Keep recycling going: New approaches to reduce LDL-C.

Hypercholesterolemia represents a leading cause in the development of atherosclerotic plaques, increasing the risk for ACVS. It actually counts as a major cause of cardiovascular disease etiopathogenesis. The causes of hypercholesterolemia are multifactorial, spanning from genetic constitution, age, sex, to sedentary lifestyle and diets rich in sugars and lipids. Although dietary restriction in saturated fats, increased exercise, and other modification in lifestyle represent a first-line approach to treat very initial stages in hypercholesterolemia, most patients will require the addition of pharmacological agents. Pharmacological approaches include inhibition of cholesterol synthesis, decreased fat absorption from the GI tract, and increased degradation of FA. These strategies present a series of side effects, low therapeutic efficiency in some patients, and reduced tolerability. One of the major goals in treatment for hypercholesterolemia is to decrease the levels of low density lipoproteins (LDL), while maintaining those of high density lipoproteins (HDL). LDL particles contain about 80% of lipids, most of it cholesterol and cholesteryl esters, and 20% of the ApoB-100 protein. LDL carries cholesterol to the tissues, to be incorporated to biological membranes, or to be transformed to steroids. Excess of LDL translates into increased levels of circulating cholesterol particles and accumulation in certain tissues, especially vascular tissue, initiating a fatty streak, which may evolve to an atheroma, causing a series of cardiovascular problems, including impaired circulation, high blood pressure, increased cardiac workload, and coronary artery disease. It is essential to prevent LDL accumulation into the bloodstream to avoid the formation of these fatty streaks and the initiation of a cascade that will lead to the development of atherosclerosis. In healthy individuals. Under physiological conditions, LDL is effectively removed from circulation through receptor-mediated endocytosis. LDL clearance involves binding to its receptor, LDLR, which enables the internalization of the LDL particle and drives its degradation in lysosomes. Once the LDL particle is degraded, the free receptor recycles to the plasma membrane, and captures new LDL particles. Adequate levels of LDLR are essential to remove the excess of cholesterol-laden LDL. Proprotein convertase, subtilysin kexin type 9 (PCSK-9), expressed in liver and intestine, binds to LDLR, and internalized. Once inside the cell, PCSK-9 catalyzes the proteolysis of LDLR, preventing its recycling to the cell surface, and effectively decreasing the number of LDLR, notoriously decreasing the ability to clear LDL from circulation. Levels of PCSK-9 varies with age, gender, and levels of insulin, glucose, and triglycerides. Loss-of-function mutations in PCSK-9 gene invariably translates into lower levels of LDL, and decreased risk of developing coronary artery disease. Conversely, increased activity or expression of this enzyme leads to hypercholesterolemia. Inhibition of PCSK9 has proven to be successful in decreasing LDL levels and risk of the development of hypercholesterolemia with its associated higher risk for ASCVD. Patient with gain-of-function mutations in the PCSK9 undoubtedly benefit from therapies based on PCSK-9 inhibitors. However, millions of patients show statin intolerance, or cannot be efficiently controlled by statins alone- the most prevalent therapy for hypeprcholesterolemia. This commentary will evaluate the possibilities, caveats and future directions in the treatment of hypercholesterolemia, and therapies with combination of drugs.

Proprotein convertase inhibition: Paralyzing the cell's master switches.

Proprotein convertases are serine proteases responsible for the cleavage and subsequent activation of protein substrates, many of them relevant for the development of an ample variety of diseases. Seven of the PCs, including furin and PACE4, recognize and hydrolyze the C-terminal end of the general sequence RXRR/KXR, whereas PCSK-9 recognizes a series of non-basic amino acids. In some systems, PC-mediated substrate activation results in the development of pathological processes, such as cancer, endocrinopathies, and cardiovascular and infectious diseases. After establishing PCs as relevant contributors to disease processes, research efforts were directed towards the development of inhibition strategies, including small and large molecules, anti-sense therapies, and antibody-based therapies. Most of these inhibitors mimic the consensus sequence of PCs, blocking the active site in a competitive manner. The most promising inhibitors were designed as bioengineered proteins; however, some non-protein and peptidomimetic agents have also proved to be effective. These efforts led to the design of pre-clinical studies and clinical trials utilizing inhibitors to PCs. Although the initial studies were performed using non-selective PCs inhibitors, such as CMK, the search for more specific, and compartmentalized selective inhibitors resulted in specific activities ascribed to some, but not all of the PCs. For instance, PACE4 inhibitors were effective in decreasing prostate cancer cell proliferation, and neovascularization. Decreased metastatic ovarian cancer utilizing furin inhibitors represents one of the major endeavors, currently in a phase II trial stage. Antibodies targeting PCSK-9 decreased significantly the levels of HDL-cholesterol, in a phase III trial. The study of Proprotein convertases has reached a stage of maturity. New strategies based on the alteration of their activity at the cellular and clinical level represent a promising experimental pharmacology field. The development of allosteric inhibitors, or specific agents directed against individual PCs is one of the challenges to be unraveled in the future.

Targeting proprotein convertases in furin-rich lung cancer cells results in decreased in vitro and in vivo growth.

Proprotein convertases (PCs) are serine proteases with an active role in the post-translational processing of numerous inactive proteins to active proteins including many substrates of paramount importance in cancer development and progression. Furin (PCSKC3), a well-studied member of this family, is overexpressed in numerous human and experimental malignancies. In the present communication, we treated two furin-overexpressing non-small cell carcinoma (NSCLC) cell lines (Calu-6 and HOP-62) with the PC inhibitor CMK (Decanoyl-Arg-Val-Lys-Arg-chloromethylketone). This resulted in a diminished IGF-1R processing and a simultaneous decrease in cell proliferation of two NSCLC lines. Similarly, growth of subcutaneous xenografts of both cell lines, were partially inhibited by an in vivo treatment with the same drug. These observations point to a potential role of PC inhibitors in cancer therapy. © 2016 Wiley Periodicals, Inc.

Enhanced aggressiveness of benzopyrene-induced squamous carcinomas in transgenic mice overexpressing the proprotein convertase PACE4 (PCSK6).

PACE4 (PCSK6) is a proprotein convertase (PC) capable of processing numerous substrates involved in tumor growth, invasion, and metastasis. Because of the human relevancy of the tobacco-associated carcinogen benzo[a]pyrene (B(a)P) we investigated whether transgenic mice in which this PC is targeted to the epidermis (K5-PACE4) may be more susceptible to B(a)P complete carcinogenesis than wild type (WT) mice. In an in vitro experiment, using cell lines derived from skin tumors obtained after B(a)P treatment, we observed that PACE4 overexpression and activity accounts for an increased proliferation rate, exaggerated sensitivity to the PC inhibitor CMK, and interference with IGF-1R autophosphorylation. Squamous cell carcinomas, obtained from K5-PACE4 mice subjected to complete chemical carcinogenesis, were characterized by a 50% increase in cell proliferation, when compared with similar tumors from WT mice. In addition, tumors from K5-PACE4 mice showed deeper invasion into the underlying dermis. Thus, mice overexpressing PACE4 exhibited tumors of increased growth rate and invasive potential when exposed to the human carcinogen B(a)P, further supporting the significance of PCs in tumor growth and progression.

Enhanced UV-induced skin carcinogenesis in transgenic mice overexpressing proprotein convertases.

The proprotein convertases (PCs) furin and PACE4 process numerous substrates involved in tumor growth, invasion, and metastasis. We have previously shown that PCs increase the susceptibility to chemical skin carcinogenesis. Because of the human relevancy of UV radiation in the etiopathogenesis of human skin cancer, we investigated whether or not transgenic mice overexpressing either furin alone or both furin and PACE4 show increased susceptibility to UV carcinogenesis. After backcrossing our previously described furin and PACE4 transgenic lines, targeted to the epidermis, into a SKH-1 background, we exposed both single and double transgenic mice to UV radiation for 34 weeks. The results showed an increase in squamous cell carcinoma (SCC) multiplicity of approximately 70% in the single furin transgenic mouse line SF47 (P < .002) and a 30% increase in the other single transgenic line SF49 when compared to wild-type (WT) SKH-1 mice. Interestingly, there was also an increase in the percentage of high histologic grade SCCs in the transgenic lines compared to the WT mice, i.e., WT = 9%, SF47 = 15%, and SF49 = 26% (P < .02). Targeting both furin and PACE4 to the epidermis in double transgenic mice did not have an additive effect on tumor incidence/multiplicity but did enhance the tumor histopathologic grade, i.e., a significant increase in higher grade SCCs was seen in the bigenic mouse line SPF47 (P < .02). Thus, we observed an increased susceptibility to UV in single furin transgenic mice that was not substantially enhanced in the double furin/PACE4 transgenic mice.

Transgenic overexpression of the proprotein convertase furin enhances skin tumor growth.

Furin, one of the members of the family of proprotein convertases (PCs), ubiquitously expressed as a type I membrane-bound proteinase, activates several proteins that contribute to tumor progression. In vitro studies using cancer cell lines and clinical specimens demonstrated that furin processes important substrates such as insulin-like growth factor 1 receptor (IGF-1R) and transforming growth factor ß, leading to increased tumor growth and progression. Despite the numerous studies associating furin with tumor development, its effects in preclinical models has not been comprehensively studied. In this study, we sought to determine the protumorigenic role of furin in vivo after a two-stage chemical carcinogenesis protocol in transgenic mice in which furin expression was targeted to the epidermal basal layer. We found that processing of the PC substrate IGF-1R and the proliferation rate of mouse epidermis was enhanced in transgenic mice when compared with their WT counterparts. Histopathologic diagnoses of the tumors demonstrated that furin transgenic mice (line F47) developed twice as many squamous carcinomas as the control, WT mice (P < .002). Similarly, tumors cells from transgenic mice were able to process PC substrates more efficiently than tumor cells from WT mice. Furthermore, furin expression resulted in a higher SCC volume in transgenic mice as well as an increase in the percentage of high-grade SCC, including poorly differentiated and spindle cell carcinomas. In conclusion, expression of furin in the basal layer of the epidermis increased tumor development and enhanced tumor growth, supporting the consideration of furin as a potential target for cancer treatment.

The mesenchymal tumor microenvironment: a drug-resistant niche.

Drug and radiation resistance represent a challenge for most anticancer therapies. Diverse experimental approaches have provided evidence that the tumor-associated microenvironment constitutes both a protective shell that impedes drug or radiation access and a permissive or promotive microenvironment that encourages a nurturing cancer (i.e., cancer stem cell) niche where tumor cells overcome treatment- and cancer-induced stresses. Better understanding of the effects of the tumor microenvironment on cancer cells before, during and immediately after chemo- or radiotherapy is imperative to design new therapies aimed at targeting this tumor-protective niche. This review summarizes some of the known mesenchymal stromal effects that account for drug resistance, the main signal transduction pathways associated with this resistance and the therapeutic efforts directed to increase the success of current therapies. Special emphasis is given to environment-mediated drug resistance in general and to cell adhesion-mediated drug resistance in particular.

Elevated expression of stromal palladin predicts poor clinical outcome in renal cell carcinoma.

The role that stromal renal cell carcinoma (RCC) plays in support of tumor progression is unclear. Here we sought to determine the predictive value on patient survival of several markers of stromal activation and the feasibility of a fibroblast-derived extracellular matrix (ECM) based three-dimensional (3D) culture stemming from clinical specimens to recapitulate stromal behavior in vitro. The clinical relevance of selected stromal markers was assessed using a well annotated tumor microarray where stromal-marker levels of expression were evaluated and compared to patient outcomes. Also, an in vitro 3D system derived from fibroblasts harvested from patient matched normal kidney, primary RCC and metastatic tumors was employed to evaluate levels and localizations of known stromal markers such as the actin binding proteins palladin, alpha-smooth muscle actin (α-SMA), fibronectin and its spliced form EDA. Results suggested that RCCs exhibiting high levels of stromal palladin correlate with a poor prognosis, as demonstrated by overall survival time. Conversely, cases of RCCs where stroma presents low levels of palladin expression indicate increased survival times and, hence, better outcomes. Fibroblast-derived 3D cultures, which facilitate the categorization of stromal RCCs into discrete progressive stromal stages, also show increased levels of expression and stress fiber localization of α-SMA and palladin, as well as topographical organization of fibronectin and its splice variant EDA. These observations are concordant with expression levels of these markers in vivo. The study proposes that palladin constitutes a useful marker of poor prognosis in non-metastatic RCCs, while in vitro 3D cultures accurately represent the specific patient's tumor-associated stromal compartment. Our observations support the belief that stromal palladin assessments have clinical relevance thus validating the use of these 3D cultures to study both progressive RCC-associated stroma and stroma-dependent mechanisms affecting tumorigenesis. The clinical value of assessing RCC stromal activation merits further study.

Proprotein convertase inhibition results in decreased skin cell proliferation, tumorigenesis, and metastasis.

PACE4 is a proprotein convertase (PC) responsible for cleaving and activating proteins that contribute to enhance tumor progression. PACE4 overexpression significantly increased the susceptibility to carcinogenesis, leading to enhanced tumor cell proliferation and premature degradation of the basement membrane. In the present study, we sought to evaluate a novel approach to retard skin tumor progression based on the inhibition of PACE4. We used decanoyl-RVKR-chloromethylketone (CMK), a small-molecule PC inhibitor, for in vitro and in vivo experiments. We found that CMK-dependent blockage of PACE4 activity in skin squamous cell carcinoma cell lines resulted in impaired insulin-like growth factor 1 receptor maturation, diminished its intrinsic tyrosine kinase activity, and decreased tumor cell proliferation. Two-stage skin chemical carcinogenesis experiments, together with topical applications of CMK, demonstrated that this PC inhibitor markedly reduced tumor incidence, tumor multiplicity, and metastasis, pointing to a significant delay in tumor progression in wild-type and PACE4 transgenic mice. These results identify PACE4, together with other PCs, as suitable targets to slow down or block tumor progression, suggesting that PC inhibition is a potential approach for therapy for solid tumors.

Physico-mechanical aspects of extracellular matrix influences on tumorigenic behaviors.

Tumor progression in vitro has traditionally been studied in the context of two-dimensional (2D) environments. However, it is now well accepted that 2D substrates are unnaturally rigid compared to the physiological substrate known as extracellular matrix (ECM) that is in direct contact with both normal and tumorigenic cells in vivo. Hence, the patterns of interactions, as well as the strategies used by cells in order to penetrate the ECM, and migrate through a three-dimensional (3D) environment are notoriously different than those observed in 2D. Several substrates, such as collagen I, laminin, or complex mixtures of ECM components have been used as surrogates of native 3D ECM to more accurately study cancer cell behaviors. In addition, 3D matrices developed from normal or tumor-associated fibroblasts have been produced to recapitulate the mesenchymal 3D environment that assorted cells encounter in vivo. Some of these substrates are being used to evaluate physico-mechanical effects on tumor cell behavior. Physiological 3D ECMs exhibit a wide range of rigidities amongst different tissues while the degree of stromal stiffness is known to change during tumorigenesis. In this review we describe some of the physico-mechanical characteristics of tumor-associated ECMs believed to play important roles in regulating epithelial tumorigenic behaviors.

VILIP-1 expression in vivo results in decreased mouse skin keratinocyte proliferation and tumor development.

VILIP-1, a member of the neuronal Ca(2+) sensor protein family, is able to act as a tumor suppressor in carcinoma cells by inhibiting cell proliferation and migration. In order to study the role of VILIP-1 in skin carcinogenesis we generated transgenic mice overexpressing VILIP-1 in epidermis under the control of the bovine keratin K5 promoter (K5-VILIP-1). We studied the susceptibility of FVB wild type and VILIP-1 transgenic mice to chemically mediated carcinogenesis. After 30 weeks of treatment with a two-stage carcinogenesis protocol, all animals showed numerous skin tumors. Nevertheless, K5-VILIP-1 mice showed decreased squamous cell carcinoma (SCC) multiplicity of approximately 49% (p<0.02) with respect to the corresponding SCC multiplicity observed in wild type (WT) mice. In addition, the relative percentage of low-grade cutaneous SCCs grade I (defined by the differentiation pattern according to the Broders grading scale) increased approximately 50% in the K5-VILIP1 mice when compared with SCCs in WT mice. Similar tendency was observed using a complete carcinogenesis protocol for skin carcinogenesis using benzo(a)pyrene (B(a)P). Further studies of tumors and primary epidermal keratinocyte cultures showed that matrix metalloproteinase 9 (MMP-9) levels and cell proliferation decreased in K5-VILIP-1 mice when compared with their wild counterparts. In addition tissue inhibitor of metalloproteinase 1 (TIMP-1) expression was higher in K5-VILIP-1 keratinocytes. These results show that VILIP-1 overexpression decreases the susceptibility to skin carcinogenesis in experimental mouse cancer models, thus supporting its role as a tumor suppressor gene.

VILIP-1 downregulation in non-small cell lung carcinomas: mechanisms and prediction of survival.

VILIP-1, a member of the neuronal Ca++ sensor protein family, acts as a tumor suppressor gene in an experimental animal model by inhibiting cell proliferation, adhesion and invasiveness of squamous cell carcinoma cells. Western Blot analysis of human tumor cells showed that VILIP-1 expression was undetectable in several types of human tumor cells, including 11 out of 12 non-small cell lung carcinoma (NSCLC) cell lines. The down-regulation of VILIP-1 was due to loss of VILIP-1 mRNA transcripts. Rearrangements, large gene deletions or mutations were not found. Hypermethylation of the VILIP-1 promoter played an important role in gene silencing. In most VILIP-1-silent cells the VILIP-1 promoter was methylated. In vitro methylation of the VILIP-1 promoter reduced its activity in a promoter-reporter assay. Transcriptional activity of endogenous VILIP-1 promoter was recovered by treatment with 5'-aza-2'-deoxycytidine (5'-Aza-dC). Trichostatin A (TSA), a histone deacetylase inhibitor, potently induced VILIP-1 expression, indicating that histone deacetylation is an additional mechanism of VILIP-1 silencing. TSA increased histone H3 and H4 acetylation in the region of the VILIP-1 promoter. Furthermore, statistical analysis of expression and promoter methylation (n = 150 primary NSCLC samples) showed a significant relationship between promoter methylation and protein expression downregulation as well as between survival and decreased or absent VILIP-1 expression in lung cancer tissues (p<0.0001). VILIP-1 expression is silenced by promoter hypermethylation and histone deacetylation in aggressive NSCLC cell lines and primary tumors and its clinical evaluation could have a role as a predictor of short-term survival in lung cancer patients.

Increased expression of the pro-protein convertase furin predicts decreased survival in ovarian cancer.

BACKGROUND: Proprotein convertases (PCs) are serine proteases that after restricted proteolysis activate many proteins that play a crucial role in cancer such as metalloproteinases, growth factors and growth factor receptors, adhesion molecules, and angiogenic factors. Although the expression of several PCs is increased in many tumors, their expression in primary ovarian tumors has not been studied in detail. We sought to determine if there was an association between the expression of the ubiquitously expressed PCs, furin, PACE-4, PC-5 and PC-7, and ovarian tumor progression. METHODS: We assessed their expression by RT-PCR, Real-time PCR, Western blot, and immunohistochemistry using cells derived from normal human ovarian surface epithelium (HOSE) and cancer cell lines as well as ovarian epithelial cancer specimens (45 RT-PCR/Real-time PCR, and 120 archival specimens for Immunohistochemistry). RESULTS: We found that furin expression was restricted to the cancer cell lines. In contrast, PACE-4 and PC-7 showed expression only in normal HOSE cells lines. Furthermore, furin was predominantly expressed in primary tumors from patients who survived for less than five years. The other PCs are either expressed in the group of survivors (PC-7 and PACE4) or expressed in low amounts (PC-5). CONCLUSIONS: Our studies point to a clear relationship between furin and ovarian cancer. In addition, these results show that furin exhibits the closest association with ovarian cancer among the ubiquitously expressed PCs, arguing against the redundancy of these proteases. In summary, furin may constitute a marker for ovarian tumor progression and could contribute to predict the outcome of this disease.

Inhibition of proprotein convertases: approaches to block squamous carcinoma development and progression.

Most proprotein convertase (PC) inhibitors are compounds that act as competitive inhibitors. All of them contain the general cleavage motif RXK/RR that binds to the PC's active site impairing further interactions with their physiological substrates. The first inhibitors synthesized were the acyl-peptidyl-chloromethyl ketones that bind to the PC's active site through its peptidyl group and are able to transverse the plasma membrane due to the acyl moiety. For instance, one of the members of this family that exhibits reduced toxicity and has been widely used as an effective general PCs inhbitor is the derivative decanoyl-RVKR-chloromethylketone (CMK). Another approach to PC inhibition is based on proteins that contain either a natural or a bioengineered PC cleavage consensus site. In this context, the bioengineered serpin, alpha-1-antitrypsin Portland (alpha 1-PDX or PDX), proved to be a potent inhibitor of furin, the most studied of the cancer-related PCs. Both PDX and CMK were able to inhibit invasiveness of squamous cell carcinoma cell lines by blocking activation of cancer-associated PC substrates such as MT-MMPs, IGF-1R, and VEGF-C. A similar effect was produced by inhibiting PC-mediated processing using furin prosegment. PDX and CMK have also been assayed in vivo using skin carcinogenesis models. Newer promising small molecules and RNA interference approaches are also being developed to inhibit PCs.

Carcinogen-induced animal models of head and neck squamous cell carcinoma.

This unit provides two models of oral carcinogenesis (hamster cheek pouch and rat tongue), as well as more simple procedures to induce squamous cell carcinomas (SCCs) in mouse skin. The most significant usage of these models has been their application in studying the molecular pathology of the genesis and multi-step progression of tobacco-associated SCC. Nevertheless, these protocols have been used frequently as in vivo bioassays to demonstrate the chemopreventive and, to a lesser extent, the chemotherapeutic effects of numerous compounds. Several examples, using four different carcinogenesis protocols, are presented with detailed steps on how to elicit squamous lesions and when and how to apply test compounds that could have either cancer chemopreventive or chemotherapeutic effects.

Proprotein convertases: "master switches" in the regulation of tumor growth and progression.

Proprotein convertases (PCs) are a group of Ca2+-dependent serine proteases that have homology to the endoproteases subtilisin (bacteria) and kexin (yeast). This group is comprised of less than a dozen members, known as furin/PACE, PC1/PC3, PC2, PC4, PACE4, PC5/PC6, PC7/PC8/LPC, SKI/S1P, and NARC-1/PCSK9. Four PCs (Furin, PACE4, PC5, and PC7) have been localized to several different tissues and epithelial or nervous system tumors. PCs activate their cognate substrates by limited proteolysis at the consensus sequence RXR/KR downward arrow. Many PC substrates are well known cancer-associated proteins such as growth factors, growth factor receptors, integrins, and matrix metalloproteases (MMPs). For example, IGF-1 and its receptor, TGF-beta, VEGF-C, and MT-MMPs have direct roles in tumor progression and metastasis. Furin, a well-studied member of the PC family, has been associated with enhanced invasion and proliferation in head and neck, breast, and lung cancer. Conversely, inhibition of PC activity by PDX or several PC pro-segments, resulted in reduced processing of these key cancer-related substrates in human squamous cell carcinomas (SCC), colon adenocarcinoma, and astrocytoma cell lines. In parallel to these changes in cell proliferation and invasiveness as well as metastatic ability were markedly impaired. By controlling the maturation/activation of key cancer-associated proteins, PCs act as "master switches" at different levels during tumor development and progression. The manifold effects of PCs, influencing tumor cell proliferation, motility, adhesiveness, and invasiveness, should be exploited by further developing competitive/inhibitory therapeutic strategies that would be able to neutralize simultaneously the most salient cancer cell properties.

PACE4 expression in mouse basal keratinocytes results in basement membrane disruption and acceleration of tumor progression.

Collagen type IV degradation results in disruption and breakdown of the normal basement membrane architecture, a key process in the initiation of tumor microinvasion into the connective tissue. PACE4, a proprotein convertase, activates membrane type matrix metalloproteinases (MT-MMPs) that in turn process collagenase type IV. Because PACE4 is overexpressed in skin carcinomas and in vitro overexpression of PACE4 resulted in enhanced invasiveness, we investigated whether or not in vivo PACE4 expression leads to the acquisition of invasiveness and increased tumorigenesis. Two transgenic mouse lines were designed by targeting PACE4 to the epidermal basal keratinocytes. Transgenic keratinocytes showed increased processing of MT1-MMP and MT2-MMP resulting in collagenase IV activation and collagen type IV degradation. Higher collagenolytic activity partially disrupted normal basement membrane architecture favoring epithelial endophytic growth into the dermis and accelerating invasion and metastasis after chemical carcinogenesis. PACE4 overexpression resulted in enhanced susceptibility to carcinogenesis and tumor progression pointing to a new target for blocking tumor cell invasiveness.

Stroma-derived three-dimensional matrices are necessary and sufficient to promote desmoplastic differentiation of normal fibroblasts.

Stromagenesis is a host reaction of connective tissue that, when induced in cancer, produces a progressive and permissive mesenchymal microenvironment, thereby supporting tumor progression. The stromal microenvironment is complex and comprises several cell types, including fibroblasts, the primary producers of the noncellular scaffolds known as extracellular matrices. The events that support tumor progression during stromagenesis are for the most part unknown due to the lack of suitable, physiologically relevant, experimental model systems. In this report, we introduce a novel in vivo-like three-dimensional system derived from tumor-associated fibroblasts at diverse stages of tumor development that mimic the stromagenic features of fibroblasts and their matrices observed in vivo. Harvested primary stromal fibroblasts, obtained from different stages of tumor development, did not retain in vivo stromagenic characteristics when cultured on traditional two-dimensional substrates. However, they were capable of effectively maintaining the tumor-associated stromal characteristics within three-dimensional cultures. In this study, we demonstrate that in vivo-like three-dimensional matrices appear to have the necessary topographical and molecular information sufficient to induce desmoplastic stroma differentiation of normal fibroblasts.