Preparation of "tissue-isolated" rat tumors for perfusion: a new surgical technique that preserves continuous blood flow

A surgical technique for preparing "tissue-isolated" rat tumors for perfusion was developed that ensures continuous blood flow to the tissue. The tumor may be perfused in situ or ex vivo. Tumor venous blood flow, tumor glucose and lactic acid metabolism, as well as host animal respiration and core temperature, were unchanged during the procedure. Following addition of 14C-D-Glucose to the tumor afferent blood, all radioactivity appeared in the tumor venous blood. None appeared in the host systemic blood, indicating complete separation of tumor and host vasculature during in situ perfusion.     

Dietary factors and growth and metabolism in experimental tumors

Development of a diet that provides adequate nutrition and effective cancer prevention is an important goal in nutrition and cancer research. A confounding aspect of dietary control of tumor growth is the fact that some nutrients may up-regulate tumor growth, whereas other nutrients and nonnutrients down-regulate growth. Both up- and down-regulators may be present in the same foodstuff. Identification of these substances, determination of their mechanisms of action and potencies, as well as the interactions among the different mechanisms are topics of ongoing research. In this review, we describe results obtained in vivo or during perfusion in situ using solid tissue-isolated rodent tumors and human cancer xenografts in nude rats. Linoleic acid (LA), an essential n-6 polyunsaturated fatty acid (PUFA), was identified as an agent in dietary fat that is responsible for an up-regulation of tumor growth in vivo. Tumor LA uptake, mediated by high intratumor cAMP, stimulated formation of the mitogen, 13-hydroxyoctadecadienoic acid (13-HODE) and also increased ERK1/2 phosphorylation, [(3)H]thymidine incorporation and growth. A mechanism for control of this growth-promoting pathway was revealed during studies of the effects of dietary nutrients and nonnutrients known to inhibit tumor growth. These included four groups of lipophilic agents: n-3 fatty acids, melatonin, conjugated LA isomers and trans fatty acids. Each of these agents activated an inhibitory G protein-coupled receptor-mediated pathway that specifically suppressed tumor uptake of saturated, monounsaturated and n-6 PUFAs, thereby inhibiting an early step in the LA-dependent growth-promoting pathway.     

Light during darkness,melatonin suppression and cancer progression

Over the past few years, we have shown that the surge of melatonin in the circulation during darkness represents a potent oncostatic signal to tissue-isolated rat hepatoma 7288CTC, which is an ER+ adenocarcinoma of the liver. This oncostatic effect occurs via a melatonin receptor-mediated suppression of tumor cAMP production that leads to a suppression of the tumor uptake of linoleic acid (LA), an essential fatty acid with substantial oncogenic properties. The ability of LA to promote cancer progression is accomplished by its intracellular metabolism to 13-hydroxyoctadecadienoic acid (13-HODE) which amplifies the activity of the epidermal growth factor receptor/mitogen-activated protein kinase pathway leading to cell proliferation. By blocking tumor LA uptake, melatonin effectively blocks the production of 13-HODE and thus, markedly attenuates tumor growth. A similar effect of melatonin is observed in tissue-isolated, ER+ MCF-7 human breast cancer xenografts and nitrosomethylurea (NMU)-induced rat mammary cancers. When male rats bearing tissue-isolated hepatomas are exposed either to constant bright light (300 lux) or dim light (0.25 lux) during the dark phase of a 12L:12D photoperiod, the latency to onset was significantly reduced while the growth of tumors was markedly increased over a 4 wk period as compared with control tumors in 12L:12D-exposed rats. In constant light- and dim light during darkness-exposed rats, melatonin levels were completely suppressed while tumor growth, LA uptake and 13-HODE production were markedly increased. Similar results were obtained in constant bright light-exposed female rats bearing tissue-isolated NMU-induced mammary cancers or MCF-7 human breast cancer xenografts. To date, these studies provide the most definitive experimental evidence that light exposure during darkness increases the risk of cancer progression via elimination of the nocturnal melatonin signal and its suppression of tumor LA uptake and metabolism to 13-HODE.     

Inhibition of fatty acid transport and proliferative activity in tissue-isolated human squamous cell cancer xenografts perfused in situ with melatonin or eicosapentaenoic or conjugated linoleic acids

Melatonin and eicosapentaenoic and 10t,12c-conjugated linoleic acids suppress the growth-stimulating effects of linoleic acid (LA) and its metabolism to the mitogenic agent 13-(S)-hydroxyoctadecadienoic acid (13-(S)-HODE) in established rodent tumors and human cancer xenografts. Here we compared the effects of these 3 inhibitory agents on growth and LA uptake and metabolism in human FaDu squamous cell carcinoma xenografts perfused in situ in male nude rats. Results demonstrated that these agents caused rapid inhibition of LA uptake, tumor cAMP content, 13-(S)-HODE formation, extracellular signal-regulated kinase p44/ p42 (ERK 1/2) activity, mitogen-activated protein kinase kinase (MEK) activity, and [3H]thymidine incorporation into tumor DNA. Melatonin's inhibitory effects were reversible with either the melatonin receptor antagonist S20928, pertussis toxin, forskolin, or 8-bromoadenosine-cAMP, suggesting that its growth-inhibitory effect occurs in vivo via a receptor-mediated, pertussis-toxin-sensitive pathway.     

Systemic perfusion: a method of enhancing relative tumor uptake of radiolabeled monoclonal antibodies

We evaluated the feasibility of systemic vascular perfusion with saline (mimicking plasmapheresis) as a method to enhance tumor-specific monoclonal antibody (MoAb) tumor/background ratios. Initially, groups of rats were injected intravenously (i.v.) with ¹³¹I-5G6.4 MoAb (murine IgG2aK reactive with ovarian carcinoma). These animal's radioactivity levels were determined by dose calibrator and they were imaged before and after perfusion which was conducted at 4 or 24 h post-antibody injection. Animals were sacrificed after perfusion, as were controls, and normal organ radioactivity levels determined. In addition, nude mice bearing HTB77 ovarian cancers subcutaneously were injected i.v. with ¹³¹I-5G6.4 MoAb and were imaged before and after systemic perfusion with saline 24 h post-5G6.4 injection. Perfusion in rats dropped whole-body 5G6.4 levels significantly at both perfusion times (P < 0.0005). The drop in whole-body radioactivity with perfusion was significantly greater for the animals perfused at 4 h post i.v. 5G6.4 antibody injection (48.3 ± 5.1%) than for those perfused at 24 h post i.v. antibody injection (32.9 ± 2.9%) (P < 0.025). In the nude mice with ovarian cancer xenografts, γ camera images of tumors were visually and quantitatively (by computer image analysis) enhanced by perfusion, with a 2.33-fold greater decline in whole body uptake than in the tumor (P < 0.05). These studies show that (1) much background antibody radioactivity can be removed using whole-body perfusion with saline, (2) that the decline in whole body activity is larger with 4 than 24 h perfusion and (3) tumor imaging can be enhanced by this approach. This and similar approaches that increase relative tumor antibody uptake such as plasmapheresis may be useful in imaging and therapy with radiolabeled antibodies.     

Light Exposure at Night Disrupts Host/Cancer Circadian Regulatory Dynamics: Impact on the Warburg Effect,Lipid Signaling and Tumor Growth Prevention

The central circadian clock within the suprachiasmatic nucleus (SCN) plays an important role in temporally organizing and coordinating many of the processes governing cancer cell proliferation and tumor growth in synchrony with the daily light/dark cycle which may contribute to endogenous cancer prevention. Bioenergetic substrates and molecular intermediates required for building tumor biomass each day are derived from both aerobic glycolysis (Warburg effect) and lipid metabolism. Using tissue-isolated human breast cancer xenografts grown in nude rats, we determined that circulating systemic factors in the host and the Warburg effect, linoleic acid uptake/metabolism and growth signaling activities in the tumor are dynamically regulated, coordinated and integrated within circadian time structure over a 24-hour light/dark cycle by SCN-driven nocturnal pineal production of the anticancer hormone melatonin. Dim light at night (LAN)-induced melatonin suppression disrupts this circadian-regulated host/cancer balance among several important cancer preventative signaling mechanisms, leading to hyperglycemia and hyperinsulinemia in the host and runaway aerobic glycolysis, lipid signaling and proliferative activity in the tumor.     

Growth of human tumor xenografts implanted under the renal capsule of normal immunocompetent mice.

The subrenal capsule technique proved effective in demonstrating that the growth of human tumors in normal, immunocompetent animals for 6 days was quantifiable in ocular micrometer units. Positive growth was demonstrable not only with human tumors that had been established in serial transplantation in athymic nude mouse hosts, but also with primary surgical explants. Growth rates of transplantation-established xenograft systems were similar whether implanted in athymic nude or in normal immunocompetent animals indicating that the 6-day time-frame successfully evades growth inhibitory effects of immunologic origin. Immunosuppression with a single dose of cyclophosphamide did not appear to affect growth rate, but permitted the tumors to grow larger extending the time to reach peak size. Significantly, xenografts of primary surgical explants showed positive growth more frequently in 6 days (82%) in the immunocompetent animal than in 11 days (30%) in the immunodeficient athymic nude mouse.     

Uptake of lipoproteins by in situ perfused rat ovaries: identification of binding sites for high density lipoproteins

We have examined the uptake and distribution of 125I-labeled human high density lipoprotein, apolipoprotein E-free (hHDL3), 125I-rat high density lipoprotein (HDL), and human HDL (hHDL) reconstituted with [3H]cholesteryl linoleate after their in situ vascular perfusion to ovaries of gonadotropin-primed immature rats on days 6-9 post human chorionic gonadotropin (hCG)-injection. Some rats were treated with 4-aminopyrazolopyrimidine to reduce plasma lipoproteins and ovarian cholesteryl ester stores. Perfused ovaries were analyzed biochemically and autoradiographically, and progestin content of the ovarian effluent was quantified. Infusion of ovine luteinizing hormone and hHDL increased ovarian progestin secretion severalfold, indicating that the perfused ovary was functional. After perfusion with HDL reconstituted with [3H]cholesteryl linoleate, radioactive progestin appeared in the effluent; thus, sterol carried by exogenous HDL was converted to steroid. At 37 degrees C, uptake of 125I-hHDL3 was greatest after 15 min of perfusion with label. This was decreased by 80% when the perfusion was carried out at 4 degrees C and by 70-95% when excess unlabeled hHDL, but not human low density lipoprotein (hLDL), was included in the perfusate with 125I-hHDL. Aminopyrazolopyrimidine treatment enhanced 125I-hHDL uptake twofold. After perfusion for 15 min with 125I-hHDL3, radioactivity in the ovary was high for 3-30 min of HDL-free wash, then declined 75% by 30-60 min. With light and electron microscope autoradiography, 125I-hHDL3 was localized to corpora lutea, both along luteal cell surfaces and over their cytoplasm. The plasma membrane grains appeared to be associated with segments that lacked bristle coats. Perfusion with 125I-rat HDL produced a similar pattern of labeling. In ovaries perfused with 125I-BSA, silver grains were concentrated over macrophage-like cells but were sparse over luteal cells. We conclude that the in situ perfused rat ovary takes up 125I-hHDL3 by a temperature-dependent, lipoprotein-specific process, and that this lipoprotein is accumulated by luteal cells.     

A transgenic Tie2-GFP athymic mouse model; a tool for vascular biology in xenograft tumors

We report the generation of a transgenic Tie2-GFP athymic nude mouse, carrying green fluorescent blood vessels throughout the body. This transgenic mouse is a tool for studies in vascular biology, and is especially of interest for imaging of tumor angiogenesis and the study of anti-angiogenesis strategies in (human) xenografts. Intravital microscopy identified the presence of blood conducting structures that are not lined by endothelial cells. Dedifferentiation of aggressive tumor cells can lead to acquisition of endothelial characteristics. This process of tumor cell plasticity, also referred to as vasculogenic mimicry, has been suggested to contribute to the circulatory system in a tumor. In plastic EW7 Ewing sarcoma tumors in these Tie2-GFP mice, we observed blood flow in both regular blood vessels and non-fluorescent tumor cell-lined channels, visualizing in vivo hemodynamics in vasculogenic channels. These results demonstrate that the transgenic Tie2-GFP athymic mouse model is a valuable tool for vascular biology research.     

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.     

Targeting of Drugs to Solid Tumors Using Anti-Her2 Immunoliposomes

Abstract

Cancer therapy would clearly benefit from a carrier system capable of intracellular delivery of systemically administered drugs to cancer cells in solid tumors. Sterically stabilized immunoliposomes specific to the cells expressing HER2 protooncogene (anti-HER2 SIL), were designed by conjugating Fab’ fragments of a recombinant humanized anti-HER2 MAb to the distal termini of poly(ethylene glycol) chains on the surface of unilamellar liposomes (size 90–100 nm) of phosphatidylcholine, cholesterol, and poly (ethylene glycol)—derivatized phosphatidylethanolamine. Anti-HER2 SIL avidly and specifically bound to cultured HER2-overexpressing cancer cells (8,000–23,000 vesicles per cell) and became endocytosed (k_e = 0.022–0.033 min.^?1) via the coated pit pathway. Anti-HER2 SIL showed prolonged circulation lifetime in rats (blood MRT approx. 24 hours) and significantly increased antitumor activity of encapsulated doxorubicin against HER2-overexpressing human breast cancer xenografts in nude mice. Although the accumulation of anti-HER2 SIL in HER2-overexpressing tumor xenografts was not increased over that of non-targeted sterically stabilized liposomes (SL), microscopic examination revealed abundance of anti-HER2 SIL in the interstitial spaces, as well as within the cytoplasm of cancer cells, while identical liposomes lacking anti-HER2 Fab’ were located predominantly within tumor-resident macrophages. Anti-HER2 SIL, a targeted vehicle capable of in vivo intracellular delivery of substances to HER2-overexpressing solid cancers, enhances the potential for tumor targeting and opens new avenues for better treatment of cancer.     

Production of acetone and conversion of acetone to acetate in the perfused rat liver

The utilization of millimolar concentrations of [2-14C]acetone and the production of acetone from acetoacetate were studied in perfused livers from 48-h starved rats. We devised a procedure for determining, in a perfused liver system, the first-order rate constant for the decarboxylation of acetoacetate (0.29 +/- 0.09 h-1, S.E., n = 8). After perfusion of livers with [2-14C]acetone, labeled acetate was isolated from the perfusion medium and characterized as [1-14C]acetate. No radioactivity was found in lactate or 3-hydroxybutyrate. After 90 min of perfusion with [2-14C]acetone, the specific activity of acetate was 30 +/- 4% (n = 13) of the initial specific activity of acetone. We conclude that, in perfused livers from 2-day starved rats, acetone metabolism occurs for the most part via free acetate.     

Regulation of mucin secretion from human bronchial epithelial cells grown in murine hosted xenografts

Studies of regulated mucin secretion from goblet cells in primary cultures of human bronchial epithelial (HBE) cells have suffered, generally, from poor signal-to-noise ratios, with reported secretory responses of <100% (less than onefold) relative to baseline. Using, instead, HBE cells grown as xenografts in the backs of nude mice, we found that UTP (100 micro M) stimulated strong mucin secretory responses from isolated, luminally perfused preparations. The peak response (10 min) for 11 control experiments (37 xenografts) was 3.3 +/- 0.05-fold relative to baseline, and the time-integrated response (60 min) was 23.4 +/- 0.5-fold. Because responses to ATP and UTP were approximately equal, an apical membrane P2Y(2)-receptor (R) is suggested. Additionally, ADP activated mucin release from HBE xenografts, whereas UDP and 2-methlythio-ADP did not, a pattern of response inconsistent with known purinoceptors. Hence, either a novel receptor to ADP is suggested or there is significant conversion of ADP to ATP by ecto-adenylate kinase activity. Adenosine and a nitric oxide donor were without effect. Consistent with P2Y(2)-R coupling to phospholipase C, HBE xenografts responded to ionomycin and PMA; however, they were recalcitrant to forskolin and chlorophenylthio-cAMP, and to 8-bromo-cGMP. Hence, human airway goblet cells, like those of other species, appear to be regulated primarily via phospholipase C pathways, activated particularly by apical membrane P2Y(2)-R agonists.     

A comparison of the effects of different perfusion regimes on the structure of the isolated human placental lobule

Summary Isolated lobules of normal term human placentas were perfused using two different procedures. In the first more conventional system, open-circuit perfusion of both the maternal and the fetal circulations with Earle's solution containing dextran was established and maintained for either 30 min or 1 h. In the second series of experiments both circulations were perfused in separate closed circuits with a mixture of fresh autologous fetal blood and Earle's solution for 0, 1, 2 or 3 h. In both series the lobule was then fixed by perfusion through the fetal circulation.Light and electron-microscopic examination of a set of tissue samples from each perfused lobule showed substantial differences between the effects of these two types of perfusion procedure. Tissue from lobules perfused by the open-circuit blood-free procedure showed patchy but severe cell swelling and vacuolation of the trophoblast after only one hour's perfusion. Particularly striking was swelling and disruption of a large proportion of the mitochondria in all placental cell types. By contrast, placental tissue from the closed-circuit perfusion with blood-containing medium showed little change over a period of two hours, while after three hours it showed oedema and microvillous damage, but no sign of cell swelling and little mitochondrial damage.It is concluded that the viability of the perfused human placental lobule depends on the type of perfusate used, and that the use of a fetal blood-enriched perfusate is of considerable value in maintenance of the preparation as assessed by structural criteria.     

Antiferritin antibodies in immunoscintigraphic detection of human tumor xenografts

Affinity-purified antibodies against human placental ferritin and their F(ab)2 fragments labeled with 131I were examined for suitability for radioimmunodetection of ferritin-containing tumors. The nude mouse model (BALB/c, nu/nu) with xenografts of HeLa cell tumors and human adenocarcinoma of the rectum (with proven ferritin content) was used. Gamma-camera imaging and tissue distribution studies revealed that both kinds of tumor selectively accumulate antiferritin antibodies and their fragments. In large necrotic tumors nonspecific uptake of radiolabeled normal IgG occurred, but otherwise there was no tumor localisation. This study, in accordance with the literature, confirms the utility of antiferritin antibodies for the detection of human tumors in an animal model.     

Host-supplied connective tissue as a guide for proliferating tumor cells in human tumor xenografts

The morphologic alterations of 7 human tumors of different origin and various histologic types, heterotransplanted to athymic nude mice, were investigated in the present study. Constant patterns of histologic and ultrastructural changes were observed in all 7 tumors. Following the initial dying of most inoculated tumor cells, host-supplied inflammatory cells invaded the xenografts and phagocytosed necrotic tumor cells. Fibroblasts which vivaciously produced collagenous material invaded the xenografts and built up solid strands of connective tissue which tightly contacted surviving tumor cells. These stands were used as guide-rails for ingrowing blood vessels. Immediately after their immigration, the first mitoses of tumor cells occurred in close proximity to capillary-conducting strands of connective tissue resulting in a revival of tumor cell proliferation near to the fibrous cords and a spreading of newly formed tumor cells along the strands of connective tissue. These results point to the host-supplied connective tissue as playing an important role for tumor proliferation and local tumor expansion.     

Decalcification by perfusion. A new method for rapid softening of temporal bones.

We describe a new technique, decalcification by perfusion, for the softening of bony tissue. The blood circulatory system was perfused in 16 rats via a cannula through the left heart ventricle with a fixative followed by New Decalc (an acidic demineralizer) for 30-240 minutes. Perfusion decalcification for 120 minutes softened all heads and middle ear specimens could be easily sampled and prepared for studies by both light and electron microscope. For comparison, a conventional immersion technique required 72 hours of decalcification to accomplish softening. The perfusion technique considerably reduced the time needed to decalcify the tissue and preserved the morphology better than did the immersion procedure.     

99mTc-labeled cyclic RGDfK dimer: initial evaluation for SPECT imaging of glioma integrin alphavbeta3 expression

This report describes the evaluation of biodistribution properties of three radiotracers, [(99m)Tc(SQ168)(EDDA)], [(99m)Tc(SQ168)(tricine)(PDA)], and [(99m)Tc(SQ168)(tricine)(TPPTS)] (SQ168 = [2-[[[5-[carboonyl]-2-pyridinyl]hydrazono]methyl]benzenesulfonic acid]-Glu(cyclo{Lys-Arg-Gly-Asp-d-Phe})-cyclo{Lys-Arg-Gly-Asp-d-Phe}; EDDA = ethylenediamine-N,N'-diacetic acid; PDA = 2,5-pyridinedicarboxylic acid; TPPTS = trisodium triphenylphosphine-3,3',3' '-trisulfonate), and their potential to image the glioma integrin alpha(v)beta(3) expression in BALB/c nude mice bearing the U87MG human glioma xenografts. It was found that all three radiotracers were able to localize in glioma tumors with a relatively high tumor uptake and long tumor retention time by binding to the integrin alpha(v)beta(3) expressed on both tumor cells and endothelial cells of tumor neovasculature. It seems that the coligand has minimal effect on integrin alpha(v)beta(3) targeting capability of the (99m)Tc-labeled RGDfK dimer, but it has a significant impact on their biodistribution properties. For example, the complex [(99m)Tc(SQ168)(tricine)(TPPTS)] has the lowest liver uptake and the highest metabolic stability in normal BALB/c nude mice. Results from SPECT imaging studies show that the glioma tumors can be clearly visualized with all three radiotracers at 4 h postinjection. Among the three radiotracers evaluated in this study, [(99m)Tc(SQ168)(tricine)(TPPTS)] has the best imaging quality and is a promising candidate for more preclinical evaluations in the future.     

Changes in blood perfusion and hypoxia after irradiation of a human squamous cell carcinoma xenograft tumor line

The effect of irradiation depends on the oxygenation status of the tissue, while irradiation itself also changes the oxygenation and perfusion status of tissues. A better understanding of the changes in tumor oxygenation and perfusion over time after irradiation will allow a better planning of fractionated radiotherapy in combination with modifiers of blood flow and oxygenation. Vascular architecture (endothelial marker), perfusion (Hoechst 33342) and oxygenation (pimonidazole) were studied in a human laryngeal squamous cell carcinoma tumor line grown as xenografts in nude mice. The effect of a single dose of 10 Gy X rays on these parameters was evaluated from 2 h to 11 days after irradiation. Shortly after irradiation, there was an 8% increase in perfused blood vessels (from 57% to 65%) followed by a significant decrease, with a minimum value of 42% at 26 h after irradiation, and a subsequent increase to control levels at 7 to 11 days after irradiation. The hypoxic fraction showed a decrease at 7 h after treatment from 13% to 5% with an increase to 19% at 11 days after irradiation. These experiments show that irradiation causes rapid changes in oxygenation and perfusion which may have consequences for the optimal timing of radiotherapy schedules employing multiple fractions per day and the introduction of oxygenation- and perfusion-modifying drugs.