First-time-in-human study with GSK1018921, a selective GlyT1 inhibitor: relationship between exposure and dizziness.

The pharmacokinetics (PK), safety, and tolerability of GSK1018921, a glycine transporter 1 (GlyT-1) inhibitor, were assessed in this first-time-in-human (FTIH) study. Single oral doses ranging from 0.5 to 280 mg and placebo were administered to 25 healthy subjects in a five-period, two-cohort, crossover study. GSK1018921 showed dose-proportional PK with a terminal half-life of ~17 h. The subjects reported dizziness with a dose-dependent frequency of 22-88% at doses of 70-280 mg. The time course of the dizziness paralleled the PK of the drug, with peak response at 2 h after the dose, consistent with time to maximum plasma concentration (T(max)). The dizziness was resolved by 10-12 h in all subjects. A Markov-chain logistic regression model was implemented in NONMEM to determine the probability of developing dizziness as a function of the plasma concentration of the compound. Frequency, onset (<1 h), and offset (4 h) were well described by the model. Exposure resulting in 80% receptor occupancy is predicted to be well tolerated.

Cold-induced apoptosis in isolated rat hepatocytes: protective role of glutathione.

Liver conservation for transplantation is usually made at 2-4 degrees C. We studied the effect of rewarming to 37 degrees C for up to 3 h of rat hepatocytes kept at 4 degrees C for 20 h, modulating intracellular glutathione (GSH) concentration either with a GSH precursor (N-acetyl-L-cysteine, NAC), or with GSH depleting agents (diethylmaleate and buthionine sulfoximine, DEM/BSO). Untreated hepatocytes showed time-dependent production of reactive oxygen species (ROS), lipid peroxidation, chromatin condensation and membrane blebbing, decrease in GSH concentration, and protein sulfhydryl groups. Fluorochromatization with Propidium Iodide (PI) and Annexin V (AnxV) of cells rewarmed for 1 h caused an increase of AnxV-positive cells without PI staining and any observed lactate dehydrogenase leakage. TUNEL and DNA-laddering tests were negative for all times and treatments, indicating that apoptosis may occur without DNA fragmentation. Cold preservation and rewarming in the presence of NAC induced a significant improvement in the morphology, less oxidative stress and apoptosis. Conversely, DEM/BSO caused a marked deterioration of morphology, increase of oxidative stress and apoptosis. These results suggested that marked changes in GSH status might play a critical role in triggering apoptosis during cold preservation of isolated rat hepatocytes. NAC, added before rewarming, might represent a therapeutic approach for preventing the early events of apoptosis during cold storage.

Promotion of colon cancer metastases in rat liver by fish oil diet is not due to reduced stroma formation.

Recently, it was demonstrated that dietary omega-3 polyunsaturated fatty acids (PUFAs) induce 10-fold more metastases in number and 1000-fold in volume in an animal model of colon cancer metastasis in rat liver. It was observed that tumors of rats on a fish oil diet lacked peritumoral stroma unlike tumors in livers of rats on a low fat diet or a diet containing omega-6 PUFAs. In the present study, only one-third of the tumors in livers of rats on omega-3 PUFA diet contained peritumoral stroma, whereas peritumoral stroma was present in 87% of the tumors in livers of rats on low fat diet. To explain these findings, we tested the hypothesis that fish oil exerts a direct inhibiting effect on the formation of extracellular matrix in tumor stroma as a consequence of blocking transformation of fat storing cells into myofibroblasts. It was found with immunohistochemical analysis of desmin as marker for fat storing cells and alpha-smooth muscle actin as marker for myofibroblasts that numbers of myofibroblasts were higher in tumors containing intratumoral stroma only than in tumors containing both peritumoral and intratumoral stroma. As most of the tumors in fish oil-treated rats contained intratumoral stroma only, this suggests that transformation of fat storing cells into myofibroblasts was highest in tumor stroma of fish oil-treated rats. Therefore, it is unlikely that the lack of stroma around tumors in fish oil-treated rats is due to inhibition of transformation of fat storing cells into myofibroblasts, but lack of peritumoral stroma is rather a consequence of rapid development of tumors in livers of fish oil-treated rats.

Dietary omega-3 polyunsaturated fatty acids promote colon carcinoma metastasis in rat liver.

The effects of omega-3 polyunsaturated fatty acids (PUFAs) and omega-6 PUFAs on the development of experimentally induced colon carcinoma metastasis in rat liver were investigated quantitatively in vivo. Rats were kept on either a low-fat diet or on a fish oil (omega-3 PUFAs) or safflower oil (omega-6 PUFAs) diet for 3 weeks before the administration of colon cancer cells to the portal vein, until they were sacrificed at 1 or 3 weeks after tumor transplantation. At 1 week after transplantation, the fish oil diet had induced 7-fold more metastases (in terms of number and size) than had the low-fat diet, whereas the safflower oil diet had not affected the number and total volume of metastases. At 3 weeks after tumor transplantation, the fish oil diet and the safflower oil diet had induced, respectively, 10- and 4-fold more metastases (number) and over 1000- and 500-fold more metastases (size) than were found in the livers of rats on the low-fat diet. These differences were sex independent. Immunohistochemical analysis revealed that the immune system in the liver (Kupffer cells, pit cells, T cells, newly recruited macrophages, and the activation state of macrophages) did not play a significant role in this diet-dependent outgrowth of tumors. In conclusion, omega-3 and omega-6 PUFAs promote colon cancer metastasis in the liver without down-regulating the immune system. This finding has serious implications for the treatment of cancer patients with fish oil diet to fight cachexia.

Long-lasting antiamnesic effect of a novel anticholinesterase inhibitor (MF268).

In the present study a short (120 min) and long-lasting (360 min) antagonism of scopolamine-induced amnesia in rats was investigated in an eight-arm radial maze, by (3a S, 8a R)-1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethylpyrrolo[2,3-b]indol-5-o l[8-(cis2,6-dimethyl-morpholin-4-yl)octyl]-carbamate L-bitartrate hydrate (MF268), a new cholinesterase inhibitor. Upon completing the training session, the rats were orally administered increasing doses of MF268 (2, 3, 6, 7, and 8 mg/kg) 60 min prior to s.c. injection of scopolamine (0.25 mg/kg). Following a further 60 min the rat was placed in the maze. The reversal of scopolamine-induced impairment was characterized by an inverted U-shaped dose-response curve. A significant reduction in the number of errors, and time taken to complete the maze was observed with a dose of 6 mg/kg. The compound improved memory retention without affecting scopolamine-induced hypermotility. When the same dose was administered 360 min prior to the test a significant reduction in the number of amnesic animals was observed, whereas no cognitive improvement was detected when either 1-Benzil-4-[(5,6-dimethoxy-1-indanon)-2-yl]-methyl piperidine hydrochloride (E2020) (0.25 mg/kg) or tacrine (0.5 mg/kg) were administered 360 min prior to the test. The kinetics of whole-brain cholinesterase confirmed the long-lasting activity for MF268. A clinical relevance for the use of MF268 in AD treatment is suggested.

Heterogeneous suppression of experimentally induced colon cancer metastasis in rat liver lobes by inhibition of extracellular cathepsin B.

Metastatic rat colon cancer cells but not normal rat hepatocytes showed activity of cathepsin B on their plasma membranes. Activity was visualized in living cells with a new fluorogenic substrate, [Z-Arg]2-cresyl violet, and confocal microscopy. When these cancer cells were injected into the portal vein of rats, the animals developed tumors in the liver in a heterogeneous fashion. Three- to four-fold more tumors were found in the small caudate lobe than in the other three large lobes of the liver. Oral treatment with a selective water-soluble inhibitor of extracellular cathepsin B, Mu-Phe-homoPhe-fluoromethylketone, resulted in 60% reduction of the number of tumors and 80% reduction of the volume of tumors in the three large lobes whereas tumor development was not affected in the small caudate lobe. This study supports the conclusions that (a) extracellular cathepsin B plays a crucial but complex role in liver colonisation by rat colon carcinoma cells in vivo, (b) its selective inhibition suppresses tumor growth heterogeneously in the liver and (c) the caudate lobe of the liver is a relatively large risk factor for tumor development.

Three-dimensional reconstruction of colon carcinoma metastases in liver.

Resection of liver metastases in patients with colon cancer increases survival but success depends on removal of all tumour tissue. For this purpose, understanding of spatial relationships between metastases and liver architecture is essential. Because metastatic cancer growth is essentially a three-dimensional (3D) event, we decided to apply 3D reconstruction techniques to study these spatial relationships between metastases and liver structures such as blood vessels, stroma and the liver capsule (Glisson's capsule). Colon carcinoma metastases were experimentally induced in rat liver by injection of colon cancer cells (CC531) into the portal vein. Three weeks later, livers from these animals and control livers were removed and immediately frozen in liquid nitrogen. Thirty-seven to 110 consecutive sections were used for each 3D reconstruction of 26 metastases in eight livers. Contours of different structures were stained by (immuno)histochemical means, traced in each section and stored in a database. From the contour model, a volume model was generated. Among the 26 metastases, seven were found to grow distantly from the liver capsule. They were small and consisted of well-differentiated cancer cells that were totally surrounded by a basement membrane and stroma which was always connected with adjacent blood vessels of a portal tract. The remaining 19 metastases showed a more advanced pattern of development. Infiltration of poorly differentiated colon cancer cells progressed through the stroma at various sites and areas of direct contact between cancer cells and hepatocytes were frequently found. This type of outgrowth of cancer cells was only found when metastases had made contact with the liver capsule. However, some areas in sections of these advanced stages still resembled small metastases. On the basis of these findings, we conclude that stroma-affects the differentiation pattern of cancer cells and has at least a dual role in tumour growth. On the one hand it limits invasion of cancer cells in the surrounding host tissue. On the other hand, stroma formation at the capsule, which consists mainly of granulation tissue, facilitates outgrowth of the tumours. Furthermore, our 3D reconstructions demonstrate the spatial heterogeneity of larger metastases and the importance of a 3D approach to understand growth and development of metastases in general and colon cancer metastases in the liver in particular.

Tumor interstitial fluid: misconsidered component of the internal milieu of a solid tumor.

The tumor interstitial fluid (TIF) is a fluid phase present in the extracellular space of all tumors whose importance in oncology is seldom recognized. In order to stimulate other researchers to give it the due importance, a review of the available data (including our own) is provided. An hypothesis is presented for the genesis, fate and role of the TIF in the processes of invasion, growth and metastatization. Open questions regarding the TIF's role in tumor response to therapy are raised.

Kupffer cells and pit cells are not effective in the defense against experimentally induced colon carcinoma metastasis in rat liver.

The present study was performed to investigate processes involved in circumvention of the immune system by advanced stages of tumor growth in the liver. The efficacy of Kupffer cells and pit cells against cancer cells was tested in vivo in an experimental model of colon carcinoma metastasis in rat liver. Liver tumors were induced by administration of CC531 colon cancer cells into the vena portae. After 3 weeks, livers were obtained and partly fixed for electron microscopic procedures or frozen in liquid nitrogen for enzyme and immunohistochemistry at the light microscope level. The activation status of Kupffer cells was studied by expression of Ia-antigen (MHC class II) and by measurement of glucose-6-phosphate dehydrogenase (G6PDH) activity in the cells in situ as a measure of production of reactive oxygen species. Large numbers of Kupffer cells were found in liver parenchyma surrounding colon carcinomas when compared with levels in control livers, but these cells were not activated. Large numbers of activated monocytes and macrophages, cytotoxic T cells but only a few pit cells were found to be recruited to the boundary between liver parenchyma and tumors or their stroma. In those areas where cancer cells invaded liver parenchyma, only newly recruited macrophages and some Kupffer cells were present but few cytotoxic T cells or pit cells were found. The low activation status of Kupffer cells both in terms of production of reactive oxygen species and Ia-antigen expression and the absence of significant numbers of pit cells at tumor sites suggest that Kupffer cells and pit cells do not play a significant role in advanced stages of tumor growth. High levels of prostaglandin E2 were detected in the parenchyma of livers containing tumors and transforming growth factor beta was detected in the stroma of the tumors, therefore suggest that cytotoxicity of newly recruited monocytes, macrophages and cytotoxic T cells may be limited in these stages because of local production of these immunosuppressive factors.

Characterization of the metabolism of perinecrotic cells in solid tumors by enzyme histochemistry.

Hypoxic tumor cells resist most therapies and cause tumor regrowth when their environment improves. Identifying the adaptation strategies to hypoxia would help develop better tailored cancer therapies. Ehrlich carcinomas implanted on mice were analyzed histochemically for the following enzyme activities: lactate, succinate and glucose-6-phosphate dehydrogenases, dihydrofolate reductase, purine nucleoside phosphorylase, xanthine oxidoreductase, and acid phosphatase. With the exception of xanthine oxidoreductase, which was not active in tumor cells, and of succinate dehydrogenase the activity of which was not significatively altered, all other activities were much higher in perinecrotic cells with respect to cells close to blood vessels. These data suggest the integration of metabolic paths allowing purine and lipid biosyntheses. Degradation products from the necrosis are presumed to be employed as surrogates of blood-borne nutritive substances by cells distant from the vascularization.

An inverted U-shaped curve for heptylphysostigmine on radial maze performance in rats: comparison with other cholinesterase inhibitors.

The potential of heptylphysostigmine tartrate (pyrrolo [2,3b] indol-5-ol, 3,3a,8,8a-hexahydro-1,3a,8-trimethylheptylcarbamate [ester, (3aS-cis)]) (MF201), a new second-generation cholinesterase inhibitor, to antagonize scopolamine-induced amnesia in rats was assessed in an 8-arm radial maze. Upon completing the training session, the rats were orally administered increasing doses of MF201 (2, 3, 4, 6 and 8 mg/kg) 60 min prior to a s.c. injection of scopolamine (0.25 mg/kg). 9-Amino-1,2,3,4-tetrahydroamino-acridine hydrochloride hydrate (tacrine) (0.25, 0.37, 0.5, 1 and 2 mg/kg), 1-benzil-4-[(5,6-dimethoxy-1-indanon)-2-yl]-methyl piperidine (E2020) (0.125, 0.18, 0.25 and 0.5 mg/kg) and physostigmine (0.15, 0.25, 0.5 and 1 mg/kg) were orally administered and rats were tested in the same task. As previously described, scopolamine induced an impairment in radial maze performance, measured in terms of total number of errors, total time taken to complete the task and the percentage of amnesic animals. The reversal of scopolamine-induced impairment was characterized by the presence of an inverted U-shaped dose-response curve. A significant antagonistic effect was achieved with a dose (mg/kg) of 0.25 for E2020, 0.5 for tacrine and physostigmine and 3, 4 and 6 for MF201, the latter manifesting a broader spectrum of activity (3-6 mg/kg). While the maximal active doses restored the scopolamine-induced modified pattern of arm entry, they were ineffective in reducing hypermotility, suggesting the drugs have a specific effect on cognitive function.

alpha2-Macroglobulin is mainly produced by cancer cells and not by hepatocytes in rats with colon carcinoma metastases in liver.

Localization and production of alpha2-macroglobulin (alpha2M), a multifunctional binding protein with protease and cytokine scavenging properties, was studied in situ in rat livers containing experimentally induced colon carcinoma metastases by means of immunocytochemistry and in situ hybridization methods. The study was performed to investigate whether alpha2M production by hepatocytes plays a role in the defense against the growth of metastases on the basis of its protease inhibiting capacity. It was found that colon cancer cells in all developmental stages of the metastases contained large amounts of messenger RNA (mRNA) of alpha2M but hardly any alpha2M protein. Cancer cells in culture contained large amounts of both mRNA and protein of alpha2M. In contrast, stromal cells and liver cells did not show positivity for alpha2M mRNA above background levels. The exception was a few layers of hepatocytes around the latest stage of metastases. Hepatocytes contained both alpha2M mRNA and protein only when Kupffer cells were present, indicating that alpha2M mRNA production was induced via Kupffer cells. On the other hand, alpha2M protein was found in high amounts in the sinusoids and stroma of all metastases, irrespective of their developmental stage. Increased levels of alpha2M could not be detected in serum in all but one rat tested (n=8). It is concluded that production of alpha2M by hepatocytes occurs only around the latest developmental stage of metastases and that alpha2M does not play a significant role in the defense against metastatic cancer growth in rat liver. In contrast, cancer cells produce and secrete large amounts of alpha2M, which seems to be linked with their tumorigenicity. We suggest that this alpha2M captures cytokines rather than proteases by complex formation. These complexes were observed using immunocytochemical staining for alpha2M protein indicating that it was captured by either stromal cells, sinusoidal cells, or hepatocytes that are in direct contact with cancer cells, Therefore, changes in serum levels of alpha2M were limited, indicating that these levels do not reflect local production and effects of alpha2M.

Enzyme histochemical studies on tumor blood vessels.

The oxygenation, the growth rate and the metastatic potential of a solid tumor depend on its vascularization and, in particular, on angiogenesis; a therapeutic approach affecting angiogenesis has been suggested as an alternative to conventional ones. Especially the study of the metabolism in the cells of the vessel wall should be a useful prerequisite for this approach. In this connection, an enzyme histochemical study was performed to characterize the blood vessels in a solid tumor (Ehrlich carcinoma). The following enzymes were considered: (a) alkaline phosphatase, involved in the transcellular phosphate transport and in the response to inflammatory and growth promoting factors; (b) dihydrofolate reductase, involved in the metabolism of tetrahydrofolate (for the synthesis of nucleic acids and the metabolism of serine and glycine); (c) purine nucleoside phosphorylase, involved in the degradation of purines and, in particular, of extracellular ATP and ADP; (d) xanthine oxidoreductase, engaged in the same degradation path and leading to the formation of urate, a strong antioxidant. Various patterns of enzyme activities were observed in the vessel wall. In particular, thin linear capillaries (presumed to be host capillaries penetrating the tumor) were identified for the intense positivity of alkaline phosphatase, dihydrofolate reductase and purine nucleoside phosphorilase; tortuous capillaries with variable diameters (presumed to be induced by angiogenesis from the host vessels) were negative for the alkaline phosphatase and expressed an heterogeneous pattern for the dihydrofolate reductase. All the data suggest a different vessel behaviour concerning the response to cytokines and to inflammatory stimuli.

Changes in the zonation of lactate dehydrogenase activity in lobules of rat liver after experimentally induced colon carcinoma metastases.

Visualization of lactate dehydrogenase (LDH) activity with Neotetrazolium as final electron acceptor under anaerobic conditions and an incubation medium containing polyvinyl alcohol showed that under normal physiological conditions a zonal distribution of LDH activity is present in the liver lobule of male rats. Periportal hepatocytes contain more LDH activity than pericentral hepatocytes. This difference is due to the role of LDH both in gluconeogenesis (periportal cells) and glycolysis (pericentral cells). In livers containing metastases from colon carcinoma, areas of the parenchyma which are not affected by tumour growth maintain such zonation in the lobule, whereas areas close to metastatic foci show increased activity which is distributed uniformly over the lobule. This change may be explained by a Cori's cycle-like relationship between malignant cells and the surrounding hepatocytes due to glucose consumption and lactate production by the tumour cells. Within the metastatic foci, a zonation of LDH activity was also observed. Malignant cells close to the edge of the tumours contained the lowest activity, whereas activity increased inwards. Cancer cells directly surrounding necrotic areas showed the highest activity. Such patterns are in line with increasing anaerobic glycolysis towards the inner metastatic regions. Anaerobic glycolysis supplies limited amounts of ATP with concomitant lactate production but also large amounts of metabolites for RNA, DNA, lipid and complex carbohydrate synthesis. Lactate that is produced by the metastases induces adaptive changes in surrounding hepatocytes to convert this excess of lactate effectively.

The histochemical G6PDH reaction but not the LDH reaction with neotetrazolium is suitable for the oxygen sensitivity test to detect cancer cells.

We used the oxygen sensitivity of the histochemical reaction to detect glucose-6-phosphate dehydrogenase (G6PDH) activity based on neotetrazolium (NT) reduction to discriminate cancer cells from normal cells. Formazan generation was strongly reduced in normal but not in malignant cells when the incubation was performed in oxygen instead of nitrogen. Competition for reductive equivalents between NT and oxygen via superoxide dismutase (SOD) has been suggested. Since SOD activity is usually decreased in cancer cells, NT reduction would not be hampered in these cells. We tested this hypothesis by demonstrating NAD-dependent lactate dehydrogenase (LDH) activity instead of NADP-dependent G6PDH activity in normal rat liver and colon, in human colon carcinoma, and in experimentally induced metastases of colon carcinoma in rat livers. Reactions for both enzymes were determined cytophotometrically in an atmosphere of pure oxygen or nitrogen. G6PDH acted as described previously, showing distinct activity in cancer cells but strongly reduced activity in normal cells after incubation in oxygen, but this was not the case with LDH because formazan was also generated in normal tissue in oxygen. It appeared that after 5 min of incubation at 37 degrees C the residual activity of G6PDH in an atmosphere of oxygen compared with nitrogen was 0% in normal liver tissue and 15% in normal colon epithelium, whereas in colon carcinoma and in colon carcinoma metastasis in liver it was 48% and 33%, respectively. The residual activity of LDH in oxygen was 30% in normal female rat liver, 75% in normal male rat liver, and 38% in normal colon epithelium, whereas the residual activity in colon carcinoma and metastases in liver was 54% and 24%, respectively. These experiments clearly indicate that the oxygen sensitivity phenomenon is not solely an effect of competition for reducing equivalents between NT and oxygen via SOD, because NADPH generated by G6PDH and NADH generated by LDH have a similar redox potential. Apparently the system is more complex. The role of specifically NADPH-converting cellular systems such as NADPH-cytochrome P450 reductase was excluded because incubations in the presence of exogenous NADPH as substrate for these systems revealed oxygen sensitivity. Involvement of NADPH-dependent lipid peroxidation in the oxygen sensitivity test is discussed.

Quantitative comparison between the gel-film and polyvinyl alcohol methods for dehydrogenase histochemistry reveals different intercellular distribution patterns of glucose-6-phosphate and lactate dehydrogenases in mouse liver.

The precise histochemical localization and quantification of the activity of soluble dehydrogenases in unfixed cryostat sections requires the use of tissue protectants. In this study, two protectants, polyvinyl alcohol (PVA) and agarose gel, were compared for assaying the activity of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) in normal female mouse liver. Quantification of enzyme activity was determined cytophotometrically in periportal (PP), pericentral (PC) and midzonal (MZ) areas. No coloured reaction product was present in PVA media after the incubation period. In contrast, the agarose gels appeared to be highly coloured after incubation. As a consequence, sections incubated with gel media were less intensely stained than those incubated in PVA-containing media. The specific G6PDH reaction (test minus control) yielded approximately 75% less formazan in sections incubated by the agarose gel method than with the PVA method. Further, the amount of formazan deposits attributable to G6PDH activity was highest in the midzonal and pericentral zones of the liver lobule with PVA media, and Kupffer cells could be discriminated easily because of their high G6PDH activity. Significant zonal differences or Kupffer cells could not be observed when agarose gel films were used for the detection of G6PDH activity. The LDH localization patterns appeared to be more uniform after incubation with both methods: no significant differences in specific test minus control reactions were seen between PP, PC and MZ. However, less formazan production (33%) was detected in sections incubated with agarose gels when compared with those incubated with PVA media.(ABSTRACT TRUNCATED AT 250 WORDS)

Omega-3 fatty acids can improve radioresponse modifying tumor interstitial pressure, blood rheology and membrane peroxidability.

Several studies provide evidence that hypoxic cells present in animal and human solid tumors, may be critical for the successful treatment of cancer. In particular hypoxic cells are resistant to ionizing radiation, photodynamic treatment and the large majority of chemotherapeutic drugs. Hypoxia is generally due to the inadequacy of vascular beds supporting the tumor and to an abnormal microcirculation. Three parameters, tumor interstitial fluid, hemorheological factors and lipoperoxidation, are considered and tentatively associated as playing a role in hypoxic cell treatment. Omega three fatty acids modify these factors and are discussed for their possible ability to enhance tumor cells susceptibility to radiotherapy.

Tumor angiogenesis: evidence of new blood channels from plasma infiltrations.

Stroma formation in Ehrlich carcinoma, studied with histochemical and TEM techniques, is similar to wound healing. In this tumour mast cells, macrophages, adipocytes, platelets and fibrin seem to co-operate locally with malignant cells in regulating stroma formation. The gaps opened in the tumor parenchyma by plasma outpouring from local blood vessels seem to offer easy routes for endothelial cell migration towards ill-nourished areas, and may explain the irregular aspect of tumor microvascularity.

In situ lactate dehydrogenase patterns as markers of tumour oxygenation.

The histochemical patterns of lactate dehydrogenase, LDH, are here proposed as indicators of the local levels of oxygenation of malignant tissue. This parameter has outstanding importance in determining the tumour aggressiveness and response to treatment. The tetrazolium salt reaction previously proposed for the mapping of hypoxia has been improved by the use of polyvinyl alcohol as a tissue stabilizer. The intracellular coloured products of this reaction appear in two distinct forms, diffuse and granular, which we previously postulated to be indicative of LDH isoenzymes soluble and bound, respectively. Solubility is promoted by H-LDH subunits preferentially synthesized under good oxygenation; binding to membranes is favoured by the presence of M-LDH subunits preferentially active under poor oxygeneration. A reversible shift between the two forms apparently regulates the cells' metabolic adaptation to different stress situations. We assume that the anoxic shock protein LDHk exists exclusively in the bound form. In the Ehrlich carcinoma model previously employed, we verify a drift towards the exclusive presence of the granular form as the section's depth increases and/or when the cuff width decreases. This trend is ascribed to a progressive worsening of the local oxygenation levels. At the tumour interface, a chronic inflammatory tissue (notoriously highly hypoxic) is characterized by a granular LDH activity. New models of hypoxia are proposed and discussed for explaining the patterns here described and observed also in other studies, namely those derived from hyperviscosaemia, damaged endothelia, fibrosis, anaemia, poor ventilation and impaired cardio-vascular system.

Stroma formation in Ehrlich carcinoma. I. Oedema phase. A mitosis burst as an index of physiological reoxygenation?

Tumor stroma induction has been shown closely to resemble wound repair process, both involving the replacement of a fibrin gel by vascularized connective tissue. In such a process the initial phase of hyperpermeability of blood vessels leads to diffuse oedema. It is here reported that cell loosening and a remarkably high mitotic burst were observed in Ehrlich carcinoma in regions in contact with the exudate, particularly at the perinecrotic (hypoxic) region. This suggests both an enhanced cell detachment from the tumour parenchyma and an improvement of the microenvironment, the exudate thus appearing as beneficial to the malignant cells contributing to the reoxygenation of formerly hypoxic regions. The temporary and well-localized concentration of mitoses in inner tumour areas has perhaps been disregarded by the pathologists engaged in mitosis counting for tumour grading. Peripheric and intraparenchymal concentrations of mast cells, lipid pools and platelets were seen in apparently key geometric disposition for controlling fibrin deposition and angiogenesis. Hypoxia is known to cause resistance to oxygen-dependent treatments and to facilitate cell detachment; normal fibroblasts respond and survive under hypoxic conditions by exhibiting features of the malignant phenotype. During reoxygenation, gene instability, cellular heterogeneity and increased drug resistance and metastatic spread have been reported. A reoxygenation process can also be deduced from several other histochemical and morphological patterns observed in this study. The findings here reported thus suggest that the oedema phase is a crucial phase regulating growth, invasion and dissemination of tumour cell populations, that should be specifically addressed therapeutically.