Recruitment of CD34(+) fibroblasts in tumor-associated reactive stroma: the reactive microvasculature hypothesis.

Reactive stroma co-evolves with cancer, exhibiting tumor-promoting properties. It is also evident at sites of wound repair and fibrosis, playing a key role in tissue homeostasis. The specific cell types of origin and the spatial/temporal patterns of reactive stroma initiation are poorly understood. In this study, we evaluated human tumor tissue arrays by using multiple labeled, quantitative, spectral deconvolution microscopy. We report here a novel CD34/vimentin dual-positive reactive fibroblast that is observed in the cancer microenvironment of human breast, colon, lung, pancreas, thyroid, prostate, and astrocytoma. Recruitment of these cells occurred in xenograft tumors and Matrigel plugs in vivo and was also observed in stromal nodules associated with human benign prostatic hyperplasia. Because spatial and temporal data suggested the microvasculature as a common site of origin for these cells, we analyzed microvasculature fragments in organ culture. Interestingly, fibroblasts with identical phenotypic properties and markers expanded radially from microvasculature explants. We propose the concept of reactive microvasculature for the evolution of reactive stroma at sites of epithelial disruption common in both benign and malignant disorders. Data suggest that the reactive stroma response is conserved among tissues, in normal repair, and in different human cancers. A more clear understanding of the nature and origin of reactive stroma is needed to identify novel therapeutic targets in cancer and fibrosis.

Compression garments as countermeasures to orthostatic intolerance.

INTRODUCTION: All astronauts experience some degree of orthostatic intolerance following spaceflight, ranging from tachycardia to orthostatic hypotension and syncope. The purpose of this study was to evaluate the ability of two compression garments, the National Aeronautics and Space Administration's inflatable antigravity suit (AGS) and the Russian Federal Space Agency's non-inflatable compression garment (Kentavr), to prevent hypovolemia-related orthostatic intolerance. METHODS: To mimic the plasma volume loss experienced by astronauts during spaceflight 19 healthy subjects received an intravenous dose of a diuretic, furosemide (0.5 mg x kg(-1)), and then consumed a low-salt diet for 36 h. Thereafter, subjects participated in a 15-min 80 degrees head-up tilt test wearing either the AGS (N = 9) or Kentavr (N = 10). Compression garments were used in the fashion recommended by the respective agencies, delivering approximately 78 mmHg and approximately 30 mmHg of compression in the AGS and Kentavr, respectively. Incidence of presyncope and hemodynamic responses during upright tilt were compared to a separate group of hypovolemic control subjects (N = 16). RESULTS: Subjects wearing the AGS or Kentavr completed the full 15 min of upright tilt without incidence of orthostatic hypotension or presyncope. In contrast, only 9 control subjects (56%) were able to complete the tilt test. In addition, both types of compression garments maintained systolic blood pressure and significantly reduced tilt-induced tachycardia and reductions in stroke volume. CONCLUSIONS: Although both garments successfully countered hypovolemia-induced orthostatic intolerance, the Kentavr provided protection by using lower levels of compression. Determining the optimal compression level required for protection of intolerance may improve crewmember comfort and decrease restrictions on physical activities after spaceflight.

Elevated epithelial expression of interleukin-8 correlates with myofibroblast reactive stroma in benign prostatic hyperplasia.

OBJECTIVES: Numerous inflammatory diseases display elevated interleukin (IL)-8, and most are associated with a reactive stroma. IL-8 expression is also elevated in benign prostatic hyperplasia (BPH), yet little is known about reactive stroma in BPH. Whether a reactive stroma response exists in BPH, whether this correlates with elevated IL-8, and whether IL-8 can induce a reactive stroma phenotype have not been determined. This study was designed to specifically address these issues. METHODS: Normal prostate transition zone tissue and BPH specimens, as identified by the Baylor College of Medicine pathology department, were examined by quantitative immunohistochemistry to correlate IL-8, smooth muscle alpha-actin, vimentin, calponin, and tenascin-C. In addition, human prostate stromal cell cultures were used to evaluate the effect of IL-8 on the expression of reactive stroma biomarkers. RESULTS: BPH nodules exhibited elevated epithelial IL-8 immunoreactivity, and this correlated with elevated smooth muscle alpha-actin, reduced calponin, and altered deposition of tenascin-C, relative to the normal prostate transition zone tissue (P <0.05). Multiple vimentin-positive prostate stromal fibroblast cultures were induced by IL-8 to also co-express smooth muscle alpha-actin and tenascin-C, typical of a reactive stroma myofibroblast phenotype. CONCLUSIONS: These data show that BPH reactive stroma is fundamentally different from normal prostate fibromuscular stroma and is typified by the emergence of a reactive stroma myofibroblast phenotype. This reactive stroma pattern correlated spatially with IL-8 elevation in adjacent epithelium. Additionally, IL-8 induced expression of myofibroblast markers in human prostate fibroblasts in vitro. These results suggest that IL-8 acts as a regulator of BPH reactive stroma and is therefore a potential therapeutic target.

Stromal expression of connective tissue growth factor promotes angiogenesis and prostate cancer tumorigenesis.

Our previous studies have defined reactive stroma in human prostate cancer and have developed the differential reactive stroma (DRS) xenograft model to evaluate mechanisms of how reactive stroma promotes carcinoma tumorigenesis. Analysis of several normal human prostate stromal cell lines in the DRS model showed that some rapidly promoted LNCaP prostate carcinoma cell tumorigenesis and others had no effect. These differential effects were due, in part, to elevated angiogenesis and were transforming growth factor (TGF)-beta1 mediated. The present study was conducted to identify and evaluate candidate genes expressed in prostate stromal cells responsible for this differential tumor-promoting activity. Differential cDNA microarray analyses showed that connective tissue growth factor (CTGF) was expressed at low levels in nontumor-promoting prostate stromal cells and was constitutively expressed in tumor-promoting prostate stromal cells. TGF-beta1 stimulated CTGF message expression in nontumor-promoting prostate stromal cells. To evaluate the role of stromal-expressed CTGF in tumor progression, either engineered mouse prostate stromal fibroblasts expressing retroviral-introduced CTGF or 3T3 fibroblasts engineered with mifepristone-regulated CTGF were combined with LNCaP human prostate cancer cells in the DRS xenograft tumor model under different extracellular matrix conditions. Expression of CTGF in tumor-reactive stroma induced significant increases in microvessel density and xenograft tumor growth under several conditions tested. These data suggest that CTGF is a downstream mediator of TGF-beta1 action in cancer-associated reactive stroma and is likely to be one of the key regulators of angiogenesis in the tumor-reactive stromal microenvironment.

Regulation of rat prostate stromal cell myodifferentiation by androgen and TGF-beta1.

BACKGROUND: Myodifferentiation of stromal cells is a key step in prostate development and is a hallmark of reactive stroma in prostate cancer. Little is known about regulatory mechanisms, however, prostate stromal cells are androgen-regulated and TGF-beta1 is a known stimulator of stromal myodifferentiation. The PS-1 rat prostate stromal cell line expresses androgen receptor, and exhibits androgen-regulated gene expression and proliferation. TGF-beta1 inhibits androgen action in PS-1 cells through translocation of androgen receptor from the nucleus to the cytoplasm. The present study was conducted to determine whether myodifferentiation of PS-1 cells is regulated by androgen and TGF-beta1, and how myodifferentiation affects androgen receptor localization and cell proliferation. METHODS: PS-1 cell cultures were exposed to physiological concentrations of dihydrotestosterone, TGF-beta1, and combinations of both in chemically defined medium. Immunocytochemistry and Western blotting for smooth muscle alpha-actin filament formation, smooth muscle alpha-actin protein levels, calponin expression, PCNA index, and androgen receptor localization were performed. RESULTS: Dihydrotestosterone (DHT) and TGF-beta1 each separately promoted PS-1 myodifferentiation. A combination did not affect the rate of differentiation, however, the level of alpha-actin protein was elevated and PCNA was decreased in co-stimulated conditions. TGF-beta1 induction resulted in a transient translocation of androgen receptor from the nucleus to the cytoplasm during differentiation followed by a resumed nuclear localization in myodifferentiated cells. CONCLUSIONS: These data indicate that a complex cross-talk mechanism exists between androgen and TGF-beta1 signaling in prostate stromal cells that affects cell proliferation and myodifferentiation. These findings also suggest that androgen and TGF-beta1 interactions may cooperatively regulate myodifferentiation of stromal cells in the stromal response in prostate cancer.

Reactive stroma as a predictor of biochemical-free recurrence in prostate cancer.

Extensive scientific literature data point to reciprocal interactions between prostate stromal cells and prostate cancer cells that likely regulate tumor progression. To investigate whether these intratumoral-reactive stromal cells in human prostate cancer are predictive of survival, tumor stroma volume and specific stroma markers were quantitated by using tissue microarrays (index tumors of 847 patients), and the results were analyzed relative to the recurrence-free survival data set for these patients. Tumor tissue was evaluated with Masson's trichrome stains and by immunohistochemistry with antibody probes to smooth muscle alpha-actin, desmin, vimentin, pro-collagen type I, and calponin. The relative volume of intratumor stroma (5% stroma, grade 0; 5-15%, grade 1; 15-50%, grade 2; >50%, grade 3) and the expression index of stromal marker (staining intensity grade x percentage of positive cells per field) were quantitated and analyzed. Interpretable data were obtained from 545 patients. Statistical analysis of the survival data set showed that the volume of reactive stroma in the tumor was a significant predictor of disease-free survival. Stroma volume was most optimal as an independent predictor in tumors containing stroma, defined as Gleason 7 and lower grades. Of interest, tumors with either little to no stroma or tumors with abundant stroma each showed reduced recurrence-free survival. For specific stromal markers, reduced desmin and smooth muscle alpha-actin were hallmarks of cancer-associated reactive stroma relative to normal fibromuscular stroma. Quantitative analysis of desmin and smooth muscle alpha-actin expression showed both to be significant and independent predictors of recurrence-free survival. This is the first study to demonstrate that nonepithelial-reactive stroma elements in prostate cancer tumors can be used as prognostic indicators. These data also add to the concept that tumors are not purely epithelial and the tumor-reactive stroma must be considered an important biological component of the cancer.

Promotion of angiogenesis by ps20 in the differential reactive stroma prostate cancer xenograft model.

Human prostate cancer is associated with a reactive stroma typified by an increase in the proportion of myofibroblast type cells and elevated synthesis of extracellular matrix proteins. Increased vascular density has been identified in the reactive stroma compartment adjacent to both precancerous and cancerous prostate lesions. The differential reactive stroma (DRS) prostate cancer xenograft model has been developed to investigate the role of reactive stroma in prostate cancer progression. Using this model, we have shown that human prostate stromal cells promote angiogenesis and growth of LNCaP human prostate carcinoma cell tumors, and that these increases are transforming growth factor (TGF) beta1 regulated. Our laboratory isolated and identified previously the ps20 protein (WFDC1 gene) as a prostate stromal cell secreted protein. The ps20 protein contains a whey acidic protein-type four-disulfide core domain, which is a functional motif characterized by serine protease inhibition activity in a number of whey acidic protein domain-containing proteins. In the present study, we show ps20 expression by normal human prostate stromal smooth muscle cells and vascular smooth muscle cells indicating a possible role of ps20 in vessel wall biology. Using in vitro assays, we show that ps20 promotes endothelial cell motility but has no effect on endothelial cell proliferation. To address the potential effects of ps20 in a tumor microenvironment, we used the DRS model to evaluate both angiogenesis and tumorigenesis of tumors generated under either ps20 or control conditions. DRS tumors generated with LNCaP and human prostate stromal cells in the presence of ps20 showed a 67% increase in microvessel density compared with control tumors. Elevated DRS tumor growth in the ps20-treated tumors was reflected by a 29% increase in wet weight and a 58% increase in volume compared with controls. Similar tumors composed of GeneSwitch-3T3 cells engineered to express ps20-V5-His under mifepristone regulation showed a 129% increase in microvessel density after induction of ps20-V5-His. GeneSwitch-3T3 cells expressing ps20-V5-His were localized to vessel walls in a mural cell (pericyte) position indicating a possible direct stabilizing interaction with endothelial cells. In addition, we show that ps20 mRNA synthesis is induced by TGF-beta1, a known regulator of endothelial cell-pericyte interactions and of stromal cell-induced angiogenesis in DRS tumors. These findings suggest that ps20 may be a TGF-beta1-induced regulator of angiogenesis that functions by either promoting endothelial cell migration or by contributing to pericyte stabilization of newly formed vascular structures.

Inhibition of transforming growth factor-beta activity decreases angiogenesis in a human prostate cancer-reactive stroma xenograft model.

We have shown previously that reactive stroma promotes angiogenesis and growth of LNCaP human prostate tumors in the differential reactive stroma xenograft model. Regulators of reactive stroma are not known, but transforming growth factor (TGF)-beta1 is a likely candidate. Three-way differential reactive stroma tumors were generated in the presence of TGF-beta1 latency-associated peptide (LAP) or TGF-beta1 neutralizing antibody. Tumors treated with either of those TGF-beta inhibitors exhibited a reduction in blood vessels, and blood lakes were observed in some areas. The microvessel density of LAP-treated tumors was decreased 3.5-fold relative to control tumors. Moreover, the average wet-weight of LAP-treated tumors was reduced 46% compared with control tumors. The results of this study suggest that TGF-beta regulates reactive stroma and its ability to promote angiogenesis and tumor growth.

Reactive stroma in human prostate cancer: induction of myofibroblast phenotype and extracellular matrix remodeling.

PURPOSE: Generation of a reactive stroma environment occurs in many human cancers and is likely to promote tumorigenesis. However, reactive stroma in human prostate cancer has not been defined. We examined stromal cell phenotype and expression of extracellular matrix components in an effort to define the reactive stroma environment and to determine its ontogeny during prostate cancer progression. EXPERIMENTAL DESIGN: Normal prostate, prostatic intraepithelial neoplasia (PIN), and prostate cancer were examined by immunohistochemistry. Tissue samples included radical prostatectomy specimens, frozen biopsy specimens, and a prostate cancer tissue microarray. A human prostate stromal cell line was used to determine whether transforming growth factor beta1 (TGF-beta1) regulates reactive stroma. RESULTS: Compared with normal prostate tissue, reactive stroma in Gleason 3 prostate cancer showed increased vimentin staining and decreased calponin staining (P < 0.001). Double-label immunohistochemistry revealed that reactive stromal cells were vimentin and smooth muscle alpha-actin positive, indicating the myofibroblast phenotype. In addition, reactive stroma cells exhibited elevated collagen I synthesis and expression of tenascin and fibroblast activation protein. Increased vimentin expression and collagen I synthesis were first observed in activated periacinar fibroblasts adjacent to PIN. Similar to previous observations in prostate cancer, TGF-beta1-staining intensity was elevated in PIN. In vitro, TGF-beta1 stimulated human prostatic fibroblasts to switch to the myofibroblast phenotype and to express tenascin. CONCLUSIONS: The stromal microenvironment in human prostate cancer is altered compared with normal stroma and exhibits features of a wound repair stroma. Reactive stroma is composed of myofibroblasts and fibroblasts stimulated to express extracellular matrix components. Reactive stroma appears to be initiated during PIN and evolve with cancer progression to effectively displace the normal fibromuscular stroma. These studies and others suggest that TGF-beta1 is a candidate regulator of reactive stroma during prostate cancer progression.

Stromal cells promote angiogenesis and growth of human prostate tumors in a differential reactive stroma (DRS) xenograft model.

Reactive stroma has been reported in many cancers, including breast, colon,and prostate. Although changes in stromal cell phenotype and extracellular matrix have been reported, specific mechanisms of how reactive stroma affects tumor progression are not understood. To address the role of stromal cells in differential regulation of tumor incidence, growth rate, and angiogenesis, LNCaP xenograft tumors were constructed in nude mice with five different human prostate stromal cell lines as well as GeneSwitch-3T3 cells engineered to express lacZ under mifepristone regulation. Alone, LNCaP prostate carcinoma cells were essentially nontumorigenic, whereas combinations of LNCaP cells with three different human prostate stromal cell lines (L/S tumors) resulted in a tumor incidence (50-63%) similar to that of control LNCaP plus Matrigel (L/M) tumors over a 9-week period. In contrast, LNCaP combinations with two other human prostate stromal cell lines were nontumorigenic, illustrating that stromal cell effects are differential. L/S tumors exhibited well-developed blood vessels at 2 weeks, whereas control L/M tumors were avascular at 2 weeks and exhibited blood lakes in lieu of extensive vessels at later time points. Xenografts constructed under three-way conditions (LNCaP, Matrigel, and stromal cells; L/M/S tumors) exhibited a 100% tumor incidence and showed rapid blood vessel formation as early as day 7 with mature vessels formed by day 10. L/M/S tumors exhibited a 10.3-fold increase in microvessel density, and the corresponding hemoglobin:tumor weight ratio was increased 2-fold relative to L/M control tumors at day 10. L/M/S tumor wet weight and volume increased by 1.6- and 2.4-fold, respectively, by day 21, compared with control L/M tumors. L/M/S tumors made with LNCaP cells plus GeneSwitch-3T3-pGene/lacZ stromal cells showed similar results. Mifepristone-regulated gene expression was observed in stromal cells immediately adjacent to clusters of carcinoma cells and in vessel walls in a mural cell (pericyte) position. This study shows that regulation of angiogenesis is one mechanism through which stromal cells affect LNCaP tumor incidence and growth rate. This regulation may be mediated through direct recruitment and interactions of stromal cells with endothelial cells. Furthermore, this study describes for the first time a model system with regulated transgene expression in the stromal compartment of an experimental carcinoma. These findings point to the stromal compartment as a potential source of new prognostic markers and therapeutic targets and show the utility of the carcinoma-stromal xenograft model system in dissecting specific mechanisms of reactive stroma.