Un-biodegradable and biodegradable plastic sheets modify the soil properties after six months since their applications.

Nowadays, microplastics represent emergent pollutants in terrestrial ecosystems that exert impacts on soil properties, affecting key soil ecological functions. In agroecosystems, plastic mulching is one of the main sources of plastic residues in soils. The present research aimed to evaluate the effects of two types of plastic sheets (un-biodegradable and biodegradable) on soil abiotic (pH, water content, concentrations of organic and total carbon, and total nitrogen) and biotic (respiration, and activities of hydrolase, dehydrogenase, ß-glucosidase and urease) properties, and on phytotoxicity (germination index of Sorghum saccharatum L. and Lepidium sativum L.). Results revealed that soil properties were mostly affected by exposure time to plastics rather than the kind (un-biodegradable and biodegradable) of plastics. After six months since mesocosm setting up, the presence of un-biodegradable plastic sheets significantly decreased soil pH, respiration and dehydrogenase activity and increased total and organic carbon concentrations, and toxicity highlighted by S. saccharatum L. Instead, the presence of biodegradable plastic sheets significantly decreased dehydrogenase activity and increased organic carbon concentrations. An overall temporal improvement of the investigated properties in soils covered by biodegradable plastic sheets occurred.

Synthesis of cartilage matrix by mammalian chondrocytes in vitro. I. Isolation, culture characteristics, and morphology.

We describe the isolation and the ultrastructural characteristics of adult bovine articular chondrocytes in vitro. Slices of bovine articular cartilage undergo sequential digestions with pronase and collagenase in order to release cells. Chondrocytes are plated at high density (1 x 10(5) cells/cm2) in culture dishes or roller bottles with Ham's F-12 medium, supplemented with 10% fetal bovine serum. Before culture, chondrocytes are freed of surrounding territorial matrix. Within the first few days of culture they re-establish a territorial matrix. As time progresses, chondrocytes synthesize both territorial and extraterritorial matrices. The matrices are rich in collagen fibrils and ruthenium red-positive proteoglycans. These features are most apparent in mass roller cultures in which aggregates of cells and matrix appear as long streaks and nodules. This morphology reveals an organization of chondrocytes and their matrices that is similar to that of the parent articular cartilage in vivo.

Synthesis of cartilage matrix by mammalian chondrocytes in vitro. II. Maintenance of collagen and proteoglycan phenotype.

The in vitro phenotype of bovine articular chondrocytes is described. Chondrocytes plated at high density in roller-bottle and dish cultures were maintained in vitro. The major matrix macromolecules, collagen and proteoglycan, synthesized by these cells were characterized during the course of the culture period. The chondrocytes synthesized mainly Type II collagen, which was found predominantly in the cell-associated matrix. The media contained a mixture of Type II and Type III collagens. Type I collagen was detectable in neither the medium nor the cell-associated matrix. The proteoglycan monomers found in media and cell-associated matrix had the same hydrodynamic sizes as monomers synthesized by cartilage slices or those extracted from adult articular cartilage. The majority of proteoglycans synthesized by the cells were found in high molecular weight aggregates which were readily recovered from the media and were extractable from cell-associated matrix with low ionic strength buffers. The results demonstrate the long-term in vitro phenotypic stability of the bovine articular chondrocytes. The advantages of the in vitro system as a model for studying the effects of external agents, such as drugs and vitamins, are discussed.

Selective emergence of differentiated chondrocytes during serum-free culture of cells derived from fetal rat calvaria.

Cells dispersed from the chondrocranial portions of fetal rat calvaria proliferated and performed specialized functions during primary culture in a chemically defined medium. Mature cultures were typified by multilayered clusters of redifferentiating cartilage cells. Flattened cells that lacked distinguishing features occupied areas between the clusters. Alkaline phosphate-enriched, ultrastructurally typical chondrocytes within the clusters were encased in a dense extracellular matrix that stained prominently for chondroitin sulfate proteoglycans. This matrix contained fibrils measuring 19 nm in diameter, which were associated with proteoglycan granules that preferentially bound ruthenium red. A progressive increase in the number of cells indicated the proliferation of certain elements in the primary culture. The cells in primary culture were biochemically as well as morphologically heterogeneous since they were found to synthesize type I and type II collagens. Homogeneous populations of redifferentiated chondrocytes were recovered as floating cells and were shown to express the chondrocyte phenotype in secondary culture. Subcultured cells synthesized type II collagen and its precursors almost exclusively and incorporated 35SO4 into proteoglycan monomer and aggregates to a greater degree than the cells in primary culture. The pattern of proteoglycan monomer and aggregate labeling resembled that of intact cartilage segments and bovine articular chondrocytes. Skin fibroblasts harvested from the same rat fetuses failed to proliferate when maintained under identical conditions. Hence, exogenous hormones, growth factors, and protein are not required for chondrocyte growth and maturation.

Frequent requirement of hedgehog signaling in non-small cell lung carcinoma.

Although it had previously been suggested that the hedgehog (HH) pathway might be activated in some lung tumors, the dependence of non-small cell lung carcinomas (NSCLC) for HH activity had not been comprehensively studied. During a screen of a panel of 60 human tumor cell lines with an HH antagonist, we observed that the proliferation of a subset of NSCLC cell lines was inhibited. These NSCLC cell lines express HH, as well as key HH target genes, consistent with them being activated through an autocrine mechanism. Interestingly, we also identified a number of NSCLC cell lines that express high levels of the downstream transcription factor GLI1 and harbor enhanced levels of HH activity, but appear insensitive to known HH antagonists. We hypothesized that the high levels of GLI1 in these cells would function downstream of the HH antagonist target, allowing them to bypass the antagonist-mediated block in proliferation. Consistent with this hypothesis, when the levels of GLI1 are knocked down in such cells, they become sensitive to these inhibitors. We go on to show that a large percentage of primary NSCLC samples express GLI1, consistent with constitutive activation of the HH pathway in these samples. Taken together, these results establish the involvement of the HH signaling pathway in a subset of NSCLCs.

Regulation of tumor invasion by cartilage-derived anti-invasion factor in vitro.

The resistance of cartilage to tumor invasion was studied with the use of a novel in vitro culture system. Articular cartilage obtained from fresh metacarpophalangeal joints of preadolescent bovines was used as a growth surface for human TE-85 osteosarcoma cells and foreskin fibroblasts. Cartilage disks formed the bottoms of stainless-steel cylinders, providing closed growth chambers for these cells. Both invasive osteosarcoma cells and normal fibroblasts were unable to penetrate viable, unextracted cartilage during a 2-week culture period. When cartilage was devitalized by freezing and thawing, the tissue remained resistant to invasion. Cartilage, extracted with either 1 or 3 M guanidine hydrochloride, was invaded by osteosarcoma cells, but not by control fibroblasts. Invasion by osteosarcoma cells into salt-extracted cartilage was abolished when low concentrations of a cartilage-derived, anti-invasion factor were added to the culture medium. These data provided evidence that the resistance of cartilage to tumor invasion is regulated in part by tissue-derived proteinase inhibitors.

Tumor cell procoagulant and urokinase expression in carcinoma of the ovary.

BACKGROUND: An association between cancer and increased blood coagulation has been observed for many years. Generally, there is an equilibrium between the coagulation system (fibrin deposition) and the fibrinolytic system (degradation of fibrin by enzymes). However, in malignant disease such as ovarian carcinoma, this equilibrium is disrupted, resulting in the abnormal activation of coagulation or hypercoagulability. Also, evidence indicates that various components of these pathways may contribute to the disorderly characteristics of malignancy, such as proliferation, invasion, and metastasis. PURPOSE: Our purpose was to define the mode of interaction of tumor cells in ovarian carcinoma with both the coagulation (procoagulant-initiated) and fibrinolysis (urokinase-type plasminogen activator-initiated) (u-PA) pathways. METHODS: Studies were performed on acetone-methylbenzoate-xylene-fixed tissue prepared from fresh resected primary tumor specimens from 15 patients with cystic epithelial ovarian carcinoma. None of the patients had received prior treatment. Antibodies were tested on control and tumor tissues in concentrations that provided maximum staining intensity with minimum background staining. Laboratory immunohistochemical techniques used purified, monospecific antibodies to detect coagulant antigens. Tests were performed utilizing antibodies to recombinant human tissue factor; factor VII; factor X; factor XIIIA; high-molecular-weight and low-molecular-weight forms of u-PA; tissue-type plasminogen activator; plasminogen; and the plasminogen activator inhibitors 1, 2, and 3. Monoclonal antibodies used for specific antigen detection included 1-8C6 (fibrinogen), T2G1 (fibrin), and EBM-11 (macrophage-specific). RESULTS: The ovarian tumor cells expressed urokinase-type plasminogen activator in a pattern that was variable in intensity and distribution. Tumor cell plasminogen was not detected. Tumor cells also expressed tissue factor and coagulation pathway intermediates that resulted in local thrombin generation as evidenced by the conversion of fibrinogen (present in tumor connective tissue) to fibrin that was found to hug the surfaces of tumor nodules and individual tumor cells. Detected fibrin could not be accounted for on the basis of necrosis or a local inflammatory cell infiltrate. CONCLUSIONS: These results are consistent with the existence of a dominant tumor cell-associated procoagulant pathway that leads to thrombin generation and hypercoagulability in carcinoma of the ovary. IMPLICATIONS: In ovarian carcinoma the procoagulant pathway may contribute to tumor progression. Clinical trials of therapeutic drugs capable of limiting local coagulability (anticoagulants, protease inhibitors) are indicated in this tumor type.

A novel monoclonal antibody, SCCL 175, with specificity for small cell neuroendocrine carcinoma of the lung.

The murine monoclonal antibody SCCL 175, which is one of several monoclonal antibodies directed against small cell neuroendocrine carcinoma developed by one of us (E.D.B.), was studied for its immunohistochemical reactivity against normal human tissues and a spectrum of bronchopulmonary and metastatic carcinomas using the avidin-biotin complex technique. SCCL 175 reacted with 40 of 44 small cell carcinomas including both primary and metastatic sites and was distributed both on the cell surface and intracytoplasmically. Staining was seen in fresh frozen tissues, cytology preparations, and in a limited number of paraffin-embedded tissue sections after trypsin pretreatment. It was nonreactive with all non-small cell lung carcinomas, neuroendocrine carcinomas from other primary sites, and nonpulmonary carcinomata studied to date. Its distribution in normal adult human tissues was limited to some hypothalamic neurons and the apical membranes of renal proximal tubular epithelium. Cytotrophoblastic and syncytotrophoblastic cells from placental tissue demonstrated variable SCCL 175 immunoreactivity. Of choriocarcinomas studied, one of three demonstrated focal staining. These findings demonstrate the diagnostic utility of SCCL 175 in phenotyping small cell carcinoma of lung, and its specificity suggests a potential role in the therapy of this disease.

In vitro determination of tumor invasiveness using extracted hyaline cartilage.

Our previous observation that salt-extracted, devitalized cartilage could be penetrated by malignant tumor cells but was nonpermissive to fibroblastic ingrowth led us to postulate that this matrix might be used as a test connective tissue to discriminate in vitro between noninvasive and invasive tumor cell lines. In a novel in vitro system, salt-extracted, bovine articular cartilage was therefore used as a growth surface for defined noninvasive, invasive, and metastatic carcinoma cell lines, derived from chemical carcinogen-induced tumors of the rat urinary bladder. As monitored by thin-section electron microscopy, salt-extracted cartilage was readily penetrated by the invasive and metastatic rat bladder carcinoma cell lines. The metastatic cell line could be differentiated from the invasive, nonmetastatic cell line by its greater depth of invasion. In contrast, noninvasive carcinoma cells as well as normal bladder epithelial cells lacked the capacity to erode and penetrate the extracted matrix of the articular cartilage. Using these defined cell lines, salt-extracted cartilage can be used to reproducibly discriminate between carcinomas having different invasive potentials. This assay system may have diagnostic application for the in vitro staging of tumors.

Development of an in vitro and in vivo epithelial tumor model for the study of invasion.

Three continuous cell lines were isolated from N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)-induced carcinoma of the Fischer rat urinary bladder by standard explant techniques. RBTCC-2 carcinoma cells were derived from a noninvasive FANFT tumor of Stage 0, RBTCC-5 carcinoma cells were from an invasive FANFT tumor of Stage B2, and RBTCC-8 carcinoma cells were from a s.c. metastasis of a FANFT tumor of Stage D2. Invasive and metastatic carcinoma cells were differentiated from their noninvasive counterparts by cellular and nuclear pleomorphism, cell size, nuclear:cytoplasmic ratio, number of nucleoli, and abnormalities of occludens junctions. Using low (less than 10) and high (greater than 80) passages of these cell strains, tumorigenicity experiments in syngeneic rats showed that the normal in vivo progression of FANFT tumors was interrupted by the isolation of carcinoma cells to cell culture. Histological appearance and biological behavior of tumor isografts closely resembled those of the original FANFT tumors. This was best demonstrated when tumor cells were inoculated adjacent to rat femurs. The destruction of bone, monitored radiographically and histologically, served as a measure of the invasive potential of the tumor cells. Destruction and deep invasion were observed only with isografts of invasive and metastatic carcinoma cells, presumably due to collagenolytic activity. Despite rapid degradation of bone by these isografts, the natural resistance of cartilage to tumor invasion could not be overcome. These carcinoma cell lines, together with their normal epithelial counterparts and the major supporting cells of connective tissue characterized previously by our laboratory, provide a unique system to study tumor invasion.

Reactivity of anti-CD15 monoclonal antibody PM-81 with breast cancer and elimination of breast cancer cells from human bone marrow by PM-81 and immunomagnetic beads.

The CD15 carbohydrate antigen, lacto-N-fucopentaose III is expressed on a variety of human cancer cells including acute myeloid leukemia, small cell carcinoma of the lung, and colorectal carcinomas. We have found that cells from breast cancer cell lines and patient-derived tissue are strongly CD15 positive, as seen by binding to the PM-81 monoclonal antibody. In this report we show that monoclonal antibody PM-81 and immunomagnetic beads can remove breast cancer cells from bone marrow and thus be used as "purging" agents for autologous bone marrow transplantation. PM-81 and immunomagnetic beads removed up to 3 log of SK-BR-3 and MCF7 breast carcinoma cell line cells while minimally affecting normal hematopoietic progenitor cells. This technique may be useful for purging marrow for autologous bone marrow transplantation in breast cancer.

Immunohistochemical staining for transforming growth factor beta 1 associates with disease progression in human breast cancer.

The transforming growth factor beta s (TGF-beta) comprise a family of M(r) 25,000 pluripotent growth factors which have been implicated in the development and progression of human breast cancer. Conflicting data suggest that TGF-beta has the potential to either inhibit or promote the progression of mammary neoplasia. We therefore examined a pathological library of malignant breast biopsy specimens to determine the prevalence and distribution of immunoreactivity with antibodies specific for the three mammalian isoforms of TGF-beta (beta 1, beta 2, and beta 3). We found that intense staining for TGF-beta 1 was positively associated with rate of disease progression, and that this was independent of age, stage, nodal status, or estrogen receptor status (P = 0.009).

Fibrinogen deposition without thrombin generation in primary human breast cancer tissue.

The occurrence and distribution of components of coagulation pathways in situ were determined using immunohistochemical techniques applied to 10 cases of primary carcinoma of the breast, normal breast tissue obtained from two patients undergoing reductive mammoplasty, and three patients with benign breast tumors. Tumor cells stained for factor X and thrombomodulin but not for tissue factor, factor V, factor VII, or factor XIII. Rare nonneoplastic duct epithelial cells stained for thrombomodulin, but these tissues did not otherwise stain for any of these antigens. Macrophages within the tumor stroma stained for tissue factor, factor VII, and factor XIII but not for factor V or factor X. These features of macrophages were the same in malignant and nonmalignant breast tissue. Fibrinogen was present in abundance throughout the connective tissue in breast cancer but not in nonmalignant tissues. By contrast, no staining was observed using fibrin-specific antibodies. These results suggest that an intact coagulation pathway does not exist in breast cancer tissue and that thrombin capable of transforming fibrinogen to fibrin is not generated in significant amounts in this tumor type. While fibrin is not a feature of the connective tissue stroma in breast cancer, it is conceivable that the abundant fibrinogen present in the tumor connective tissue (and factor XIII present in connective tissue macrophages) might contribute to the structural integrity of breast tumor tissues.

Occurrence of components of fibrinolysis pathways in situ in neoplastic and nonneoplastic human breast tissue.

The occurrence and distribution of components of fibrinolysis pathways were determined using immunohistochemical techniques applied to 10 cases of primary carcinoma of the breast, normal breast tissue obtained from two patients undergoing reductive mammoplasty, and three cases of benign breast tumors. Tumor cells stained for urokinase- and tissue-type plasminogen activators, plasminogen activation inhibitor-1, plasminogen, and plasmin-antiplasmin complex neoantigen. The tumor connective tissue stained for fibrinogen and its D fragment plasmin digestion product. By contrast, only occasional nonneoplastic duct epithelial cells stained for urokinase- and tissue-type plasminogen activators and there was little or no staining for the other antigens tested. These results are consistent with the existence of local amplification of expression of enzymatically active plasminogen activators, and particularly of urokinase-type plasminogen activator, in situ in primary breast cancer tissue. These features distinguish malignant from benign breast tissue and may modulate neoplastic progression through an effect on tumor cell proliferation, invasion, and metastatic dissemination.

Phase Ia/Ib trial of bispecific antibody MDX-210 in patients with advanced breast or ovarian cancer that overexpresses the proto-oncogene HER-2/neu.

PURPOSE: MDX-210 is a bispecific antibody that binds simultaneously to type I Fc receptors for immunoglobulin G (IgG) (Fc gamma RI) and to the HER-2/neu oncogene protein product. MDX-210 effectively directs Fc gamma RI-positive effector cells such as monocytes and macrophages to phagocytose or kill tumor cells that overexpress HER-2/neu. The goals of this phase Ia/Ib trial were to determine the maximum-tolerated dose (MTD) and/or the optimal biologic dose (OBD) of MDX-210. PATIENTS AND METHODS: Patients with advanced breast or ovarian cancer that overexpressed HER-2/neu were eligible for treatment. Cohorts of three patients received a single intravenous (IV) infusion of MDX-210 at increasing dose levels from 0.35 to 10.0 mg/m2. RESULTS: Treatment was well tolerated, with most patients experiencing transient grade 1 to 2 fevers, malaise, and hypotension only. Two patients experienced transient grade 3 hypotension at 10.0 mg/m2. Transient monocytopenia and lymphopenia developed at 1 to 2 hours, but no other hematologic changes were observed. Doses of MDX-210 > or = 3.5 mg/m2 saturated > or = 80% of monocyte Fc gamma RI and produced peak plasma concentrations > or = 1 microgram/mL, which is greater than the concentration for optimal monocyte/macrophage activation in vitro. Elevated plasma levels of the monocyte products tumor necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), and neopterin were observed with maximal levels at doses > or = 7.0 mg/m2. Localization of MDX-210 in tumor tissue was demonstrated in two patients. One partial and one mixed tumor response were observed among 10 assessable patients. CONCLUSION: MDX-210 is immunologically active at well-tolerated doses. The MTD and OBD is 7 to 10 mg/m2.

Pulmonary hemorrhage in systemic lupus erythematosus.

The clinicopathological features of four patients with systemic lupus erythematosus and pulmonary hemorrhage are described. Our study confirms that pulmonary hemorrhage may be a dominant clinical expression of lung involvement in this disease. Its clinical manifestations are usually quite characteristic. However, hemoptysis may be absent. Radiographically, bilateral alveolar infiltrates resembling pulmonary edema or infection may be seen. Pulmonary hemorrhage was a major contributing factor to the death of three of our patients. The possible pathogenetic mechanisms responsible for pulmonary hemorrhage in our patients and other patients previously recorded in the literature are reviewed. Evidence supporting an immune complex pathogenesis is presented. Our immunopathological and ultrastructural studies demonstrate deposition of immune aggregates in the lungs in the alveolar septa, large blood vessels, and bronchioles in a manner similar to that which has been observed in the experimental serum sickness model of immune complex mediated pulmonary injury. The histological abnormalities, although nonspecific, are consistent with this interpretation, and collectively show diffuse alveolar lining cell and endothelial cell injury. However, an immune complex pathogenesis may not completely explain the occurrence of pulmonary hemorrhage in SLE. Other factors, including bleeding disorders, pulmonary infection, oxygen toxicity, and the "shock lung" syndrome, may also have contributed to lung hemorrhage in some of these patients.

Soluble normal and mutated DNA sequences from single-copy genes in human blood.

Healthy individuals have soluble (extracellular) DNA in their blood, and increased amounts are present in cancer patients. Here we report the detection of specific sequences of the cystic fibrosis and K-ras genes in plasma DNA from normal donors by amplification with the polymerase chain reaction. In addition, mutated K-ras sequences are identified by polymerase chain reaction utilizing allele-specific primers in the plasma or serum from three patients with pancreatic carcinoma that contain mutated K-ras genes. The mutations are confirmed by direct sequencing. These results indicate that sequences of single-copy genes can be identified in normal plasma and that the sequences of mutated oncogenes can be detected and identified with allele-specific amplification by polymerase chain reaction in plasma or serum from patients with malignant tumors containing identical mutated genes. Mutated oncogenes in plasma and serum may represent tumor markers that could be useful for diagnosis, determining response to treatment, and predicting prognosis.

Fibrillin-1 in human cartilage: developmental expression and formation of special banded fibers.

The molecular basis for Marfan's syndrome (MS), a heritable disorder of connective tissue, is now known to reside in mutations in FBN1, the gene for fibrillin-1. Classic phenotypic manifestations of MS include several skeletal abnormalities associated primarily with overgrowth of long bones. As a first step towards understanding how mutations in FBN1 result in skeletal abnormalities, the developmental expression of fibrillin-1 (Fib-1) in human skeletal tissues is documented using immunohistochemistry and monoclonal antibodies demonstrated here to be specific for Fib-1. At around 10-11 weeks of fetal gestation, Fib-1 is limited in tissue distribution to the loose connective tissue surrounding skeletal muscle and tendon in developing limbs. By 16 weeks, Fib-1 is widely expressed in developing limbs and digits, especially in the perichondrium, but it is apparently absent within cartilage matrix. Fib-1 appears as a loose meshwork of fibers within cartilage matrix by 20 weeks of fetal gestation. Until early adolescence, Fib-1 forms loose bundles of microfibrils within cartilage. However, by late adolescence, broad banded fibers composed of Fib-1 are found accumulated pericellularly within cartilage. Because these fibers can be extracted from cartilage using dissociative conditions, we postulate that they are laterally packed and crosslinked microfibrils. On the basis of these findings, we suggest that the growth-regulating function of Fib-1 may reside persistently within the perichondrium. In addition, the accumulation of special laterally crosslinked Fib-1 microfibrils around chondrocytes during late adolescence suggests that growth-regulating activities may also be performed by Fib-1 at these sites.

Cellular localization of enzymatically active thrombin in intact human tissues by hirudin binding.

Cellular sites of coagulation activation within complex, intact tissues have been studied by immunohistochemical techniques. Hirudin, a specific and high affinity inhibitor of the active site of thrombin, together with antibody to hirudin were applied to sections of AMeX-fixed specimens of normal lung, kidney, placenta, freshly incised skin and unperturbed skin obtained at fresh autopsy; to rheumatoid synovial tissue; and to malignant tissue from a variety of tumor types. Staining for thrombin was observed selectively on pulmonary alveolar, rheumatoid synovial, and placental macrophages that express an intact extrinsic coagulation pathway. Staining was also observed restricted to the endothelium of capillaries in freshly incised skin but not in either unperturbed skin or in aged incisions. Staining of tumor cell bodies was observed in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma tissues that we found previously to show tumor cell-associated procoagulant activity. This staining occurred commonly on cells within the tumor mass that were distant from stromal fibrinogen/fibrin. By contrast, tumor-associated macrophage but not tumor cell staining was seen in adenocarcinoma and squamous cell carcinoma of the lung, and little or no staining was seen colon cancer tissue. Negative controls in which either the hirudin probe or its antibody were omitted failed to show staining. These results are in accord with previous findings and suggest that such techniques may be useful for studying the cellular sites of thrombin generation in intact tissues. We postulate that administration of potent and specific thrombin antagonists, such as hirudin, to patients with relevant tumor types might be followed by homing of hirudin to tumor cells in vivo so that effects of local thrombin generation on malignant progression can be determined.

Cellular localization of activated factor X by Xa-specific probes.

A probe, recombinant antistasin, that reacts specifically with the activated form of factor X (Xa) was used in immunohistochemical procedures to detect cellular sites of Xa generation within intact tissues. Factor Xa was detected on tumor cells in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma. Tumor-associated macrophages (but not tumor cells) expressed Xa in adenocarcinoma and squamous cell carcinoma of the lung, and Hodgkin's disease. Factor Xa in these locations corresponded to evidence reported previously for an intact coagulation pathway and thrombin formation associated with these tumor cells and macrophages. By contrast, only rare connective tissue cells stained for Xa in breast and colon cancer, tumor types shown previously to lack an intratumoral coagulation pathway and thrombin generation, and in normal liver, lung, breast, kidney, and placental tissues. Hepatocytes did not stain. These results suggest that such probes may be useful for studying the activation state of cell-associated factor X in situ within intact tissues.