Plasminogen activator inhibitor-type 1 in Lewis lung carcinoma

Summary Plasminogen activator inhibitor-type 1 (PAI-1) was identified in extracts of Lewis lung carcinoma, and its immunohistochemical localization was studied together with that of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators. All primary tumors (n=11) contained heterogeneously distributed immunoreactivity against each of the three components. Most often, areas that contained u-PA immunoreactivity also contained PAI-1 immunoreactivity. However, several areas showed a strong u-PA immunoreactivity, but no or low PAI-1 immunoreactivity. The latter staining pattern was only found in periferal areas, and usually in areas with histological signs of tissue destruction. Lung metastases always contained u-PA immunoreactivity, while PAI-1 immunoreactivity was found in most, but not all, metastases. t-PA immunoreactivity was found in a few scattered tumor cells, in primary carcinomas as well as metastases. Controls that included absorption with highly purified antigen preparations and immunoblotting, indicated that all the immunoreactivity represented genuine PAI-1, u-PA and t-PA, respectively. The results are consistent with an assumption that the plasminogen activation system, and particularly u-PA and PAI-1, plays a role in regulation of breakdown of extracellular matrix proteins during invasive growth in this carcinoma.     

Plasminogen activator inhibitor-type 1 in Lewis lung carcinoma

Plasminogen activator inhibitor-type 1 (PAI-1) was identified in extracts of Lewis lung carcinoma, and its immunohistochemical localization was studied together with that of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators. All primary tumors (n = 11) contained heterogeneously distributed immunoreactivity against each of the three components. Most often, areas that contained u-PA immunoreactivity also contained PAI-1 immunoreactivity. However, several areas showed a strong u-PA immunoreactivity, but no or low PAI-1 immunoreactivity. The latter staining pattern was only found in peripheral areas, and usually in areas with histological signs of tissue destruction. Lung metastases always contained u-PA immunoreactivity, while PAI-1 immunoreactivity was found in most, but not all, metastases. t-PA immunoreactivity was found in a few scattered tumor cells, in primary carcinomas as well as metastases. Controls that included absorption with highly purified antigen preparations and immunoblotting, indicated that all the immunoreactivity represented genuine PAI-1, u-PA and t-PA, respectively. The results are consistent with an assumption that the plasminogen activation system, and particularly u-PA and PAI-1, plays a role in regulation of breakdown of extracellular matrix proteins during invasive growth in this carcinoma.     

immunocytochemical localization of urokinase-type plasminogen activator in lewis lung carcinoma

The invasively growing and metasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.     

The respiratory behavior of Lewis carcinoma cells--existence of a cyanide-resistant respiration

The respiratory O2 consumption of vegetative, non-necrotic, Lewis lung carcinoma cells was found to be very low compared with that of non-tumor tissues and was highly resistant to cyanide. However, the resistant rate was inhibited by salicylhydroxamic acid (SHAM) in either isolated cells or tissue fragments. In addition, this compound did not affect their cytochrome-c oxidase activity. The results support the existence of an alternative oxidase system that significantly contributes to oxygen uptake in Lewis carcinoma cells. To our knowledge, this is the first report showing significant SHAM-sensitivity of tumor respiration and perhaps of higher animal cell respiration.     

Localization of retinoic acid-binding protein in nuclei

Retinoic acid-binding component has been detected in the nuclei of chick embryo skin. The physicochemical properties of this macromolecule are in agreement with the properties of the retinoic acid-binding protein isolated from tissue cytosol. Although no binding protein could be detected in normal colon or lung tissue, nuclei isolated from a transplantable colon tumor and Lewis lung carcinoma contained this protein.     

ADAM12 produced by tumor cells rather than stromal cells accelerates breast tumor progression

Expression of ADAM12 is low in most normal tissues but is markedly increased in numerous human cancers, including breast carcinomas. We have previously shown that overexpression of ADAM12 accelerates tumor progression in a mouse model of breast cancer (PyMT). In this study, we found that ADAM12 deficiency reduces breast tumor progression in the PyMT model. However, the catalytic activity of ADAM12 seems to be dispensable for its tumor-promoting effect. Interestingly, we show that ADAM12 endogenously expressed in tumor-associated stroma in the PyMT model does not influence tumor progression, but that ADAM12 expression by tumor cells is necessary for tumor progression in these mice. This finding is consistent with our observation that in human breast carcinoma, ADAM12 is almost exclusively located in tumor cells and, only rarely, seen in the tumor-associated stroma. We hypothesized, however, that the tumor-associated stroma may stimulate ADAM12 expression in tumor cells, on the basis of the fact that TGF-β1 stimulates ADAM12 expression and is a well-known growth factor released from tumor-associated stroma. TGF-β1 stimulation of ADAM12-negative Lewis lung tumor cells induced ADAM12 synthesis, and growth of these cells in vivo induced more than 200-fold increase in ADAM12 expression. Our observation that ADAM12 expression is significantly higher in the terminal duct lobular units (TDLU) adjacent to human breast carcinoma compared with TDLUs found in normal breast tissue supports our hypothesis that tumor-associated stroma triggers ADAM12 expression.     

Analysis of metallothionein and vimentin immunoreactivity in pharyngeal squamous cell carcinoma and its microenvironment

Metallothionein (MT) has been shown to have pro-proliferative anti-apoptotic activity and to be involved in microenvironment remodeling. The aim of this study has been to determine whether the changes in MT and vimentin immunoreactivity observed in cancer and its microenvironment are related to the local spread of the disease. The immunoreactivity levels of both MT and vimentin were evaluated together with CD56 and CD57 antigens in 49 tissue samples taken from patients with squamous cell carcinoma originating from the palatine tonsils and in 20 tissue samples derived from patients with chronic tonsillitis (the reference group). MT immunoreactivity levels were statistically significantly higher in the tissue samples from squamous cell carcinoma than in those of the reference group and also higher in the squamous cell carcinoma samples compared with the stromal samples. Moreover, stromal fibroblasts exhibited high vimentin and MT immunoreactivity levels. Statistically significantly higher MT immunoreactivity levels within the tumor cells were identified in patients with the presence of lymph node metastases in contrast to those patients without such metastases. Vimentin was detected in both the tumor and the stromal tissue samples and presented an interesting pattern of staining strongly expressed within the stroma and the septal architecture of the tumor. The number of CD56- and CD57-positive lymphocytes identified in tissue samples both from squamous cell carcinoma and from the stroma was statistically significantly lower than that in the reference group. MT expression by tumor cells is thus associated with an aggressive phenotype of the tumor and the ability to create metastases.     

Urokinase induces expression of its own receptor in Beas2B lung epithelial cells

Interaction between the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) localizes cellular proteolysis and promotes cellular proliferation and migration. The interaction between uPA and uPAR at the surface of epithelial cells thereby contributes to the pathogenesis of lung inflammation and neoplasia. In this study, we sought to determine if uPA itself alters uPAR expression by lung epithelial cells. uPA enhanced uPAR expression as well as (125)I-uPA binding in Beas2B lung epithelial cells in a time- and concentration-dependent manner. The uPA-mediated induction of uPAR is not accomplished through its receptor and requires enzymatic activity. The low molecular weight fragment of uPA, lacking the receptor binding domain, was as potent as intact two-chain uPA in inducing expression of uPAR at the cell surface. Plasmin, the end product of plasminogen activation, did not alter uPA-mediated uPAR expression. Induction of uPAR by uPA represents a novel pathway by which epithelial cells can regulate uPAR-dependent cellular responses that may contribute to stromal remodeling in lung injury or neoplasia.     

Morphological appearance, content of extracellular matrix and vascular density of lung metastases predicts permissiveness to infiltration by adoptively transferred natural killer and T cells

We have recently shown that adoptively transferred, IL-2-activated natural killer (A-NK) cells are able to eliminate well-established B16-F10.P1 melanoma lung metastases. However, some B16-F10.P1 lung metastases were resistant to infiltration by the A-NK cells and also resistant to the A-NK cell treatment. The infiltration-resistant (I-R) B16-F10.P1 metastases had a unique “compact” morphology compared to the “loose” morphology of the infiltration-permissive (I-P) metastases. Here, we show that I-P loose tumors and I-R compact tumors are also found in lung metastases of mouse Lewis lung carcinoma (3LL), MCA-102 sarcoma, and MC38 colon carcinoma as well as rat MADB106 mammary carcinoma origin. Furthermore, the infiltration resistance of the compact tumors is not restricted to A-NK cells, since PHA and IL-2 stimulated CD8+ T-cells (T-LAK cells) also infiltrated the compact tumors poorly. Analyses of tumors for extracellular matrix (ECM) components and PECAM-1⁺ vasculature, revealed that the I-R lesions are hypovascularized and contain very little laminin, collagen and fibronectin. In contrast, the I-P loose tumors are well-vascularized and they contain high amounts of ECM components. Interestingly, the distribution pattern of ECM components in the I-P loose tumors is almost identical to that of the normal lung tissue, indicating that these tumors develop around the alveolar walls which provide the loose tumors with both a supporting tissue and a rich blood supply. In conclusion, tumor infiltration by activated NK and T cells correlates with the presence of ECM components and PECAM-1⁺ vasculature in the malignant tissue. Thus, analysis of the distribution of ECM and vasculature in tumor biopsies may help select patients most likely to benefit from cellular adoptive immunotherapy.     

Sphinganine in Sphingomyelins of Tumors and Mouse Regenerating Liver

Contents of sphingenine (sphingosine) and sphinganine were studied in sphingomyelins of transplantable mouse tumors (hepatoma-22, melanoma B16, Lewis lung carcinoma, intestine carcinoma) and rat nephroma RA. The content of sphinganine was increased in sphingomyelins of hepatoma-22 and nephroma RA compared to sphingomyelins of liver and kidneys. Significant contents of sphinganine were also found in sphingomyelins of other studied tumors. The content of sphinganine in regenerating mouse liver (30 h after hepatectomy) was normal. The data suggest that disorders should exist in biosynthesis of sphingoid bases in tumors but not in normal rapidly proliferating tissue.     

Collagenase inhibitors retarding invasion of a human tumor in nude mice

Tumor invasion has been correlated with the ability of tumor cells to produce collagenolytic enzymes which are capable of degrading normal host tissues. However, the human small cell carcinoma implanted subcutanouesly and growing progressively in athymic (nude) mice produced large quantities of collagenase but did not appear to significantly infultrate adjacent host tissue. In comparison, subcutaneously implanted murine Lewis lung tumors produced similar quantities of collagenase and were locally invasive. The human tumors were surrounded by a compact layer of fibroblast cells in a fibrous matrix. This fibrous sheath exhibited anticollagenase activity and indicated a mechanism of host tissue resistance to invasion via the formation of inhibitors to degradative enzymes produced by tumor cells.     

Immunohistochemical evaluation of urokinase plasminogen activator receptor in noninvasive and early invasive urothelial papillary neoplasia

OBJECTIVE: To investigate urokinase plasminogen activator receptor (uPAR) expression in urothelial papilloma and in noninvasive and early invasive papillary carcinoma. STUDY DESIGN: The study included 40 cases of papillary neoplasia of the urinary bladder subdivided into 10 cases of urothelial papilloma (UP); 10 of papillary carcinoma, grade 1; 10, grade 2 (G2); and 10, grade 3 (G3). Invasion of the subepithelial connective tissue was present in 5 cases of G2 and 7 cases of G3. According to the 2002 revision of the TNM system, these cases were defined as T1 and the others as Ta. uPAR expression was evaluated with an immunohistochemical technique in 5-microns-thick tissue sections. RESULTS: Difference in the distribution of positive cases in the 4 groups were statistically significant and greatest in G3. Statistically significant differences were also observed between Ta and T1 cases in terms of uPAR intensity. CONCLUSION: Detection of immunoreactivity for uPAR was associated with high grade UP carcinomas. These data indicate that uPAR is potentially an important prognostic factor in bladder carcinoma.     

Immunohistochemical distribution of inter-alpha-trypsin inhibitor chains in normal and malignant human lung tissue.

The inter-alpha-trypsin inhibitor (ITI) family is a group of plasma proteins built up from heavy (HC1, HC2, HC3) and light (bikunin) chains synthesized in the liver. In this study we determined the distribution of ITI constitutive chains in normal and cancerous lung tissues using polyclonal antibodies. In normal lung tissue, H2, H3, and bikunin chains were found in polymorphonuclear cells, whereas H1 and bikunin proteins were found in mast cells. Bikunin was further observed in bronchoepithelial mucous cells. In lung carcinoma, similar findings were obtained on infiltrating polymorphonuclear and mast cells surrounding the tumor islets. Highly differentiated cancerous cells displayed strong intracytoplasmic staining with H1 and bikunin antiserum in both adenocarcinoma and squamous cell carcinoma. Moreover, weak but frequent H2 expression was observed in adenocarcinoma cells, whereas no H3-related protein could be detected in cancer cells. Local lung ITI expression was confirmed by RT-PCR. Although the respective role of inflammatory and tumor cells in ITI chain synthesis cannot be presently clarified, these results show that heavy chains as well as bikunin are involved in malignant transformation of lung tissue.(J Histochem Cytochem 47:1625-1632, 1999)     

Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues

We have characterized the normal human tissue distribution and tumor expression of the human multidrug resistance gene (MDR1) product P-glycoprotein (Pgp) by immunohistochemical staining of frozen tissue sections of human normal and tumor tissues, using three mouse monoclonal antibodies (MAb) which recognize at least two different epitopes of Pgp. Pgp expression on normal human tissues was detected in specialized epithelial cells with secretory/excretory functions, trophoblasts in the placenta, and on endothelial cells of capillary blood vessels at blood-tissue barrier sites. There were significant differences in the staining patterns of these MAb. Mouse MAb HYB-241 and HYB-612 each recognize an extracellular epitope of Pgp, whereas mouse MAb C219 detects a carboxy terminal intracellular epitope and has recently been reported to crossreact with the MDR3 gene product. HYB-241 and HYB-612 strongly stain endothelial cells and trophoblasts, whereas C219 is weakly positive or unreactive on these cells. Likewise, C219 strongly stains the biliary pole of hepatocytes, skeletal and heart muscle fibers, whereas HYB-241 and HYB-612 are unreactive on these cells. Immunopathological studies were performed on a wide variety of human tumors. Pgp expression on human tumors was most commonly detected in colon. renal, and adrenal carcinomas; rarely in lung and gastric carcinomas and certain germ cell tumors; and was undetectable in breast and endometrial carcinomas tested. Few sarcomas and none of the melanomas, neuroblastomas, gliomas, and pheochromocytomas had detectable Pgp expression. Intensity and pattern of staining varied among different cases of a given tumor type; although homogeneous immunoreactivity was observed, heterogeneity of expression in a single histological section was more common. The finding of Pgp expression in a variety of normal tissues with diverse physiological functions suggests that the role of Pgp may not be limited to excretion of xenobiotics. Pgp expression in capillaries of the brain and testis may explain the failure of drugs such as vincristine and actinomycin-D to penetrate into these tissues, allowing them to remain as pharmacological sanctuaries for malignant cells. Although Pgp expression can now be detected in a variety of human tumors, further studies are needed to establish the possible significance of this finding.     

小鼠Lewis肺癌原位模型的建立

目的利用Matrigel与Lewis制备细胞混悬液注射于小鼠左肺内,建立小鼠Lewis肺癌原位模型,评价其肿瘤生长情况、转移情况,以期建立更稳定、更接近于人肺癌生长情况的小鼠肺癌原位模型。方法将处于对数生长期的Lewis肺癌细胞混悬于Matrigel中,接种于C57BL/6近交系小鼠左肺内。分别于第4、7、10、13、16天各处死5只小鼠,观察其局部成瘤率、肿瘤生长情况、中位生存期及肿瘤转移情况,并对各阶段小鼠行肺部,肝脏,肾脏,脾脏病理切片检查。结果术后第7天解剖的5只小鼠中,3只小鼠肺上可见小的瘤结节形成,其余2只肺上未见肉眼成瘤,行病理HE染色检查在显微镜下可见2只小鼠肺脏有小的瘤结节形成。术后第10天以后处死的所有小鼠肺上均有肉眼成瘤,术后第13天,所有小鼠肺原位成瘤并伴有血性胸腔积液、胸腔内转移。术后第25天,有1只小鼠出现上述转移的同时还出现了心包膜转移及肾脏远处转移。5只小鼠生存期分别为17 d、20 d、22 d、22 d、25 d,小鼠中位生存期为21.2 d（17~25 d）。成瘤率100%。结论利用Matrigel法成功建立小鼠Lewis肺癌原位模型,稳定性好,成瘤率高,并具有远处转移的特性,更接近于人肺癌的发生、发展过程。     

Metastatic potential of B16 melanoma cells after in vitro selection for organ-specific adherence

Heterogeneous primary tumors contain subpopulations of cells that differ in ability to metastasize to specific host organs. We have used cryostat sections of host organs to select for metastatic variants of B16 melanoma cells with increased adhesion to specific syngeneic tissues. By repeating the selection procedure with lung tissue, a subpopulation of cells was isolated that demonstrated a specific increase in binding to cryostat sections of mouse lung. This altered binding was reflected by a sixfold increase in the frequency of lung metastasis 21 d after tail vein injection of the tumor cells. In contrast, B16 melanoma cells selected on cryostat sections of mouse brain showed no increase in adhesion to brain or lung tissue and the metastatic pattern in vivo was not significantly different compared with the parent cell line. When cells selected for increased adhesion to cryostat sections of lung were further examined in vitro, they showed altered morphology and increased motility but no change in growth rate. These results demonstrate that alterations in the adhesive interactions between metastatic tumor cells and a specific host tissue can directly affect the frequency of metastasis to that tissue in vivo.     

Endocrine cells in gastric carcinoma and adjacent mucosa. An immunohistochemical and ultrastructural study

Endocrine cells are often found in human gastric carcinoma and may be recognized by the immunoreactivity of their chromogranin A, peptides and biogenic amines content. Anti-chromogranin A was used to investigate the morphology of endocrine cells using light and electron microscope immunohistochemical techniques. The hormone content of endocrine cells was examined in both tumour tissue and tumour-adjacent mucosa. It was found that the endocrine cells in tumour tissue were malignant, often had amphocrine differentiation and did not resemble a normal cell type. The hormone content of endocrine cells in tumour tissue seldom corresponded to the hormonal content of endocrine cells in tumour-adjacent mucosa. In intestinal-type carcinoma and in some parts of diffuse-type gastric carcinomas, endocrine cell hyperplasia and an alteration of the differentiation in the tumour-adjacent mucosa were discovered. The distribution of endocrine cells in the tumour tissue was different in both types of gastric carcinoma. The results reported here suggest that endocrine cell differentiation of malignant endocrine cells in human gastric carcinoma develops in a different way from that of endocrine cells in tumour-adjacent mucosa, and as a result, diverse hormonal products may appear in tumour tissue.     

Immunohistochemical Analysis of 1,25-dihydroxyvitamin D3 Receptor in Cervical Carcinoma

The immunohistochemical localization and expression of 1,25-dihydroxyvitamin D₃ receptors (VDR) has been investigated in normal human cervical tissue (n = 15) and in cervical carcinomas (n = 23). VDR immunoreactivity (monoclonal antibody 9A7r354;) was compared with the staining patterns of transglutaminase K, cytokeratin 10 and Ki-67 in these tumours. Moderate to strong nuclear immunoreactivity for VDR was detected in almost all cervical carcinomas analysed. VDR staining was homogeneous, with no visual differences between individual tumour cells. Some 60% of normal cervical tissues revealed weak immunoreactivity for VDR. In normal cervical tissue, nuclear VDR staining was confined to the lower cervical layers, predominantly to the basal cell layer. Both the intensity of VDR immunostaining and the number of VDR-positive cells were up-regulated in cervical carcinomas compared with normal cervical tissue. No visual correlation wa s found for the coexpression of VDR with markers of proliferation and differentiation. Our findings indicate that: (1) cervical tissue may be a new target organ for therapeutically applied vitamin D analogues; (2) VDR is up-regulated at the protein level in cervical carcinomas compared with normal cervical tissue; (3) up-regulation of VDR in cervical carcinoma is induced not exclusively by alterations in epithelial differentiation or proliferation, but by different, unknown mechanisms; and (4) calcitriol and new vitamin D analogues exerting fewer calcaemic side-effects may be promising new drugs for the treatment or chemoprevention of metastasizing cervical carcinomas as well as of cervical precancerous lesions.     

Distribution of radiolabelled monoclonal antibody Po66 after intravenous injection into nude mice bearing human lung cancer grafts

Monoclonal antibody Po66, produced by immunization against a patient's lung squamous cell carcinoma was found suitable for the scintigraphic detection of human tumours. Surprisingly, the cellular antigen recognized by Po66 was abundant in the cytoplasm of tumour cells but could not be detected on the surface membrane. In the present work the biodistribution of radiolabelled Po66 and of an unrelated immunoglobulin were studied comparatively after intravenous injection into nude mice bearing lung squamous cell carcinoma grafts. Radioactivity distribution among mouse organs and tumour was analysed by gamma counting and autohistoradiography. After injection, radiolabelled Po66 decreased rapidly from the blood in tumour-bearing animals whereas, in controls, it remained at a level comparable to that of the unrelated immunoglobulin. The antibody seemed slowly trapped by the tumour and, 12 days after its injection, distribution ratios between tumour and mouse organs reached values of 20-30 as against 1 in animals injected with the non-specific immunoglobulin. Autohistoradiographic investigations in the tumour confirmed the slow diffusion rate of the antibody, which remained in the vascular spaces up to the 24th hour after injection and diffused afterwards throughout the clusters of tumor cells. Furthermore, radioactivity was detected in cells which, unexpectedly, seemed morphologically unaltered. These cells, the viability of which remains to be determined, were predominant in the central area of the tumours. The results presented constitute new evidence of the ability of an in vivo injected monoclonal antibody to reach a cytoplasmic target inside non-necrotic cells and suggest that the cells permeable to the antibody might be in defective nutritional conditions.