlling tumor development and progression. Here, we report that stromal modulation induced by a biodegradable meshwork (Hyalograft 3D) inhibited tumor vascularization and invasion of the locally invasive low-grade malignant human HaCaT-ras II-4 keratinocytes in a surface xenotransplantation assay. The scaffold caused formation of an active granulation tissue that shifted to a fibrotic-type connective tissue with accumulation of myofibroblasts and collagen bundles. Most importantly, in transplants with scaffolds, the epithelial-stromal border was normalized developing an ultrastructurally complete basement membrane (BM) including hemidesmosomes. The observed reversion of the tumor phenotype was not due to decreased tumor cell proliferation but correlated with (i) normalization of epidermal differentiation, (ii) condensation of extracellular matrix (ECM) and (iii) reduction of peritumoral protease activity Furthermore, inhibited invasion was paralleled by eliminated tumor vascularization. This was substantiated by a diminished endothelial VEGF-receptor (VEGFR) expression and, in turn, by a concomitant increase in the ECM components thrombospondin-1 (TSP-1) and endostatin, known to impair angiogenesis. Even in transplants of the metastatic high-grade malignant HaCaT-ras A-5RT3 keratinocytes the anti-invasive effect of the scaffold-modulated stroma prevailed. Tumor vascularization and invasion was reduced and the epithelial tissue partially normalized including formation of stretches of BM. This clearly demonstrates that the scaffold-modulated connective tissue not only blocks tumor invasion but reverts the tumor phenotype. These novel findings underline the controlling function of tumor stroma and open new strategies of cancer therapy by targeting tumor stroma elements.

Platelet-derived growth factor-BB controls epithelial tumor phenotype by differential growth factor regulation in stromal cells.

Platelet-derived growth factor (PDGF) stimulates tumor growth and progression by affecting tumor and stromal cells. In the HaCaT skin carcinogenesis model, transfection of immortal nontumorigenic and PDGF-receptor-negative HaCaT keratinocytes with PDGF-B induced formation of benign tumors. Here, we present potential mechanisms underlying this tumorigenic conversion. In vivo, persistent PDGF-B expression induced enhanced tumor cell proliferation but only transiently stimulated stromal cell proliferation and angiogenesis. In vitro and in vivo studies identified fibroblasts as PDGF target cells essential for mediating transient angiogenesis and persistent epithelial hyperproliferation. In fibroblast cultures, long-term PDGF-BB treatment caused an initial up-regulation of vascular endothelial growth factor (VEGF)-A, followed by a drastic VEGF down-regulation and myofibroblast differentiation. Accordingly, in HaCaT/PDGF-B transplants, initially enhanced VEGF expression by stromal fibroblasts was subsequently reduced, followed by down-regulation of angiogenesis, myofibroblast accumulation, and vessel maturation. The PDGF-induced, persistently increased expression of the hepatocyte growth factor by fibroblasts in vitro and in vivo was most probably responsible for enhanced epithelial cell proliferation and benign tumor formation. Thus, by paracrine stimulation of the stroma, PDGF-BB induced epithelial hyperproliferation, thereby promoting tumorigenicity, whereas the time-limited activation of the stroma followed by stromal maturation provides a possible explanation for the benign tumor phenotype.

Epidermal homeostasis in long-term scaffold-enforced skin equivalents.

Epidermal homeostasis is understood as the maintenance of epidermal tissue structure and function by a fine tuned regulatory mechanism balancing proliferation and cell loss by desquamation and apoptosis. The lack of appropriate experimental models has largely prevented a better understanding of the regulatory mechanisms controlling epidermal tissue homeostasis in human skin. Keratinocyte culture studies had revealed a strict dependency of regular epidermal differentiation on dermal interactions only accomplishable in three-dimensional skin models. As major drawbacks, conventional models, employing collagen hydrogels as dermal equivalents (DEs) exhibit a rather poor stability and limited lifespan. Here, we present an improved stabilized in vitro-model for long-term growth and differentiation of keratinocytes providing the basis for tissue homeostasis. Keratinocytes were grown on DEs reinforced by modified hyaluronic acid fibers (Hyalograft-3D) and colonized with skin fibroblasts, producing genuine dermis-type matrix. These skin equivalents (SEs) develop superior epidermal architecture with regular differentiation and ultrastructure. Critical aspects of differentiation, still unbalanced in early stages, are renormalized, most strikingly the coexpression of keratins K1/K10, downregulation of regeneration-associated keratins (K16), and restriction of K15 to the basal layer. The strict localization of integrins to basal cells underlining restored tissue polarity, the drop of keratinocyte growth rates towards physiological levels and the rapid formation of a mature basement membrane with abundant anchoring fibrils are altogether features fulfilling the criteria of tissue homeostasis. Therefore, these scaffold-based SEs not only allow for studying homeostasis control but also for the first time provide proper experimental conditions for establishing a stem cell niche in vitro.

Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor promote malignant growth of cells from head and neck squamous cell carcinomas in vivo.

Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are used to ameliorate cancer therapy-induced neutropenia and mucositis. Yet, first data in head and neck squamous cell carcinoma (HNSCC) indicate an impaired long-term prognosis on G-CSF treatment, and previous studies showed a contribution of both factors to the progression of human epithelial tumors. Therefore, we investigate the role of G-CSF and GM-CSF in progression of tumor cells from human HNSCC. Both factors stimulated proliferation and migration of tumor cell lines established from patient tumors expressing G-CSF and GM-CSF and/or their receptors. Blockade of G-CSF and GM-CSF inhibited tumor cell invasion in a three-dimensional organotypic culture model. The contribution of both factors to tumor malignancy was further confirmed in nude mouse transplants in vivo. Invasive and malignant growth yielding a similar tumor phenotype as the original patient tumor was exclusively observed in G-CSF- and GM-CSF-expressing tumors and was associated with enhanced and persistent angiogenesis and enhanced inflammatory cell recruitment. Although factor-negative tumors grew somewhat faster, they were characterized by lack of invasion, reduced and transient angiogenesis, and large necrotic areas. These data provide evidence for a progression-promoting effect of G-CSF and GM-CSF in human HNSCC and suggest further detailed evaluation of their use in the therapy of these tumors.

Vascular anomalies in lipoid proteinosis (hyalinosis cutis et mucosae): basement membrane components and ultrastructure.

BACKGROUND: In lipoid proteinosis (LP) vascular anomalies represent severe functional defects caused by excessive deposition of basement membrane (BM)-like matrix, particularly around small subepithelial blood vessels. OBJECTIVE: Correlation of microvascular anomalies in morphology and ultrastructure with extracellular matrix composition and cell interactions for elucidating vascular involvement in LP-pathophysiology. METHODS: Biopsies from non-related LP-patients were analyzed by indirect immunofluorescence (IIF), electron microscopy (EM), and immune-EM (ImEM). RESULTS: In LP-skin and mucosa the thickened vessel walls stained strongly for the BM-components type IV collagen, laminin, perlecan, and nidogen (IIF). Integrin alpha6beta4 was regularly collocated with endothelial surface markers such as PECAM (CD31). Ultrastructure (EM) revealed highly ordered matrix deposits around microvessels, with frequently collapsed lumina, functionally compensated by increased vascular density (histology, IIF). Pericytes were trapped between these concentric BM-layers at varying distances towards the periphery (EM), contrasting their regularly close endothelial apposition. Periodic type IV collagen patterns (ImEM) corroborated the multiple BM-leaflet structure and the lack of a common 'fused' endothelial-pericyte BM, seen normally. Presumptive secretory vesicles, abundant in both cell types, implied an equal contribution to BM-synthesis, but also indicated partial loss of endothelial polarity. CONCLUSIONS: In LP thickened vessel walls, composed of multiple BM, profoundly alter microvascular properties, also by interference with endothelial-pericyte interactions. The increased microvascular density reflects compensatory restoration for disabled function. Most remarkable was the exaggerated secretory activity (also at luminal surfaces) underlining the regulatory key role of extracellular matrix protein 1 (ECM1; mutated in LP) in export or turnover of all major BM-components.

E-cadherin loss promotes the initiation of squamous cell carcinoma invasion through modulation of integrin-mediated adhesion.

Much remains to be learned about how cell-cell and cell-matrix interactions are coordinated to influence the earliest development of neoplasia. We used novel 3D human tissue reconstructs that mimic premalignant disease in normal epidermis, to directly investigate how loss of E-cadherin function directs conversion to malignant disease. We used a genetically tagged variant of Ha-Ras-transformed human keratinocytes (II-4) expressing dominant-interfering E-cadherin fusion protein (H-2k(d)-Ecad). These cells were admixed with normal human keratinocytes and tumor cell fate was monitored in 3D reconstructed epidermis upon transplantation to immunodeficient mice. Tumor initiation was suppressed in tissues harboring control- and mock-infected II-4 cells that lost contact with the stromal interface. By contrast, H-2k(d)-Ecad-expressing cells persisted at this interface, thus enabling incipient tumor cell invasion upon in vivo transplantation. Loss of intercellular adhesion was linked to elevated cell surface expression of alpha2, alpha3 and beta1 integrins and increased adhesion to laminin-1 and Types I and IV collagen that was blocked with beta1-integrin antibodies, suggesting that invasion was linked to initial II-4 cell attachment at the stromal interface. Collectively, these results outline a novel aspect to loss of E-cadherin function that is linked to the mutually interdependent regulation of cell-cell and cell-matrix adhesion and has significant consequences for the conversion of premalignancy to cancer.

Loss of intercellular adhesion activates a transition from low- to high-grade human squamous cell carcinoma.

The relationship between loss of intercellular adhesion and the biologic properties of human squamous cell carcinoma is not well understood. We investigated how abrogation of E-cadherin-mediated adhesion influenced the behavior and phenotype of squamous cell carcinoma in 3D human tissues. Cell-cell adhesion was disrupted in early-stage epithelial tumor cells (HaCaT-II-4) through expression of a dominant-negative form of E-cadherin (H-2Kd-Ecad). Three-dimensional human tissue constructs harboring either H-2Kd-Ecad-expressing or control II-4 cells (pBabe, H-2Kd-EcadDeltaC25) were cultured at an air-liquid interface for 8 days and transplanted to nude mice; tumor phenotype was analyzed 2 days and 2 and 4 weeks later. H-2Kd-Ecad-expressing tumors demonstrated a switch to a high-grade aggressive tumor phenotype characterized by poorly differentiated tumor cells that infiltrated throughout the stroma. This high-grade carcinoma revealed elevated cell proliferation in a random pattern, loss of keratin 1 and diffuse deposition of laminin 5 gamma2 chain. When II-4 cell variants were seeded into type I collagen gels as an in vitro assay for cell migration, we found that only E-cadherin-deficient cells detached, migrated as single cells and expressed N-cadherin. Function-blocking studies demonstrated that this migration was matrix metalloproteinase-dependent, as GM-6001 and TIMP-2, but not TIMP-1, could block migration. Gene expression profiles revealed that E-cadherin-deficient II-4 cells demonstrated increased expression of proteases and cell-cell and cell-matrix proteins. These findings showed that loss of E-cadherin-mediated adhesion plays a causal role in the transition from low- to high-grade squamous cell carcinomas and that the absence of E-cadherin is an important prognostic marker in the progression of this disease.

Targeting perlecan in human keratinocytes reveals novel roles for perlecan in epidermal formation.

Heparin-binding growth factors are crucial for the formation of human epidermis, but little is known about the role of heparan sulfate proteoglycans in this process. Here we investigated the role of the heparan sulfate proteoglycan, perlecan, in the formation of human epidermis, by utilizing in vitro engineered human skin. By disrupting perlecan expression either in the dermis or the epidermis, we found that epidermally derived perlecan is essential for epidermal formation. Perlecan-deficient keratinocytes formed a strikingly thin and poorly organized epidermis because of premature apoptosis and failure to complete their stratification program. Exogenous perlecan fully restored epidermal formation. Perlecan deposition in the basement membrane zone correlated with formation of multilayered epidermis. Perlecan deficiency, however, had no effect on the lining and deposition of major basement membrane components as was evident by a continuous linear staining of laminin and collagen IV. Similarly, perlecan deficiency did not affect the distribution of beta1 integrin. Addition of the perlecan ligand, fibroblast growth factor 7, protected perlecan-deficient keratinocytes from cell death and improved the thickness of the epidermis. Taken together, our results revealed novel roles for perlecan in epidermal formation. Perlecan regulates both the survival and terminal differentiation steps of keratinocytes. Our results suggested a model whereby perlecan regulates these processes via controlling the bioavailability of perlecan-binding soluble factors involved in epidermal morphogenesis.

Rapid vessel regression, protease inhibition, and stromal normalization upon short-term vascular endothelial growth factor receptor 2 inhibition in skin carcinoma heterotransplants.

Vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis, and blockade of VEGF receptor 2 (VEGFR-2), with the monoclonal antibody DC101, inhibits angiogenesis and tumor growth. To examine the short-term effects of DC101, we surface transplanted the squamous cell carcinoma cell line A5-RT3 onto nude mice. After short-term treatment with DC101, we observed rapid reduction in vascularization and reversion of the tumor phenotype. Beginning 24 hours after treatment, VEGFR-2 inhibition resulted in decreased vessel density within the tenascin-c-staining tumor-associated stroma and reduced endothelial cell proliferation. Stromal expression of matrix metalloproteinase-9 and -13 was drastically reduced 96 hours after VEGFR-2 inhibition as detected by in situ hybridization and in situ zymography. Moreover, the morphology of the tumor-stroma border changed from a highly invasive carcinoma to a well-demarcated, premalignant phenotype. The latter was characterized by the appearance of a regular basement membrane in immunostaining and ultrastructural analyses. These findings suggest that VEGFR-2 inhibition by DC101 evokes very rapid reduction of preformed vessels and decreases both stromal protease expression and gelatinolytic activity, resulting in the modulation of the tumor-stroma border zone and reversion of the tumor phenotype. Thus, short-term inhibition of VEGF signaling results in complex stromal alterations with crucial consequences for the tumor phenotype.