Accumulation of adoptively transferred adherent, lymphokine-activated killer cells in murine metastases.

While close contact between lymphokine-activated killer (LAK)/adherent, lymphokine-activated killer (A-LAK) cells and tumor cells is believed to be a prerequisite for initiating the events leading to tumor cell lysis, clear evidence for the ability of these effector cells to infiltrate tumors or tumor metastases in vivo still has to be obtained. In the present study, we report that a significant fraction of adoptively transferred A-LAK cells, labeled with fluorochromes for identification, accumulates in lung and liver metastases of the B16 melanoma, the MCA 102 sarcoma and the Lewis lung carcinoma lines. Thus, 5- to 10-fold higher numbers of A-LAK cells were found in the malignant lesions compared to the surrounding normal tissue. The infiltration seemed very heterogeneous after intravenous injection of moderate numbers of A-LAK cells (15 x 10(6)). However, after adoptive transfer of 45 million A-LAK cells, an A-LAK cell/tumor cell ratio higher than 1:1 in most metastases was observed. Surprisingly, approximately 5% of the lung metastases seemed totally resistant to infiltration even though neighboring metastases were highly infiltrated. While substantial infiltration of lung metastases was seen after i.v. injection, significant infiltration of liver metastases was seen only after intraportal injection of the A-LAK cells indicating impaired traffic of intravenous injected A-LAK cells through the lung capillaries. These results present direct evidence that A-LAK cells, upon a proper route of administration, have the potential to migrate to and heavily infiltrate metastases from murine tumors of different origin.

Pericyte loss and microaneurysm formation in PDGF-B-deficient mice.

Platelet-derived growth factor (PDGF)-B-deficient mouse embryos were found to lack microvascular pericytes, which normally form part of the capillary wall, and they developed numerous capillary microaneurysms that ruptured at late gestation. Endothelial cells of the sprouting capillaries in the mutant mice appeared to be unable to attract PDGF-Rbeta-positive pericyte progenitor cells. Pericytes may contribute to the mechanical stability of the capillary wall. Comparisons made between PDGF null mouse phenotypes suggest a general role for PDGFs in the development of myofibroblasts.

The direction of human mesenchymal stem cells into the chondrogenic lineage is influenced by the features of hydrogel carriers.

Low back pain is a major public health issue in the Western world, one main cause is believed to be intervertebral disc (IVD) degeneration. To halt/diminish IVD degeneration, cell therapy using different biomaterials e.g. hydrogels as cell carriers has been suggested. In this study, two different hydrogels were examined (in vitro) as potential cell carriers for human mesenchymal stem cells (hMSCs) intended for IVD transplantation. The aim was to investigate cell-survival and chondrogenic differentiation of hMSCs when cultured in hydrogels Puramatrix® or Hydromatrix® and potential effects of stimulation with growth hormone (GH). hMSCs/hydrogel cultures were investigated for cell-viability, attachment, gene expression of chondrogenic markers SOX9, COL2A1, ACAN and accumulation of extracellular matrix (ECM). In both hydrogel types, hMSCs were viable for 28days, expressed integrin ß1 which indicates adhesion of hMSCs. Differentiation was observed into chondrocyte-like cells, in a higher extent in hMSCs/Hydromatrix® cultures when compared to hMSCs/Puramatrix® hydrogel cultures. Gene expression analyses of chondrogenic markers verified results. hMSCs/hydrogel cultures stimulated with GH displayed no significant effects on chondrogenesis. In conclusion, both hydrogels, especially Hydromatrix® was demonstrated as a promising cell carrier in vitro for hMSCs, when directed into chondrogenesis. This knowledge could be useful in biological approaches for regeneration of degenerated human IVDs.

Establishment of cell-to-cell contact by adoptively transferred adherent lymphokine-activated killer cells with metastatic murine melanoma cells.

A murine model of pulmonary B16 melanoma was used to study the infiltration into metastases of lymphokine-activated killer (LAK) cells and adherent lymphokine-activated killer (A-LAK) cells and, specifically, to study whether A-LAK cells are able to leave the tumor microcirculation and establish cell-to-cell contact with malignant cells. Fluorescence microscopy demonstrated that A-LAK cells accumulated in metastases twice as efficiently as LAK cells during interleukin-2 stimulation. Electron microscopy of pulmonary metastases 16 hours after administration of 2.5 x 10(7) A-LAK cells revealed A-LAK cells, identified by the presence of typical two-compartment granules, in direct contact with melanoma cells. This finding was confirmed by using A-LAK cells prelabeled with polycationized ferritin. In conclusion, our observations demonstrate unambiguously the ability of adoptively transferred A-LAK cells to establish contact with extravascular metastatic melanoma cells.

Microvessel origin and distribution in pulmonary metastases of B16 melanoma: implication for adoptive immunotherapy.

To elucidate the role of tumor vascularization on the localization of adoptively transferred, interleukin 2-activated natural killer (A-NK) cells, pulmonary B16 melanoma metastases were analyzed with respect to location, morphological appearance, origin and density of microvessels, and infiltration by A-NK cells. The B16 melanoma metastases could be divided into four subtypes according to their location (superficial or deep in the lung parenchyma) and morphological appearance (compact or loose). Localization of adoptively transferred A-NK cells into the four subtypes of B16 pulmonary metastases differed significantly. More than 800 A-NK cells/mm2 were found in metastases of the deep-loose type, compared to approximately 400/mm2 A-NK cells in the superficial-loose metastases, and less than 200 A-NK cells/mm2 in the compact subtype, regardless of its location (deep or superficial). Although the origin (pulmonary or bronchial) of the blood supply to the metastatic subtypes (as revealed by electron microscopic analyses of lungs perfused with a lanthanum solution) did not account for this difference, the density of microvessels in the metastatic subtypes correlated with the number of A-NK cells that localized into these metastases. The resistance of metastases of the compact type to infiltration of adoptively transferred effector cells might explain, in part, why adoptive immunotherapy seldom results in complete eradication of disseminated cancer.

The GTPase activity of the Escherichia coli Ffh protein is important for normal growth.

The Escherichia coli (E. coli) Ffh protein is homologous to the 54kDa subunit of the eukaryotic signal recognition particle. We have examined an intrinsic GTPase activity of this protein and have created mutations in one sequence motif (GXXXXGK) of the putative GTP binding site. When glycine-112 was changed to valine (Ffh-G112V), Vmax was reduced to only 4% of the wildtype level. On the other hand, when glutamine-109 was altered to glycine (Ffh-Q109G), the major effect was a 50-fold increase in Km. These results show that the residues Q-109 and G-112 are essential for the binding and hydrolysis of GTP and that they are part of a catalytic site structurally related to that of many other GTPase proteins. Expression of the mutant protein Ffh-G112V in E. coli was highly toxic in the presence of the wildtype protein. In contrast, genetic complementation experiments showed that a viable strain could be constructed where the Ffh-Q109G mutant protein replaced wildtype Ffh. However, expression of the mutant protein had a negative effect on growth rate at 30 degrees C and resulted in elongated cells. These results demonstrate that the GTPase activity of the Ffh protein is required for proper function of the protein in vivo.

Immunocytochemical localization of multicatalytic protease complex (proteasome) during generation of murine IL-2-activated natural killer (A-NK) cells.

Murine IL-2-activated, adherent natural killer (A-NK) cells produce proteolytic activities (including a chymase and a tryptase) which appear to be components of the proteasome/multicatalytic proteinase complex and appear to represent the mouse homologues of the rat A-NK cell A-NKP 2 and A-NKP 1 protease components. The chymase is readily inhibited by Z-Gly-Gly-Phe chloromethylketone (Z-GGF) and to a lesser extent by N-tosyl-L-phenylalanyl-chloromethylketone (TPCK). In addition, this activity is inhibited by 3,4-dichloroisocoumarin (DCI), a suicide inhibitor for both chymotryptic and tryptic proteolytic enzymes. Protease inhibitors reduced A-NK cell-mediated cytotoxicity against P815 target cells, most prominently with inhibitors of chymotryptic and tryptic enzymes, including TPCK, DCI and Z-GGF. A polyclonal rabbit antibody raised against rat liver proteasome immunofluorescently labeled the cytoplasm of 4-day-cultured murine A-NK cells, multiple granules in 5 to 6-day cultures and large intracytoplasmic pools in cells cultured longer. Ultrastructurally this shift in labeling over time corresponded to an immunogold redistribution to non-membrane delineated mucoid masses. A minor nuclear labeling was noted at all time points, whereas the cisternae of the endoplasmic reticulum or Golgi region were negative. It is concluded that murine A-NK cells synthesize and accumulate proteasome in large intracytoplasmic pools, non-delineated by membranes which can occupy up to 80% of the A-NK cellular volume. The potential function of the proteasome produced by A-NK cells including cell-mediated cytotoxicity against tumor target cells remains to be elucidated.

Leptin stimulates the activity of the system A amino acid transporter in human placental villous fragments.

The activity and expression of placental nutrient transporters are primary determinants for the supply of nutrients to the fetus, and these nutrients in turn regulate fetal growth. We developed an experimental system to assess amino acid uptake in single primary villous fragments to study hormonal regulation of the amino acid transporter system A in term human placenta. Validation of the method, using electron microscopy and studies of hormone production, indicated that fragments maintained ultrastructural and functional integrity for at least 3 h. The activity of system A was measured as the Na(+)-dependent uptake of methylaminoisobutyric acid (MeAIB), and the effect of 1 h incubation in various hormones was investigated. Uptake of MeAIB into villous fragments in the presence of Na(+) was linear up to at least 30 min. Insulin (300 ng/ml, n = 14) increased system A activity by 56% (P < 0.05). This effect was also present at insulin concentrations in the physiological range (+47% at 0.6 ng/ml, n = 10, P < 0.05). Leptin (500 ng/ml, n = 14) increased Na(+)-dependent MeAIB uptake by 37% (P < 0.05). System A activity increased in a concentration-dependent fashion in response to leptin (n = 10). However, neither epidermal GF (600 ng/ml), cortisol (340 ng/ml), nor GH (500 ng/ml) altered system A activity significantly (n = 14). We conclude that primary single isolated villous fragments can be used in studies of hormonal regulation of nutrient uptake into the syncytiotrophoblast. These data suggest that leptin regulates system A, a key amino acid transporter.

Inhibition of rat mast cell protease 1 by vitronectin.

Rat mast cell protease 1 (RMCP-1) is a chymotrypsin-like serine protease specifically expressed by connective tissue-type mast cells. The enzyme is stored in the secretory granules in a macromolecular complex with heparin proteoglycan. In the present investigation it was shown that RMCP-1 is inhibited by vitronectin (VN), an RGD-containing adhesive glycoprotein with heparin-binding properties. RMCP-1 that had been separated from heparin proteoglycan was less susceptible to inhibition than RMCP-1 present in complex with heparin proteoglycan. Pre-incubation of VN with purified heparin partially blocked the RMCP-1 inhibiting activity of VN. Plasma VN had negligible RMCP-1-inhibiting activity. However, heat treatment of plasma VN, which is known to expose the heparin-binding domain, induced RMCP-1-inhibiting activity. Affinity chromatography on immobilized VN showed that RMCP-1 bound with high affinity to VN. The binding of RMCP-1 to VN was not heparin-dependent since free RMCP-1 bound with equal affinity to the immobilized VN as RMCP-1 present in complex with heparin. The inhibition of RMCP-1 by VN was shown to be reversible.

A comparison between morphological, rheological and lodgement properties of rat fibrosarcoma cells harvested from solid tumours and cultures.

Vital microscopic studies on tumour cell lodgement in the rat liver have suggested that tumour cells from culture (CTCs) and from solid tumour (STCs) have different rheological properties. In the present study CTCs and STCs from a rat fibrosarcoma were compared in respect to their morphology, rheology and lodgement properties. The ultrastructural examination showed that the CTCs had a larger mean diameter (14.9 microns) than the STCs (11.2 microns). Both cell types had a very irregular nucleus. Surface irregularities provide the CTCs and STCs with a 'membrane excess', i.e. a larger plasmalemma than required to enclose the cells as smooth spheres, amounting to 46.8% and 60.9%, respectively. These values indicate that both CTCs and STCs can be substantially deformed with preservation of the volume and area. The CTCs were stiffer than the STCs when deformed at constant pressure in 6.5 microns glass pipettes, the nucleus appearing to be a significant hindrance to CTC deformation. Five minutes after intraportal injection of radiolabelled CTCs and STCs a much higher percentage of CTCs (82%) than of STCs (20%) remained lodged in the rat liver. The results indicate that the propagation in culture of fibrosarcoma cells alters the morphology and rheology of the cells such that CTCs are not suited for studies in vivo where the spontaneous intravascular dissemination and organ lodgement of tumour cells is simulated.

Melanoma cell destruction in the microvasculature of perfused hearts is reduced by pretreatment with vitamin E.

Different mechanisms have been proposed to explain the rapid elimination of circulating malignant cells: interactions with circulating leukocytes, mechanical trauma induced by deformation, shear forces and tissue pressure variations. Based on earlier observations in an isolated heart perfusion model the present study was performed to test whether or not microvascular damage of malignant cells depends on their anti-oxidant status. Murine melanoma B16F10 cells, pretreated with 100 microM alpha-tocopherol (or solvent) for 48 h, were used. The cells were perfused into the coronary vasculature of isolated hearts from C57/BL6 mice. Passing cells were collected and their viability determined by Trypan Blue exclusion. The hearts were processed for electron microscopy and the frequency of ultrastructurally intact and damaged B16 cells trapped in capillaries was recorded. In filter perfusion experiments the effect of vitamin E pretreatment on the resistance of the melanoma cells to mechanical deformation was determined. Morphometrically, cell size and cell profile perimeter excess of the melanoma cells were computed. Vitamin E pretreatment increased perfused cell viability from 50% to 81%. Ultrastructurally 30% of the intracapillary vitamin E treated cells were damaged (plasmalemmal fragmentation or worse) as compared to 58% of control cells. These differences were statistically significant (P < 0.01) whereas no differences could be demonstrated in filterability, cell size, or cell surface excess. The data support the hypothesis that malignant cell destruction in the systemic microcirculation is at least partly dependent on an oxygen metabolite mediated process, the exact nature (e.g. superoxide, hydrogen peroxide, nitric oxide) of which remains to be determined.

Calcium depletion reduces the destruction of fibrosarcoma cells in the microvasculature of artificially perfused rat hearts.

Earlier studies have shown that most tumour cells (TCs) injected into the circulation die rapidly. The mechanisms behind this rapid elimination of TCs are not known, but some experimental data suggest that mechanical trauma to the cells in the capillary bed plays a role. In the present investigation 725,000 rat fibrosarcoma cells (250,000/ml) were infused into the coronary microcirculation of isolated and artificially perfused (flow rate approx. 6 ml/min), beating and non-beating (Ca2+ excluded from the perfusate) rat hearts. The analyses included calculations of the number of TCs retained in the myocardium 5 min after start of TC infusion and their distribution within the ventricular wall. In addition, ultrastructural studies were performed to identify possible structural changes of trapped TCs and myocardial tissue. Intact and viable TCs in the effluent perfusate were counted. In beating hearts about 20% of the infused TCs were collected microscopically intact after passage through the heart circulation, and of these cells only 32% were viable (in-flow viability 87-91%). In Ca(2+)-depleted hearts the corresponding figures were 29 and 48%, respectively. The difference in viable cell counts was statistically significant (P less than 0.001). The TCs trapped in the left ventricular wall of the myocardium of beating hearts were mainly found in the subepicardial third of the wall, whereas in non-beating hearts the trapped cells were distributed randomly. The ultrastructural appearance of trapped cells differed between the two groups: 82% of cells trapped in beating hearts showed signs of severe damage, with subcellular vacuolization and plasmalemmal disruption, whereas 65% of cells trapped in Ca(2+)-depleted hearts seemed undamaged with intact cell membranes and cytoplasmic organization. The remaining 35% showed variable subcellular disorganization. The results cannot entirely be explained by mechanical TC damage in the microcirculation due to intracapillary deformation. The observations require additional explanatory mechanisms. One possible important TC damaging mechanism, dependent on extracellular Ca2+ levels, could be endothelial cell release of reactive oxygen species.

Migration of IL-2-activated natural killer cells in vitro: influence of extracellular matrix proteins.

An experimental set-up for estimating a) cellular migration under agarose and b) response to chemoattractant gradients built up in the agarose was used in order to explore the behavior of adherent interleukin-2 (IL-2)-activated natural killer (A-NK) cells on cell culture plastic and after coating with extracellular matrix (ECM) constituents. A-NK cells were deposited in wells in the agarose and directed migration, chemotaxis, towards aggregates and suspensions of B16F10 melanoma cells, suspensions of YAC-1 cells, and tumor-conditioned media, all deposited in wells at a 2.5 mm distance, was tested. A-NK cell chemotaxis was exclusively observed when B16F10 aggregates were used as attractants. The substrate influenced chemotaxis considerably, untreated plastic surface being most favorable for a chemotactic response, followed by laminin, fibronectin, and collagen IV pretreatments. Coating with reconstituted basement membrane matrix (Matrigel) gave lesser random movements, chemokinesis, of A-NK cells than coating with the purified components laminin and collagen IV, and the least motile response was obtained after collagen I pretreatment. These in vitro observations indicate that melanoma cell aggregates release humoral factors of a probably short-lived nature with a chemoattractant effect on A-NK cells, and that ECM composition influences migratory response, both conclusions with a bearing on the understanding of A-NK cell infiltration into tumors in vivo.

Method for immunolocalization of extracellular proteins in association with the implant-soft tissue interface.

A method allowing immunolocalization of macromolecules surrounding biomaterial implants has been developed. Plugs of pure titanium were inserted into the abdominal wall of rats. One week after implantation plugs with adjacent tissue were gently fixed and cryoprotected before being rapidly frozen in LN2-cooled propane, followed by cryosubstitution, using methanol, and low temperature infiltration and UV curing of the methacrylate LR-Gold. Before sectioning, bulk metal was removed electrochemically, leaving the surface oxide intact, appearing in sections as a dense line in contact with the tissue. Postembedding immunolocalization for rat albumin, IgG, C3c, fibrinogen and fibronectin was performed at the light and electron microscopic levels. Our observations indicate that 1 wk after implant insertion, a network of fibrin and fibronectin decorates the tissue surface that faces the implant, but without any obvious concentration to the implant surface.

Immunohistochemical studies on the distribution of albumin, fibrinogen, fibronectin, IgG and collagen around PTFE and titanium implants.

Time-dependent distribution of extracellular proteins (albumin, fibrinogen, fibronectin, collagen-I and IgG) in the interface zone between implant and soft tissue has been investigated utilizing a recently developed method. Commercially pure (c.p.) titanium and polytetrafluoroethylene (PTFE) implants were inserted in the abdominal wall of rats for 1, 6 and 12 weeks followed by a mild fixation, cryoprotection, rapid freezing in LN2-cooled propane, cryosubstitution and low-temperature infiltration with UV curing of the methacrylate LR-Gold. Before sectioning, the bulk part of the titanium was removed by an electrolytical dissolution technique (electropolishing), while the PTFE implants were removed by a fracture technique. Employing a cryosubstitution method combined with postembedding immunohistochemistry, a light microscopic analysis was allowed. The selected proteins had an apparently varying distribution in the implant-close tissue and their distribution changed during the follow-up period. There was also a difference in the distribution pattern for each protein around titanium and PTFE implants. Insertion of the c.p. titanium implants elicited an inflammatory reaction in many respects similar to a normal wound healing response, while the PTFE implants caused a more pronounced, persistent inflammation.

The morphology, deformability and microvascular arrest of rat fibrosarcoma and adenocarcinoma cells.

The deformation and flow properties of tumour cells may play a role in their arrest in the microvasculature of different organs. In the present investigation the morphology, deformability and microvascular arrest in the liver of rat fibrosarcoma cells (FSCs) and adenocarcinoma cells (ACCs) were compared using electron microscopy, deformability measurements in narrow glass pipettes and isotope-labelling techniques. The ACCs had a larger mean diameter (13.9 microns) than the FSCs (10.9 microns) and showed a slower rate of deformation into 6.5 microns glass pipettes. A significantly larger percentage of ACCSs (52.4%) than of FSCs (19.9%) remained in the livers 5 min after intraportal injection. The results indicate that for the particular tumour cells studied here, there exists a relationship between cell deformability and the tendency for microvascular trapping in the liver, i.e. less deformable cells have a greater tendency for retention in the liver.

The effects of delayed nerve repair on nerve regeneration in a silicone chamber model.

The silicone chamber model for nerve regeneration is suitable to test the effects of exogenous agents or surgical manipulations on nerve regeneration. The total 16-day regeneration period used in this model makes it possible to analyze the effects of certain manipulations on the sequential advancement of the individual cellular components (circumferential perineurial-like cells, vessels, Schwann cells, axons, and myelin) into the chamber fibrin matrix. In the present study we compared the effects on cellular migration of a 7 day delayed chamber repair vs. chamber repair immediately after transection (control chambers) of the rat sciatic nerve. Regeneration was evaluated with light and electron microscopic techniques. Chambers implanted after a delay of 7 days had a statistically significant more advanced migration of vessels, Schwann cells, and axons from the proximal nerve stump and also a significantly increased vascular density as compared to control chambers. We conclude that a 7 day delayed nerve repair stimulates nerve regeneration in this specific silicone chamber model.

Opposing effects of suramin and DL-alpha-difluoromethylornithine on polyamine metabolism contribute to a synergistic action on B16 melanoma cell growth in vitro.

Polyamines are crucial for normal and neoplastic cell growth. Treatment with the polyanionic drug suramin has pronounced antigrowth activity in several tumor cell lines, but its clinical use has been hampered by its toxicity. We have earlier shown that suramin affects cellular polyamine metabolism and transport, and that these effects were, in some respects, opposite to those of alpha-difluoromethylomithine (DFMO), a specific inhibitor to ornithine decarboxylase, a key metabolic enzyme for polyamines. DFMO has been used in anticancer trials, although with limited success. Combinations of suramin and DFMO were, hence, evaluated in vitro and were found to strongly inhibit B16 melanoma cell proliferation. DFMO alone induced melanoma cell differentiation, and suramin used in combination with DFMO did not abrogate this DFMO-induced differentiation. Synergy analysis demonstrated a pronounced growth-inhibitory synergism between suramin and DFMO. The results suggest that the efficacy of combinations of DFMO with suramin or its analogues should be further explored, especially in cells requiring high levels of polyamines for their growth.

Effects of suramin on polyamine metabolism in B16 murine melanoma cells.

Polyamines and their biosynthetic enzymes, such as ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are crucial for normal and neoplastic cell growth and differentiation. Suramin inhibits the growth of several tumor cells by affecting various intracellular targets, but its effects on polyamines are not known. In this study, the effects of suramin on some parameters of polyamine metabolism in B16 melanoma cells were investigated in vitro. Suramin increased cellular ODC activity and ODC mRNA levels, whereas the drug was directly inhibitory to the enzyme. AdoMetDC was not affected. Cellular putrescine levels were enhanced by suramin, whereas spermidine and spermine pools were unaltered. Cells cultured in the presence of suramin showed decreased cellular polyamine transport, but no direct inhibitory effect on the polyamine transporter could be found. Fluorescence spectroscopy demonstrated a direct interaction between suramin and spermine. It may be concluded that suramin affects polyamine metabolism, and that its effects in some respects are opposite to those of alpha-difluoromethylomithine (DFMO), a specific inhibitor of ODC.

Matrix metalloproteinases of human NK cells.

We have previously reported that MMP-2 and MMP-9 are present in rat A-NK cells, and have recently documented that additional MMPs are present in rodent A-NK cells. To our knowledge only proMMP-9 has previously been reported for human NK and A-NK cells. Herein, we report for the first time the presence of MMP-2 and MT1-MMP in human NK cells. The importance of these enzymes for the migration of A-NK cells into tumor metastases is of great potential relevance. MMPs may be rate limiting in A-NK cells, following their adoptive transfer, to traverse basement membrane and accumulate within established cancer metastases, a likely pre-requisite to their cytolytic function. Human NK cells express and produce MMP-2, MMP-9, MT1-MMP and the inhibitor TIMP-1. Moreover, human A-NK cells degrade the extracellular matrix equivalent (Matrigel) in a seemingly IL-2 dependent manner. It is therefore likely that A-NK cell MMPs play crucial roles in contributing to A-NK cell localisation and positioning the cells in vivo to allow for triggering their cytolytic potential.