Glutathione S-transferase M1-null genotype as risk factor for SOS in oxaliplatin-treated patients with metastatic colorectal cancer.

BACKGROUND: Oxaliplatin is used as a neo-adjuvant therapy in hepatic colorectal carcinoma metastasis. This treatment has significant side effects, as oxaliplatin is toxic to the sinusoidal endothelial cells and can induce sinusoidal obstruction syndrome (SOS), which is related to decreased overall survival. Glutathione has an important role in the defence system, catalysed by glutathione S-transferase (GST), including two non-enzyme producing polymorphisms (GSTM1-null and GSTT1-null). We hypothesise that patients with a non-enzyme producing polymorphism have a higher risk of developing toxic injury owing to oxaliplatin. METHODS: In the nontumour-bearing liver, the presence of SOS was studied histopathologically. The genotype was determined by a semi-nested PCR. RESULTS: Thirty-two of the 55 (58%) patients showed SOS lesions, consisting of 27% mild, 22% moderate and 9% severe lesions. The GSTM1-null genotype was present in 25 of the 55 (46%). Multivariate analysis showed that the GSTM1-null genotype significantly correlated with the presence of (moderate-severe) SOS (P=0.026). CONCLUSION: The GSTM1-null genotype is an independent risk factor for SOS. This finding allows us, in association with other risk factors, to conceive a potential risk profile predicting whether the patient is at risk of developing SOS, before starting oxaliplatin, and subsequently might result in adjustment of treatment.

LGR6-dependent conditional inactivation of E-cadherin and p53 leads to invasive skin and mammary carcinomas in mice.

Tissue-specific inactivation of E-cadherin combined with tumor suppressor loss leads to invasive and metastatic cancers in mice. While epidermal E-cadherin loss in mice induces squamous cell carcinomas, inactivation of E-cadherin in the mammary gland leads to invasive lobular carcinoma. To further explore the carcinogenic consequences of cell-cell adhesion loss in these compartments, we developed a new conditional mouse model inactivating E-cadherin (Cdh1) and p53 (Trp53) simultaneously in cells expressing the leucine-rich repeat-containing G-protein coupled receptor 6 (Lgr6), a putative epithelial stem cell marker in the skin and alveolar progenitor marker in the mammary gland. Compound Lgr6-CreERT2;Cdh1F;Trp53F female mice containing either heterozygous or homozygous Cdh1F alleles were bred, and Lgr6-driven Cre expression was activated in pre-puberal mice using tamoxifen. We observed that 41% of the mice (16/39) developed mostly invasive squamous-type skin carcinomas, but also a non-lobular mammary tumor was formed. In contrast to previous K14cre or WAPcre E-cadherin and p53 compound models, no significant differences were detected in the tumor-free survival of Lgr6-CreERT2 heterozygous Cdh1F/WT;Trp53F/F versus homozygous Cdh1F/F;Trp53F/F mice (778 versus 754 days, p=0.5). One Cdh1F homozygous mouse presented with lung metastasis that originated from a non-lobular and ERα negative invasive mammary gland carcinoma with squamous metaplasia. In total, 2/8 (25%) Cdh1F heterozygous and 3/12 (25%) Cdh1F homozygous mice developed metastases to lungs, liver, lymph nodes, or the gastro-intestinal tract. In conclusion, we show that inducible and conditional Lgr6-driven inactivation of E-cadherin and p53 in mice causes squamous cell carcinomas of the skin in approximately 40% of the mice and an occasional ductal-type mammary carcinoma after long latency periods.

Jaw Bone Invasion of Oral Squamous Cell Carcinoma Is Associated with Osteoclast Count and Expression of Its Regulating Proteins in Patients and Organoids.

AIMS: Oral squamous cell carcinoma (OSCC) frequently invades the jaw. The exact mechanism of bone invasion remains unclear. This study investigates (premature) osteoclasts and the expression of its differentiation regulating proteins RANKL, OPG and RANK in patients with OSCC. METHODS: Resection specimens from OSCC patients were divided into NI group (No Invasion), E group (Erosion) or I group (bone Invasion). Tissue sections were stained with Cathepsin K (osteoclast-counting), RANKL, OPG and RANK. The staining intensity was scored on different regions of the tumor: front, center, back and normal mucosa. Immunohistochemistry and qPCR for RANKL/OPG/RANK were performed on five head and neck squamous cell carcinoma (HNSCC) organoids. RESULTS: The mean number of osteoclasts (I group) and premature osteoclasts (E group) was significantly higher compared to the NI group (p = 0.003, p = 0.036). RANKL expression was significantly higher in the tumor front and tumor center compared to normal mucosa (all groups). In the I group, RANKL and RANK expression was significantly higher in the tumor front compared to the tumor back and there was a trend of higher RANKL expression in the tumor front compared to the E group and NI group. qPCR showed a 20-43 times higher RANKL mRNA expression in three out of five tumor organoids compared to a normal squamous cell organoid line. There was no correlation between protein and mRNA expression in the HNSCC organoids. CONCLUSIONS: These findings suggest that OSCCs induce bone invasion by stimulating osteoclast activation by regulating the production of RANKL and RANK proteins.

Spatial collagen stiffening promotes collective breast cancer cell invasion by reinforcing extracellular matrix alignment.

The tumor micro-environment often contains stiff and irregular-bundled collagen fibers that are used by tumor cells to disseminate. It is still unclear how and to what extent, extracellular matrix (ECM) stiffness versus ECM bundle size and alignment dictate cancer cell invasion. Here, we have uncoupled Collagen-I bundling from stiffness by introducing inter-collagen crosslinks, combined with temperature induced aggregation of collagen bundling. Using organotypic models from mouse invasive ductal and invasive lobular breast cancers, we show that increased collagen bundling in 3D induces a generic increase in breast cancer invasion that is independent of migration mode. However, systemic collagen stiffening using advanced glycation end product (AGE) crosslinking prevents collective invasion, while leaving single cell invasion unaffected. Collective invasion into collagen matrices by ductal breast cancer cells depends on Lysyl oxidase-like 3 (Loxl3), a factor produced by tumor cells that reinforces local collagen stiffness. Finally, we present clinical evidence that collectively invading cancer cells at the invasive front of ductal breast carcinoma upregulate LOXL3. By uncoupling the mechanical, chemical, and structural cues that control invasion of breast cancer in three dimensions, our data reveal that spatial control over stiffness and bundling underlie collective dissemination of ductal-type breast cancers.

The size of endothelial fenestrae in human liver sinusoids: implications for hepatocyte-directed gene transfer.

Fenestrae allow the passage of gene transfer vectors from the sinusoidal lumen to the surface of hepatocytes. We have previously shown that the diameter of fenestrae correlates with species and strain differences of transgene expression following intravenous adenoviral transfer. In the current study, we demonstrate that the diameter of fenestrae in humans without liver pathology is 107+/-1.5 nm. This is similar to the previously reported diameter in New Zealand White (NZW) rabbits (103+/-1.3 nm) and is significantly smaller than in C57BL/6 mice (141+/-5.4 nm) and Sprague-Dawley rats (161+/-2.7 nm). We show that the diameter of fenestrae in one male NZW rabbit and its offspring characterized by a more than 50-fold increase of transgene expression after adenoviral gene transfer is significantly (113+/-1.5 nm; P<0.001) larger than in control NZW rabbits. In vitro filtration experiments using polycarbonate filters with increasing pore sizes demonstrate that a relatively small increment of the diameter of pores potently enhances passage of adenoviral vectors, consistent with in vivo data. In conclusion, the small diameter of fenestrae in humans is likely to be a major obstacle for hepatocyte transduction by adenoviral vectors.

Sinusoidal obstruction syndrome (SOS): A light and electron microscopy study in human liver.

AIMS: Oxaliplatin is an important chemotherapeutic agent, used in the treatment of hepatic colorectal metastases, and known to induce the sinusoidal obstruction syndrome (SOS). Pathophysiological knowledge concerning SOS is based on a rat model. Therefore, the aim was to perform a comprehensive study of the features of human SOS, using both light microscopy (LM) and electron microscopy (EM). METHODS AND RESULTS: Included were all patients of whom wedge liver biopsies were collected during a partial hepatectomy for colorectal liver metastases, in a 4-year period. The wedge biopsy were perfusion fixated and processed for LM and EM. The SOS lesions were selected by LM and details were studied using EM. Material was available of 30 patients, of whom 28 patients received neo-adjuvant oxaliplatin. Eighteen (64%) of the 28 patients showed SOS lesions, based on microscopy. The lesions consisted of sinusoidal endothelial cell detachment from the space of Disse on EM. In the enlarged space of Disse a variable amount of erythrocytes were located. CONCLUSION: Sinusoidal endothelial cell detachment was present in human SOS, accompanied by enlargement of the space of Disse and erythrocytes in this area. These findings, originally described in a rat model, were now for the first time confirmed in human livers under clinically relevant settings.

Proliferation, kinetics, and fate of monocytes in rat liver during a zymosan-induced inflammation.

The fate and kinetics of monocytes, recruited to the liver by a single zymosan injection, were investigated by light (LM) and electron (EM) microscopy combined with peroxidase cytochemistry and latex phagocytosis. Ultrastructural and cytochemical differences between these cells and resident Kupffer cells persisted during a 7-day period, demonstrating the existence of two types of hepatic mononuclear phagocytes. Both cell types exhibited a pronounced mitotic activity during their numerical increase. Next to peroxidase-positive (POP) Kupffer cells and monocytes, peroxidase-negative (PON) mononuclear phagocytes were observed. These may represent a monocyte subset and/or possibly Kupffer cell precursors.

High deformability and motility of lymphokine-activated killer cells in vitro and in vivo.

The motility and deformability of lymphokine-activated killer cells, purified by their adherence to plastic (A-LAK cells), was investigated in vivo and in vitro. In vitro, A-LAK cells were observed as intrinsically motile cells. They continuously changed their shape while forming protopods or pseudopods and crawled over the culture surface. A-LAK cells were able to migrate across micropores of 3 microns diameter, which was three times smaller than the average diameter of an A-LAK cell. Even in the absence of serum factors and of interleukin-2 (IL-2), more than half of the inoculated cells migrated across such micropore membranes within 18 h. Electron microscopic examination of these micropore membranes showed that the A-LAK cells were highly deformable. A-LAK cells also migrated across a confluent monolayer of endothelium-like 10T1/2 cells. After 24 h incubation on the monolayers, about 20% of the A-LAK cells were found underneath the monolayer. There, they actively moved in the narrow space between the monolayer and the bottom of the culture dish. In vivo IL-2-activated cells showing the large granular lymphocyte morphology accumulated in the hepatic microvasculature. Electron microscopic observations indicate that these cells did not accumulate by mechanical entrapment due to rigidity and size but rather by adherence to the endothelial lining of the sinusoidal capillaries. This also appeared to be the case for adoptively transferred A-LAK cells, which were seen to be attached to the capillary wall. These observations stress the importance of adhesive entrapment to explain the accumulation of intravenously transferred LAK cells in the vascular beds.

Local proliferation and extrahepatic recruitment of liver macrophages (Kupffer cells) in partial-body irradiated rats.

The relative significance of local proliferation and extrahepatic recruitment of Kupffer cells was investigated by partial-body irradiation before the induction of macrophage hyperplasia by zymosan. There was no difference in growth of the Kupffer cells population between nonirradiated rats and rats irradiated with the liver shielded, whereas irradiation of the liver with the rest of the body (bone marrow) shielded resulted in strong inhibition of growth (-61%). Splenectomy combined with bone marrow irradiation inhibited growth to a lesser extent as compared to liver irradiation (-38%). Monocyte and other leukocyte numbers were strongly reduced in peripheral blood and their accumulation in the liver was completely prevented by bone marrow irradiation. Our results demonstrate that local proliferation of resident Kupffer cells represents the predominant source for their increased number during hyperplasia.

Accumulation and localization of gallium-67 in various types of primary lung carcinoma.

The uptake and location of Ga-67 were investigated in 15 primary pulmonary carcinomas. The accumulation in the tumor was determined by scintigraphy of the patient, grain counts over fields of tumor cells in autoradiographs of tumor-tissue samples, and gamma counts in specimens of the tumor. Good correlation was found between the results obtained with these three methods. The relationship between accumulation of Ga-67 in the tumor and the histologic type of tumor was also studied. Undifferentiated carcinomas, and tumor cells in squamous-cell carcinomas showed significantly more Ga-67 than tumor cells in adenocarcinomas. No correlation was found between the presence of inflammatory infiltrates in or around the tumor and the grade of the scintigraphic images. In the autoradiograms, lymphocytes, plasma cells, granulocytes, and macrophages showed less radioactivity than the tumor cells--or none at all. Collagen fibers appeared to have bound some Ga-67, but necrotic areas showed no uptake.

Immunocytochemical localization of osteocalcin in developing rat teeth.

Osteocalcin was purified by gel chromatography from a crude extract obtained after decalcification of rat incisors. The apparent molecular weight, as determined by 5-15% SDS-polyacrylamide gel electrophoresis, was 18,000, and amino acid analysis revealed 60 gamma-carboxyglutamic acid residues per 1000. Antisera against osteocalcin, raised in rabbits, reacted specifically with osteocalcin when investigated by immuno-electroblotting of dentin crude extract. 4-micron cryosections of formaldehyde-fixed tooth germs showed positive immunocytochemical staining for osteocalcin in dentin and odontoblasts. The staining of the mantle dentin at the coronal sides of the tooth germs was more intense than that of the adjacent circumpulpal dentin, while the odontoblasts involved in the formation of mantle dentin showed stronger immunoreactivity than did odontoblasts involved in circumpulpal dentin formation. This marked difference was not observed on the root sides of the tooth germs. In 1-micron cryosections, osteocalcin immunoreactivity was found evenly distributed throughout the entire cell body, with the exception of the Golgi region, which was less intensely stained, while the nucleus and the cell process were negative. The positive staining reaction with anti-osteocalcin antiserum was found in dentin from the very onset of its formation in the fetus. In conclusion, our results demonstrate the presence of osteocalcin in odontoblasts and dentin. Its immunocytochemical localization may be compatible with a distinct role in early dentinogenesis.

Induction of lysosomal storage by suramin.

Isolated livers of rats injected with saline or with suramin (250 mg per kg body weight) 24h previously were perfused with a medium containing radioactively labeled formaldehyde-treated albumin. Suramin-loaded livers released breakdown products at a much lower rate than controls and contained about the double amount of undigested radioactive protein up to about 3 h after the start of the perfusion. These results show that inhibition of proteolysis by suramin as reported previously (Davies et al., 1971; Buys et al., 1973) is not caused by binding of the drug to the substrate in the bloodstream. Electron micrographs of liver sections of suramin-treated rats showed that lysosomes of sinusoidal cells resembled those seen in certain lysosomal storage diseases. The effect of suramin on lysosomal enzymes was studied in vitro. When used at a concentration corresponding to the putative concentration in lysosomes in vivo, the drug inhibited the lysosomal endopeptidases cathepsin Bl and D as well as acid phosphatase. Inhibition of acid phosphatase by suramin in vivo could also be demonstrated by histochemical methods. These results suggest that the observed storage phenomena may be mainly caused by inhibition of lysosomal enzymes.

Early detection of cytotoxic events between hepatic natural killer cells and colon carcinoma cells as probed with the atomic force microscope.

The atomic force microscope (AFM) is a powerful tool to investigate surface and submembranous structures of living cells under physiological conditions at high resolution. These properties enabled us to study the interaction between live hepatic natural killer (NK) cells, also called pit cells, and colon carcinoma cells in vitro by AFM. In addition, the staining for filamentous actin and DNA was performed and served as a reference, because actin and nuclear observations at the light microscopic level during the cytotoxic interaction between these two cell types have been presented earlier. In this study, we collected evidence that conjugation of hepatic NK cells with CC531s colon carcinoma cells results in a decreased binding of CC531s cells to the substratum as probed with the AFM in contact mode as early as 10 min after cell contact (n = 11). To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was performed on hepatic NK/CC531s cell conjugates, resulting in identical observations (n = 3). In contrast, the first cytotoxic signs, as determined with the nuclear staining dye Hoechst 33342, could be observed 3 h after the start of the co-culture. This study illustrates that the AFM can be used to probe early cytotoxic effects of effector to target cell contact in nearby physiological conditions. Other routine cytotoxicity tests detect the first cytotoxic effects after 1.5-3 h co-incubation at the earliest.

Standardized mitotic counts in breast cancer. Evaluation of the method.

Twenty-one pathologists and technicians participated in a study evaluating the variation present in mitotic counts for prognostication of breast cancer. The participants counted the mitotic figures in 20 breast cancer samples from ten high power fields (mitotic activity index, MAI, giving the results in mitotic figures per 10 fields) and also made a correction for field size and area fraction of the neoplastic epithelium to get the standardized mitotic index (volume fraction corrected mitotic index, or M/VV index, giving the result in mitotic figures per square mm of neoplastic epithelium). The difference in variation between the two methods was not big, but the standardized mitotic index (SMI) showed consistently smaller variation among all participants and different subgroups. Experienced pathologists had the highest variation in mitotic counts, and specially trained technicians, the lowest. The efficiency of the mitotic counts in grading (the grading efficiency) was used to evaluate the mitotic counts. In groups without special training for mitotic counts the mean grading efficiency was lower (experienced and training pathologists both on average had the potential to grade 88% of the cases correctly) than in the group specially trained for the purpose (trained technicians had the potential to grade 95% of the cases correctly). Among the specially trained technicians, the grading efficiency was of the same magnitude as the grading efficiency achieved in determining the S-Phase fraction of cells from paraffin embedded breast cancers by flow cytometry in different laboratories. The results suggest that special training is helpful in making mitotic counts more reproducible, and that in trained hands, the mitotic counts give results comparable to more sophisticated methods of determining proliferative activity in breast cancer.

Characterization and immunocytochemical localization of dentine phosphoprotein in rat and bovine teeth.

Dentine phosphoprotein (DPP) was isolated from unerupted bovine molars and from rat incisors. The proteins were characterized biochemically and used to immunize rabbits and guinea pigs. Antibody activity was investigated by enzyme-linked immunosorbent assay (ELISA). Guinea-pig anti-rat DPP did not cross-react with bovine DPP, but rabbit anti-bovine DPP did cross-react with rat DPP. Anti-rat DPP antiserum was applied to cryotome sections of rat molar tooth germs and DPP immunoreactivity was seen in dentine, odontoblasts, odontoblast processes and pre-ameloblasts. Anti-bovine DPP antiserum reacted positively in bovine dentine and dentinal tubules. When this antiserum was applied to rat tissue, predentine was positive but dentine was negative. Adsorption experiments with DPP, purified by methods including and excluding precipitation with calcium, suggested that non-calcium precipitable DPP is present in rat predentine. Rat and bovine DPP are thus species-specific and DPP is synthesized by the odontoblasts, transported through their processes and secreted into the dentine.

Serum and synovial fluid antibodies to collagen in rheumatic diseases: a review.

The literature on the occurrence and significance of antibodies to native or denatured collagen in rheumatic diseases is reviewed. Mainly type I and type II collagen have been investigated, both in serum and synovial fluid, with special reference to their possible role in rheumatoid arthritis. Brief results of the analysis of 40 synovial fluid samples, using an ELISA technique are included.

Subcellular abnormalities of liver sinusoidal lesions in human hepatocellular carcinoma.

Liver sinusoidal lesions in 20 cases of human hepatocellular carcinoma (HCC) were examined with the electron microscope. Kupffer cells existed in the compact type of HCC. In area with pseudoglandular and trabecular cell patterns, Kupffer cells could not be observed. Only a few macrophages were distributed in the tumor tissues. Endothelial cells were altered and showed a poly-layered arrangement and were attached to each other with desmosome-like junctional complexes. There was a general loss of endothelial fenestrae. Endocytotic vesicles could be recognized in these endothelial cells. Poly-layered endothelial cells and accompanying layers of basal laminae were variably arranged between sinusoids and tumor cells with pseudoglandular, trabecular, or compact types of tumor cell patterns. Atypical cells containing numerous lysosomes, together with altered fat-storing (Ito) cells, were located in the tissue space bordering the capillaries. Moreover, tumor cells possessed flattened sinusoidal surfaces. These alterations of the sinusoidal wall suggest that capillarization of liver sinusoids in HCC took place, by loss of fenestrations, formation of basal laminae, and loss of microvilli on the surface of tumor cells. These architectural alterations are thought to completely change the physiological pattern of exchange of metabolites between tumor cells and the sinusoidal lumen. The absence of large numbers of Kupffer cells suggests that at this stage of tumor development, local cellular defense mechanisms were inoperative.

Contributions of light and transmission electron microscopy to the study of the human fat-storing cell.

We examined the human fat-storing cell (Ito cell, lipocyte) in normal and pathologic liver biopsies using toluidine blue staining and transmission electron microscopy. We studied 5 normal patients, 6 non-alcoholic patients with liver steatosis, 8 patients with early alcoholic liver damage, 4 patients with extrahepatic cholestasis, 10 patients with chronic active hepatitis B (N = 2) or C (N = 8) with mild fibrosis, 5 patients with alcoholic cirrhosis and 4 with posthepatitic cirrhosis. We found that fat-storing cells were increased in patients with alcoholic steatofibrosis and extrahepatic cholestasis and decreased in cirrhotic patients. The mean number of lipid droplets per fat-storing cell was significantly increased in patients with alcoholic steatofibrosis. Transmission electron microscopy confirmed the light microscopic findings, especially the accumulation of lipid droplets in fat storing cells in early alcoholic liver disease. Sometimes lipid droplets with different electron density were noted. In cirrhosis there was a more prominent development of intracellular organelles, and cells often changed into a more elongated, myofibroblast-like shape.

AFM imaging of fenestrated liver sinusoidal endothelial cells.

Each microscope with its dedicated sample preparation technique provides the investigator with a specific set of data giving an instrument-determined (or restricted) insight into the structure and function of a tissue, a cell or parts thereof. Stepwise improvements in existing techniques, both instrumental and preparative, can sometimes cross barriers in resolution and image quality. Of course, investigators get really excited when completely new principles of microscopy and imaging are offered in promising new instruments, such as the AFM. The present paper summarizes a first phase of studies on the thin endothelial cells of the liver. It describes the preparation-dependent differences in AFM imaging of these cells after isolation. Special point of interest concerned the dynamics of the fenestrae, thought to filter lipid-carrying particles during their transport from the blood to the liver cells. It also describes the attempts to image the details of these cells when alive in cell cultures. It explains what physical conditions, mainly contributed to the scanning stylus, are thought to play a part in the limitations in imaging these cells. The AFM also offers promising specifications to those interested in cell surface details, such as membrane-associated structures, receptors, coated pits, cellular junctions and molecular aggregations or domains. The AFM also offers nano-manipulation possibilities, strengths and elasticity measurements, force interactions, affinity measurements, stiffness and other physical aspects of membranes and cytoskeleton. The potential for molecular approaches is there. New developments in cantilever construction and computer software promise to bring real time video imaging to the AFM. Home made accessories for the first generation of AFM are now commodities in commercial instruments and make the life of the AFM microscopist easier. Also, the combination of different microscopies, such as AFM and TEM, or AFM and SEM find their way to the market allowing comfortable correlative microscopy.