兔骨髓间充质干细胞向尿路上皮分化后构建膀胱组织工程化移植物

背景：尿路上皮细胞是泌尿系组织工程领域重要的种子细胞,但是难以体外大量扩增;有研究表明骨髓间充质干细胞可以向尿路上皮细胞分化,但关于分化后细胞在植入动物体内后上皮生成情况,以及分化后细胞在组织工程领域的具体应用研究尚不多。 目的：分离、扩增兔骨髓间充质干细胞,诱导骨髓间充质干细胞向尿路上皮细胞分化并与兔膀胱脱细胞基质构建组织工程化移植物,了解分化后细胞作为种子细胞的效果。 方法：12只8周龄雄性新西兰大白兔胫骨穿刺,抽取骨髓,密度梯度离心法分离骨髓间充质干细胞,第4或5代细胞以条件培养基培养2周,进行分化后细胞的鉴定。随后将分化后细胞种植在膀胱脱细胞基质上构建组织工程化移植物,进行膀胱修补;另12只动物作为对照组以尿路上皮细胞与膀胱脱细胞基质构建复合物行膀胱修补。 结果与结论：骨髓间充质干细胞培养成功并在体外扩增,由条件培养基培养诱导分化后,PCR检测提示分化后细胞干细胞标志物CD44表达降低,而上皮细胞标志物UP1a表达升高,行免疫荧光检测发现分化后细胞能表达尿路上皮特异性标志物UP1a,而骨髓间充质干细胞无表达。分化后细胞构建的组织工程化移植物在膀胱修补2周后可形成稳定连续的上皮覆盖,上皮层厚度与尿路上皮细胞构建的组织工程化移植物类似。提示骨髓间充质干细胞向尿路上皮诱导分化后可作为泌尿系组织工程的种子细胞,可能是尿路上皮细胞之外的又一新选择。     

Rat urinary bladder denuded of urothelium. An in vivo model for the epithelial-stromal interactions in carcinogenesis.

To investigate epithelial-stromal interactions in bladder carcinogenesis, the authors developed an experimental model using a heterotopically transplanted rat urinary bladder (HTB). A rat urinary bladder which was completely denuded of epithelial cells ex vivo with hypotonic shock and a nonionic detergent was transplanted into a syngeneic recipient. No reepithelialization occurred during the 8-week posttransplant period. The basal lamina remained intact throughout this period and showed linear immunofluorescence with antibodies against laminin, Type IV collagen, and heparan sulfate-proteoglycan. When 1 X 10(5) to 5 X 10(5) dispersed normal urothelial cells were delivered into the HTB through an attached reservoir 4 days after transplantation, complete resurfacing by inoculated cells occurred within a few days. A transient hyperplasia was followed by a normal 2-3-cell-thick urothelial organization in 4 weeks. Cultured bladder carcinoma cells also resurfaced the denuded bladder basal lamina; the progressive growth of this neoplastic epithelium resulted in carcinoma in situ as well as foci of invasive carcinoma within 4 weeks following inoculation. This in vivo system has an advantage over other in vivo and in vitro models in that complete removal of epithelial cells can be achieved easily and completely; the course after reepithelialization can be modified by subsequent treatment, progression of neoplastic development can be closely observed by a change in the color of the aspirate, its cytology, and biochemical analysis of secretions; and an adequate amount of tissue can be available for subsequent examinations. The model is potentially useful not only for studying epithelial-stromal interactions during carcinogenesis, but also for examining the mechanisms of tumor invasion and metastasis.     

胶原海绵支架体外构建组织工程膀胱黏膜***◆

背景：组织工程膀胱黏膜层的构建在组织工程膀胱修复中占有重要的地位,但目前并没有最合理的构建方法。 目的：探讨胶原海绵支架复合猪膀胱尿路上皮细胞体外构建膀胱黏膜结构的可行性。 方法：刮取猪膀胱黏膜层后用酶消化方式进行猪膀胱尿路上皮细胞原代培养,并进行尿路上皮细胞标志物免疫荧光和RT-PCR鉴定。制备疏松多孔的胶原海绵支架材料,将第3代尿路上皮细胞接种在胶原海绵支架上,体外培养4~8 d后观察尿路上皮细胞和胶原海绵材料的复合情况。 结果与结论：原代培养的猪膀胱尿路上皮细胞呈“多角形”、“铺路石”样,以克隆团形式生长。免疫荧光鉴定AE1/AE3阳性,RT-RCR检测uroplakin-ⅠA、uroplakin-Ⅱ阳性。胶原海绵复合尿路上皮细胞体外培养4~8 d后,细胞在胶原海绵支架上生长良好,覆盖胶原材料的表面并长入材料内部,保持了尿路上皮细胞的特性。体外培养6 d时,尿路上皮细胞与胶原支架复合效果最好,同时细胞数量也最多。结果初步表明了胶原海绵复合膀胱尿路上皮细胞可以构建组织工程膀胱黏膜,且体外培养6 d为最佳时间点。     

Towards defining roles and relationships for tenascin-C and TGFbeta-1 in the normal and neoplastic urinary bladder

Tenascin-C (TN-C) is an extracellular matrix glycoprotein expressed along epithelial/stromal boundaries during tissue remodelling events, such as those that occur during morphogenesis, wound healing, and tumour invasion. Using clinical specimens and a range of in vitro models that simulate homeostasis, wound healing, and malignant progression, this study sought to establish the patterns of TN-C expression in normal and neoplastic bladder and to determine the role of exogenous transforming growth factor beta-1 (TGFbeta-1), interleukin-4 (IL-4), basic fibroblast growth factor (bFGF), tumour necrosis factor alpha (TNFalpha), and interferon gamma (IFNgamma) in the induction of TN-C expression by bladder uro-epithelial cells. The findings indicate that normal urothelial cells may express TN-C, with both TGFbeta-1 and IL-4 able to induce expression. TN-C was not expressed in neoplastic urothelium, although both TN-C and TGFbeta-1 may be involved in tissue remodelling during papillary tumour formation and invasion. Furthermore, the urothelium of high-grade papillary tumours and carcinoma in situ specimens exhibited little TGFbeta-1 immunoreactivity, compared with the urothelium of low-grade tumours and normal specimens, suggesting an association between TGFbeta-1 expression and urothelial differentiation. A tumour invasion model, in which established bladder cancer cell lines were seeded onto a normal bladder stroma, corroborated the evidence from the clinical specimens and demonstrated that TN-C was strongly expressed around foci of stromal invasion. Thus, TN-C immunoreactivity may provide an additional tool in the assessment of early stromal invasion in bladder cancer.     

Constructs of electrospun PLGA,compressed collagen and minced urothelium for minimally manipulated autologous bladder tissue expansion

Bladder regeneration based on minced bladder mucosa in vivo expansion is an alternative to in vitro culturing of urothelial cells. Here, we present the design of a hybrid, electrospun poly(lactic-co-glycolide) (PLGA) – plastically compressed (PC) collagen scaffold that could allow in vivo bladder mucosa expansion. Optimisation of electrospinning was performed in order to obtain increased pore sizes and porosity to consolidate the construct and to support neovascularisation and tissue ingrowth. Tensile tests showed an increase in average tensile strength from 0.6 MPa for PC collagen to 3.57 MPa for the hybrid construct. The optimised PLGA support scaffold was placed between two collagen gels, and the minced tissue was distributed either on top or both on top and inside the construct prior to PC; this was then cultured for up to four weeks. Morphology, histology and SEM demonstrated that the construct maintained its integrity throughout cell culture. Cells from minced tissue migrated, expanded and re-organised to a confluent cell layer on the top of the construct after two weeks and formed a multilayered urothelium after four weeks. Cell morphology and phenotype was typical for urothelial mucosa during tissue culture.     

Loss of co-localization of alpha 6 beta 4 integrin and collagen VII in bladder cancer.

In normal epithelial cells, the alpha 6 beta 4 integrin co-localizes with the hemidesmosomal anchoring complex on the basolateral surface of basal cells. We studied the co-expression of alpha 6 beta 4 integrin with collagen VII, a component of the hemidesmosomal anchoring complex, in normal bladder tissues and in bladder cancers. In normal bladder, the alpha 6 beta 4 integrin co-localized with collagen VII at the junction of the basolateral surface of the basal urothelial cells and the lamina propria. In five of six noninvasive bladder cancers, the localization of collagen VII remained unchanged, found at the junction of the basal cells and the papillary connective tissue. However, in these tumors the alpha 6 beta 4 expression was not polarized and was expressed on suprabasal as well as basal cells. In invasive bladder cancers, the majority (25 of 30) showed either loss of alpha 6 beta 4 and/or collagen VII expression or showed a lack of co-localization of alpha 6 beta 4 and collagen VII. Our results show derangement of the co-localization of these two components of the hemidesmosomal anchoring complex is a consistent event in bladder cancer. Furthermore, the degree of derangement increases in invasive cancers. Loss of co-expression and co-localization of alpha 6 beta 4 and collagen VII may predispose cancer cells to local invasion and may facilitate metastasis as well.     

High-density collagen gel tubes as a matrix for primary human bladder smooth muscle cells

Tissue-engineered grafts for the urinary tract are being investigated for the potential treatment of several urologic diseases. These grafts, predominantly tubular-shaped, usually require in vitro culture prior to implantation to allow cell engraftment on initially cell-free scaffolds. We have developed a method to produce tubular-shaped collagen scaffolds based on plastic compression. Our approach produces a ready cell-seeded graft that does not need further in vitro culture prior to implantation. The tubular collagen scaffolds were in particular investigated for their structural, mechanical and biological properties. The resulting construct showed an especially high collagen density, and was characterized by favorable mechanical properties assessed by axial extension and radial dilation. Young modulus in particular was greater than non-compressed collagen tubes. Seeding densities affected proliferation rate of primary human bladder smooth muscle cells. An optimal seeding density of 10(6) cells per construct resulted in a 25-fold increase in Alamar blue-based fluorescence after 2?wk in culture. These high-density collagen gel tubes, ready seeded with smooth muscle cells could be further seeded with urothelial cells, drastically shortening the production time of graft for urinary tract regeneration.     

So-called "superficial" bladder tumors. Which classification in 2003? Part 2: Flat urothelial lesions

Flat urothelial lesions of the urinary bladder have been recently discussed by the International Society of Urological Pathology (ISUP) in 1998 and more recently redefined by an international consultation held in Ancona, Italy in 2001. This paper summarizes and illustrates the recent literature about non-papillary lesions of the urinary bladder. Flat urothelial lesions include: epithelial abnormalities (reactive urothelial atypia and flat hyperplasia), preneoplastic lesion (dysplasia) and neoplastic non invasive carcinoma (carcinoma in situ) and a new category of presumed neoplastic lesions and conditions; the latter points out to a notion of tumor biology, which may help to the understanding of urothelial carcinogenesis.     

Uropathogenic E. coli promote a paracellular urothelial barrier defect characterized by altered tight junction integrity, epithelial cell sloughing and cytokine release

The urinary bladder is a common site of bacterial infection with a majority of cases attributed to uropathogenic Escherichia coli. Sequelae of urinary tract infections (UTIs) include the loss of urothelial barrier function and subsequent?clinical morbidity secondary to the permeation of urine potassium, urea and ammonia into the subepithelium.?To date there has been limited research describing the mechanism by which this urothelial permeability?defect develops. The present study models acute uropathogenic E. coli infection in?vitro using intact canine bladder mucosa mounted in Ussing chambers to determine whether infection induces primarily a transcellular or paracellular permeability defect. The Ussing chamber sustains tissue viability while physically separating?submucosal and lumen influences, so this model is ideal for quantitative measurement of transepithelial electrical resistance (TER) to assess alterations of urothelial barrier function. Using this model, changes in both tissue ultrastructure and TER indicated that uropathogenic E. coli infection promotes a paracellular permeability defect associated with the failure of umbrella cell tight junction formation and umbrella cell sloughing. In addition, bacterial interaction with the urothelium promoted secretion of cytokines from the urinary?bladder with bioactivity capable of modulating epithelial barrier function including tumour necrosis factor-α, interleukin (IL)-6 and IL-15. IL-15 secretion by the infected bladder mucosa is a novel finding and, because IL-15 plays key roles in reconstitution of tight junction function in damaged intestine, this study points to a potential role for IL-15 in UTI-induced urothelial injury.     

Three-dimensional cardiac tissue engineering using a thermoresponsive artificial extracellular matrix

The purpose of this study was to try to reconstitute three-dimensional cardiac tissue using a thermoresponsive artificial extracellular matrix, poly (N-isopropylacrylamide)-grafted gelatin (PNIPAM-gelatin), as the scaffold. PNIPAM-gelatin solution gels almost immediately when heated above 34 degrees C. We thought this property could become advantageous as scaffolding for reconstituting three-dimensional tissue. Because PNIPAM-gelatin solution gels so quickly, all seeded cells in PNIPAM-gelatin solution would become entrapped and uniformly distributed toward three dimensions. Thus it would be possible to reconstitute three-dimensional tissue by a very simple method of mixing cells and PNIPAM-gelatin solution. Fetal rat cardiac cells were mixed with PNIPAM-gelatin solution, incubated at 37 degrees C to allow the mixture to gel, and cultured in vitro. To define suitable culture conditions the following parameters were tested: (1) PNIPAM-gelatin concentration, 0.04 approximately 0.125 mg/ml; (2) cell seeding density, 1 approximately 50 x 10(6) cells/ml; and (3) addition or not of hyaluronic acid. With a PNIPAM-gelatin concentration of 0.05 mg/ml, a cell seeding density of 50 x 10(6) cells/ml, and the addition of hyaluronic acid, tissue was reconstituted and it contracted synchronously. After hematoxylin and eosin staining, the cells reconstituted three-dimensional tissue, and the tissue cross-section was approximately 60 microm thick.     

Altered growth patterns in vitro of human papillary transitional carcinoma cells.

In vitro growth patterns and morphologic characteristics of five low-grade human papillary transitional cell carcinomas (TCCs) were compared and contrasted with those of normal human urothelial cells in culture. Biopsies of TCC were performed by transurethral resection. Specimens of normal human ureters were obtained surgically. Singly dispersed TCC cells grew in 0.3% agarose semisolid medium with a cloning efficiency ranging from 0.02% to 0.71%. Singly dispersed normal ureteral urothelial cells under the same conditions did not form colonies in 0.3% agarose. Neither singly dispersed TCC nor normal urothelial cells formed colonies when plated on collagen-gel substrates. In primary explant culture, normal human urothelial cells grew rapidly, to form tightly adherent flat sheets of apparently nonmotile cells. Autoradiographic labeling with 3H-thymidine of growing cultures of normal urothelial cells showed cell division primarily in the zones of growth near the explant. Outgrowth of TCC from primary explants was loosely adherent. One TCC explant culture gave rise to a continuous suspension culture. Numerous multilayered cellular formations of fronds, nodules, and "walls" were seen around the periphery of TCC explant colonies. Autoradiography showed that these multilayered areas of TCC growth contained actively dividing cells. The altered ability of papillary TCC to form superficial multilayered formations in vitro distinguishes them from normal human urothelium and reflects the morphologic characteristic of this tumor type in vivo.     

How to optimize seeding and culturing of human osteoblast-like cells on various biomaterials

The optimization of seeding and culturing of human osteoblast-like cells on three collagen-based biomaterials (bovine, equine and calf collagen membrane) was studied by cell proliferation and cell colonization (scanning electron microscopy) analysis. Osteoblasts of five patients were seeded onto the three biomaterials and two different parameters were varied: the time intervals between initial seeding and adding culture medium (2 h 6 h. 12 h, 24 h) and the seeding concentration (1 x 10(5), 1 x 10(6), 2 x 10(6)cells/ml) of cells onto biomaterials. The results of the study demonstrated that the time interval between seeding osteoblasts and adding culture medium as well as the seeding concentration effects the cell proliferation and the cell colonization. The best proliferation rate was achieved by adding the culture medium 2 h after initial seeding and with a seeding density of 1 x 10(5) cells/ml. Moreover, all three biomaterials resulted in different proliferation rates. The best proliferation rate resulted with the bovine collagen membrane. In conclusion, the examined parameters are very important for the development of the tissue engineering techniques and in a larger perspective also for reconstructive surgery.     

A collagen-poly(lactic acid-co-ɛ-caprolactone) hybrid scaffold for bladder tissue regeneration

Scaffold materials should favor cell attachment and proliferation, and provide designable 3D structures with appropriate mechanical strength. Collagen matrices have proven to be beneficial scaffolds for tissue regeneration. However, apart from small intestinal submucosa, they offer a limited mechanical strength even if crosslinking can enhance their mechanical properties. A more cell-friendly way to increase material strength is to combine synthetic polymer meshes with plastic compressed collagen gels. This work describes the potential of plastic compressed collagen-poly(lactic acid-co-?-caprolactone) (PLAC) hybrids as scaffolds for bladder tissue regeneration. Human bladder smooth muscle and urothelial cells were cultured on and inside collagen-PLAC hybrids in vitro. Scaffolds were analyzed by electron microscopy, histology, immunohistochemistry, and AlamarBlue assay. Both cell types proliferated in and on the hybrid, forming dense cell layers on top after two weeks. Furthermore, hybrids were implanted subcutaneously in the backs of nude mice. Host cell infiltration, scaffold degradation, and the presence of the seeded bladder cells were analyzed. Hybrids showed a lower inflammatory reaction in vivo than PLAC meshes alone, and first signs of polymer degradation were visible at six months. Collagen-PLAC hybrids have potential for bladder tissue regeneration, as they show efficient cell seeding, proliferation, and good mechanical properties.     

Tissue-engineered heart valve leaflets: an effective method for seeding autologous cells on scaffolds

BACKGROUND: A precondition for the successful formation of tissue-engineered heart valves is the generation of a proper matrix on biodegradable scaffolds over a limited period of time. The aim of this study was to find an effective method of seeding autologous cells on these scaffolds to create a new matrix for heart valves. METHODS: Myofibroblasts and endothelial cells were isolated and cultured from an ovine artery. A synthetic biodegradable scaffold consisting of polyglycolic and polylactic acids was seeded first with the myofibroblasts, then coated with endothelial cells. Three different methods of myofibroblast seeding were compared: I) daily seeding of myofibroblasts (1x10(6)) for ten days and culture for four days; II) seeding of myofibroblasts (1x10(7)) and culture for 14 days with the use of a simple medium; III) seeding of myofibroblasts (1x10(7)) with the use of a medium containing collagen and culture for 14 days. Light and electron microscopic analyses were performed. RESULTS: The group that used the medium containing collagen showed the best results in terms of seeding efficiency. CONCLUSION: Seeding autologous cells with a medium containing collagen onto the scaffold showed the largest cell population and might generate the best matrix on the scaffold.     

Intraepithelial lumina in urothelial bladder neoplasms. A histochemical, immunohistochemical and electron microscopy study

Intraepithelial lumina observed in 12 urothelial bladder neoplasms were studied histochemically, immunohistochemically and ultrastructurally. Both intercellular and intracytoplasmic lumina could be demonstrated showing an alcianophilic margin and containing non-sulphated acid mucins. The presence of secretory component (SC) was identified in neoplastic urothelial cells around or adjacent to intercellular lumina as well as in cells with intracytoplasmic lumina. The cells surrounding intercellular lumina revealed ultrastructurally tight junctions, microvilli and a prominent glycocalyx while cellular remnants were found quite often within the lumen. As similar histochemical, immunohistochemical and ultrastructural characteristics are also expressed in surface umbrella cells of normal urothelium it is suggested that a focal differentiation of neoplastic urothelial cells towards surface umbrella-like cells takes place and that this process is intimately related to the formation of lumina.     

The human urothelium consists of multiple clonal units, each maintained by a stem cell

Little is known about the clonal architecture of human urothelium. It is likely that urothelial stem cells reside within the basal epithelial layer, yet lineage tracing from a single stem cell as a means to show the presence of a urothelial stem cell has never been performed. Here, we identify clonally related cell areas within human bladder mucosa in order to visualize epithelial fields maintained by a single founder/stem cell. Sixteen frozen cystectomy specimens were serially sectioned. Patches of cells deficient for the mitochondrially encoded enzyme cytochrome c oxidase (CCO) were identified using dual-colour enzyme histochemistry. To show that these patches represent clonal proliferations, small CCO-proficient and -deficient areas were individually laser-capture microdissected and the entire mitochondrial genome (mtDNA) in each area was PCR amplified and sequenced to identify mtDNA mutations. Immunohistochemistry was performed for the different cell layers of the urothelium and adjacent mesenchyme. CCO-deficient patches could be observed in normal urothelium of all cystectomy specimens. The two-dimensional length of these negative patches varied from 2-3 cells (about 30 μm) to 4.7 mm. Each cell area within a CCO-deficient patch contained an identical somatic mtDNA mutation, indicating that the patch was a clonal unit. Patches contained all the mature cell differentiation stages present in the urothelium, suggesting the presence of a stem cell. Our results demonstrate that the normal mucosa of human bladder contains stem cell-derived clonal units that actively replenish the urothelium during ageing. The size of the clonal unit attributable to each stem cell was broadly distributed, suggesting replacement of one stem cell clone by another.     

Mesenchymal stem cell-seeded collagen matrices for bone repair: effects of cyclic tensile strain, cell density, and media conditions on matrix contraction in vitro

Type I collagen is the most abundant extracellular matrix protein in bone and contains arginine- glycine-aspartic acid sequences that promote cell adhesion and proliferation. We have previously shown that human mesenchymal stem cells (hMSCs) seeded in three-dimensional (3D) collagen gels upregulate BMP-2 mRNA expression in response to tensile strain, indicative of osteogenesis. Therefore, collagen could be a promising scaffold material for functional bone tissue engineering using hMSCs. However, high contraction of the collagen gels by hMSCs poses a challenge to creating large, tissue-engineered bone constructs. The effects of cyclic tensile strain, medium (with and without dexamethasone), and hMSC seeding density on contraction of collagen matrices have not been investigated. hMSCs were seeded in 3D collagen gels and subjected to cyclic tensile strain of 10% or 12% for 4 h/day at a frequency of 1 Hz in osteogenic-differentiating or complete MSC growth media for up to 14 days. Viability of hMSCs was not affected by strain or media conditions. While initial seeding density affected matrix contraction alone, there was a high interdependence of strain and medium on matrix contraction. These findings suggest a correlation between hMSC proliferation and osteogenic differentiation on collagen matrix contraction that is affected by media, cell-seeding density, and cyclic tensile strain. It is vital to understand the effects of culture conditions on collagen matrix contraction by hMSCs in order to consider hMSC-seeded collagen constructs for functional bone tissue engineering in vitro.     

Tissue-engineered cartilage using serially passaged articular chondrocytes. Chondrocytes in alginate, combined in vivo with a synthetic (E210) or biologic biodegradable carrier (DBM)

In vitro multiplication of isolated autologous chondrocytes is required to obtain an adequate number of cells to generate neo-cartilage, but is known to induce cell-dedifferentiation. The aim of this study was to investigate whether multiplied chondrocytes can be used to generate neo-cartilage in vivo. Adult bovine articular chondrocytes, of various differentiation stages, were suspended in alginate at densities of 10 or 50 million/ml, either directly after isolation (P0) or after multiplication in monolayer for one (P1) or three passages (P3). Alginate with cells was seeded in demineralized bovine bone matrix (DBM) or a fleece of polylactic/polyglycolic acid (E210) and implanted in nude mice for 8 weeks. The newly formed tissue was evaluated by Alcian Blue and immunohistochemical staining for collagen type-II and type-I. Structural homogeneity of the tissue, composed of freshly isolated as well as serially passaged cells, was found to be enhanced by high-density seeding (50 million/ml) and the use of E210 as a carrier. The percentage of collagen type-II positive staining P3-cells was generally higher when E210 was used as a carrier. Furthermore, seeding P3-chondrocytes at the highest density (50 million/ml) enhanced collagen type-II expression. This study shows promising possibilities to generate structurally regular neo-cartilage using multiplied chondrocytes in alginate in combination with a fleece of polylactic/polyglycolic acid.     

Immunocytochemical detection of basement membrane antigens in the histopathological evaluation of laryngeal dysplasia and neoplasia

Immunocytochemical detection of intrinsic components of the basement membrane (type IV collagen and laminin) was performed in biopsies of carcinoma in situ, dysplasia and hyperplasia of the laryngeal mucosa. We found a distinct and continuous basement membrane, containing both laminin and type IV collagen, at the border between epithelial cells and mesenchymal stroma in normal as well as in hyperplastic and dysplastic mucosa. In contrast, irregular discontinuities were found in some cases of carcinoma in situ and in invasive carcinoma. In addition, immunoreactivity for intracytoplasmic basement membrane components was noticed occasionally in neoplastic epithelial cells. In areas of inflammation, infiltration of leucocytes into the epithelium was occasionally accompanied by sharply defined small interruptions of the basement membrane. Our results indicate that immunohistochemical detection of basement membrane components can be of value for the demonstration of microinvasive growth in laryngeal cancer.     

Genetic modeling of human urinary bladder carcinogenesis

We developed a model of human urinary bladder cancer progression from in situ precursor lesions to invasive carcinoma using whole organ histologic and genetic mapping. The model represents a high-density and detailed analysis regarding allelic losses on chromosomes 4, 8, 9, 11, and 17 as revealed by testing of 234 samples obtained from five cystectomy specimens. The samples corresponded to microscopically identified intraurothelial precursor conditions ranging from dysplasia to carcinoma in situ and invasive cancer. The initial analysis of paired normal and tumor DNA samples disclosed allelic losses in 72 of 225 tested hypervariable DNA markers. Subsequent use of these markers on all mucosal samples revealed that 47 had alterations with a statistically significant relation to urothelial neoplasia. The allelic losses clustered in 33 distinct chromosomal regions, indicating the location of putative tumor suppressor genes involved in the development and progression of urinary bladder cancer. Some of the markers with statistically significant allelic losses mapped to the regions containing well-characterized tumor suppressor genes but many were located in previously unknown loci. The majority of statistically significant allelic losses (70%) occurred early in low-grade intraurothelial dysplasia, and some of them involved adjacent areas of morphologically normal mucosa preceding the development of microscopically recognizable precursor lesions. The remaining 30% of markers developed allelic losses in the later phases of urothelial neoplasia, implicating their involvement in progression to invasive disease. Markers exhibiting allelic losses in early phases of urothelial neoplasia could be used for detection of occult preclinical or even premicroscopic phases of urinary bladder cancer, whereas markers that showed allelic losses in the later phases of the process could serve as indicators of progression to invasive disease. The approach used in this study facilitates genome-wide modeling of cancer progression and provides important chromosomal landmarks for more specific studies of multistep urinary bladder carcinogenesis.