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[CANCER RESEARCH 38, 2740-2744, September 1978] 0008-5472/78/0038-0000$02.00 Prevention of Hepatic Metastases by Intravenous Radioactive Gold' Antonio E. Aifonso,2 All Hassan, Bernard Gardner,3Sidney Stein, Joseph Path, Nathan A. Solomon, Joseph McCarthy,and Joseph Steigman Divisions of Surgical Oncology (A. E. A., A. H., B. G., S. S., J. P.1 and Nuclear Medicine (N. A. S., J. M., J. S.), State University of New York, Downstate MedicalCenter,Brooklyn,New York11203 ABSTRACT This investigationwas designed to determine whether livertumorgrowthcan be preventedby localizedradioac tivfty through radioactive gold (198Au) infusion. Colloidal 198Auis phagocytosedby Kupifer cells and emits I@-and a-radiation. Measurementof a-radiation showedmarked 198Auliver concentration. Liver chemistries, histology, and isolated hepatocyte function studies that measure [14C]acetateincorporationinto bile acid and cholesterol formationafter doses from 200 to 3000 gtCiwere serially determined.A 4OO-@zCi dose (2944rads)providedsubstan tial local radiation in the liver for 9 days withouttoxicity. Three hundredtwenty-sevenmale 150-gSprague-Dawley rats received either stable (“cold―) colloidalgold, 198Au, intraportallyor through a peripheral vein within 10 mm following intraportal infusion of 10,000,000 Walker 256 sarcoma cells. In 66 other rats intraportal 198Auadminis trationwas delayed for 2 weeks after tumorimplantation. In rat livers that received ‘98Au early following tumor injection, significantly fewer tumors were observed than in controls(p < 109). When radiationwas delayed for 2 weeks after tumor infusion,the differenceswere not as significant. Intraportal or systemic 198Augiven within 10 mm follow ing tumor infusion can protect an animal from hepatic tumor growth without significant liver toxicity. That he patic radiation may prevent tumor growth may have im portant clinical implications. INTRODUCTION liver metastases that utilized adjuvant modalities of therapy (1—3,17). Previous studies demonstrating that radioactive colloids injected into either a splanchnic or peripheral vein selectively concentrate in the liver (5, 13, 18, 21, 22) have prompted our interest in utilizing prophylactic internal na dionuclide therapy to prevent clinical liven metastases. Considerable data have already been accumulated con cerning the biochemical properties, tissue distribution, ef fects, and metabolism of radioactive colloidal gold (198Au) after systemic administration in both humans (16) and experimental animal models (19). After i.v. administration 198Auremains insoluble in serum, does not cause capillary microaggregation, becomes rapidly phagocytosed by retic uloendothelial cells in the liver (Kupffer cells), and can provide a selective temporary homogeneous field of hepatic radioactivity without systemic radioactive isotope spillover. Radioactive colloidal gold was also ideally suited for our investigation since it emits both 1@-and a-radiation and possesses a relatively short half-life. /3-Radiation from ‘98Au has an ionizing tumonicidal range of 8 mm in tissue and water, and its concomitant a emission permits measure ment of the isotope distribution in other organs of the body. Preliminary studies were performed to establish the mm imum effective i.v. dose of ‘98Au that would prevent liver tumor “takes― in rats after introduction of tumor cells into the portal circulation, to determine tissue distribution of the isotope following either splanchnic or peripheral vein injec tion, and to observe for associated hepatic toxicity of the proposed treatment regimen. The main experiment was designed to evaluate the efficacy of internal hepatic radio nuclide therapy with respect to reducing the incidence of hepatic ‘ ‘metastases― after either splanchnic or systemic injection of radioactive colloidal gold. Successful treatment following an adequate wide-field colon cancer resection has often been frustrated by the subsequent occurrence of hepatic metastases (14). Unfor tunately, once overt metastases become firmly established MATERIALSAND METHODS in the liver, current treatment modalities to date have All experiments were performed on male Sprague-Dawley yielded extremely poor overall results. The likelihood of the rats weighing 200 g and kept under standard laboratory and development of hepatic metastases reflected by the ad diet conditions. Operations were performed under i.m. vanced stage of the local tumor at the time of initial surgical resection can be predicted in a majority of cases. Such sodium pentobarbital anesthesia, 0.05 mg/g body weight, and with sterile techniques. Tumor cells were prepared patients could be greatly benefitted if there were a practical from homogenates of solid i.p. Walker 256 carcinoma and effective method of preventing liver tumor “takes― without the significant local and systemic side effects of implants. The homogenates were appropriately diluted with sterile 0.9% NaCI solution, and separated cells were ob external or internal hepatic radiation and systemic or infu tamed to provide a constant dose of 10,000,000 cells! sional chemotherapy. injection. Cells were counted in a hemocytometer, viability Recent interest has focused on the prevention of clinical was confirmed by trypan blue exclusion (>90%), and sub sequent growths of aliquots were implanted s.c. Previous results from our laboratory have consistently confirmed an ‘Supported in 2 To requests whom part by Alan for Foss Memorial reprints should Fund be Grant addressed Clarkson Avenue, Brooklyn, N. Y. 11203. 3 John and Mary Markle Scholar in Academic Medicine. ReceivedJanuary27, 1978;acceptedMay17,1978. 2740 1779. at Box 40, 450 80 to 85% liver tumor ‘ ‘take― after this dose of Walker 256 carcinoma was given through a portal vein. Radioactive CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1978 American Association for Cancer Research. Prevention of Hepatic Metastasesby i.v. 198Au colloidal gold obtained from Amensham/Seanle Corp. , Ar “takes―“Au ‘Au on liver tumor Effect of increasTable ing dosesof “1 lington Heights, Ill., with an average fairly uniform particle size (20 to 40 nm), and calibrated to 25 mCi/mI was used. for tumor%Control of ratsPositive dose (MCi)No. Colloidal ‘98Au after it had been allowed to decay for at least 60 days to negligible levels of radioactivity (“cold― 4 4 100 colloidal gold) was used for the control animals. 4 4 200 4 2 300 RESULTS 48 0100 4008 100 100 50 0 Minimal Effective Dose of ‘98Au. Twenty-fourrats re ceived either “cold― colloidal gold (8) on 100 (4), 200 (4), 300 (4), or 400 (4) tributary @Ciof ‘“Au through a mesentenic 5 to 15 mm after intraportal 10,000,000 Walker 256 carcinoma cells. vein introduction All animals of were sacrificed at 28 days and examined for liver tumor growths. As shown in Table 1 , there was complete Walker 256 ‘U U) Cl) I. ‘a- 0 sarcoma tumor hepatic growth inhibition at 28 days follow 4 ing a 4OO-@Cidose of ‘98Au. Dosages of 100 and 200 @.tCi CD were completely ineffective, while the 300-MCi dose pre ‘U 0. vented liven tumor growth in only 2 of 4 rats. In the 8 control ‘U Ianimals that received “cold― gold colloid, a 100% liver z tumor “take― was observed. Isotope Tissue Distribution. Thirty-six rats were divided a. into equal groups. In 18 rats 400 @Ci of 198Auwere injected U) aintraportally through a mesentenic vein tributary. The re z mainder had a similar dose administered systemically 0 U through the femoral vein. Pained rats from each group were sacrificed at 4 h and 1, 2, 3, 5, 6, 7, 10, and 15 days. The ‘@ota 2 3 4 5 6 7 8 9 lOll 2 following organs were dissected out, weighed, and pre pared for counting: liver, spleen, lung, brain, and gastroin Chart 1. Distribution of ‘@Au in different organs after portal vein) injection. testinal tract. A sample of muscle and bone with marrow ‘U 4HR 3 45 DAYS (mesenteric was also taken. The specimenswere immediatelyplaced in appropriately shielded a-scintillation counters, and mdi vidual radioactivity in terms of cpm/g weight of tissue was determined. These data obtained on tissue distribution of ‘15Au as measured by the decay of a emissions at sequential time intervals from 4 h to 14 days are presented in Charts 1 and 2. Confirming previous work by others with radioactive colloidal gold particles, the liven and spleen preferentially concentrated the gold particles after introduction through either an intraportal or systemic route. Other phagocytic tissues, like lung, bone, and marrow, were significantly less active in the removal of gold particles from the blood. Muscle, brain, and intestines did not significantly concen trate the isotope. Interestingly, transient higher concentra tion of intestinal 198Au followed intraportal injection as compared with systemic administration. Toxicity. Following intraportal 1Au administration in increasing dose increments (400, 1200, and 2000 MCi), serum levels of bilirubin, alkaline phosphatase, SGOT,4 and SGPT were determined and liver histological examinations were performed in 18 rats that were sequentially sacrificed at 7 and 21 days. Ten other rats consisting of pairs that received 400, 1200, 2000, 3000, and 6000 @Ci“Auwere observed clinically for 6 months and then autopsied. All livers were examined microscopically for morphological alterations. Bile acid secretion from isolated hepatocytes 4 The abbreviations used are: SGOT, serum glutamic-oxaloacetic aminase; SGPT, serum glutamic-pyruvic transaminase. SEPTEMBER 1978 trans ‘U U) U) aIa. 0 4 CD ‘U a. ‘U az ‘U a. U) az 0 U 01 4CC a 2 3 4 5 6 7 8 9 0 II 2 3 4 DAYS Chart 2. Distribution of “Auin different organs after systemic (femoral vein) injection. with culture techniques previously described in our labora tory (4), after 2 h of incubation with [‘@C]acetate, was measured in 2 rat livers that were exposed to 400 pCi of internal hepatic radiation 1 week before and compared with pained normal rat livers. No significant alterations in serum levels of bilirubin, alkaline phosphatase, SGOT, and SGPT were found at 7 and 21 days after hepatic radiation dosages 2741 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1978 American Association for Cancer Research. A. E. Alfonso et al. Table 2 Liver function studies obtained Intraportal ‘“Au dose at 7 and 21 days after intraportal injections of increasing doses of of ratsBilirubinAlkaline phosphataseSGOTSGPTControl (MCi)DayNo. ±0.2@ 4007 3 0.2 ±0.1 ±76 196.6 ±40.4 ±161 147.3±99.8 ±18 57.3 ± 2.5 199.7±86.852 55.7± 812007 218 30.4 0.13±0.06231 203 13.620007 213 ±0.057 30.13 0.2 ±0180 ±58 ±45.6 190 ±26303 303 ±75109 213 ±44 ±20.5 30.17±0.057 0.17±0.057129 160 ±10152 196.7±2577.7±36.8 69 ±35a ±25237 ±10 104.7 ± Mean ±S.D. @ of 400, 1200, and 2000 @Ci (Table 2). None of the animals observed for 6 months developed any clinical signs of cirrhosis. At autopsy gross liver nodulanity, hypenemia, and hepatomegaly were observed in both rats that received 6000 MCi. These were accompanied by distortion of lobular pattern, hepatic cell ballooning, and necrosis on histologi cal examination. Similar microscopic alterations were also observed at 6 months in rats that received 3000 @tCi.None of the livers of animals that received dosages of 2000 pCi or less showed either gross or histological alterations at 6 months. The average total bile acid secretion rates in mol/g liver cells from isolated hepatocyte cultures from 2 rat livers exposed to 400 @tCiat 2 hr (18.71 ) were comparable to a pair that received “cold― colloidal gold and to values derived from normal rat hepatocytes (19.15). Table 3 Effect of “Au colloid on prevention of liver metastasesafter intraportal andadministrationPortal systemic vein Systemic Tumor Tumor “take―Control Total “take― Total 28(93)d‘“Au149 123(5,3)a.b.c 30 2(8)―Delayed ‘Au a 19(15)― 66 34 (54)C 25 < b Numbers C p 123 < in parentheses, percentages. 10-.. d ,@< Efficacyof ‘98Au. For establishmentof the effectiveness of 198Au-induced hepatic local radioactivity in preventing experimental liver tumor “takes,― 393 rats were divided into 179 control and 214 experimental animals. All animals received tumor injections of 10,000,000 Walker carcinoma cells introduced into a mesentenic vein portal tributary. All experimentally treated animals received 400 @tCiof 198Au. Five to 10 mm after tumor administration, a comparable volume of “cold― colloidal gold was injected either through a separate mesentenic vein (149 rats) or the femoral vein (30 rats). After a similar time interval, experimental rats re ceived ‘“Au colloid through either a mesentenic vein (123 rats) ora femoralvein(25rats) injection. In66 ratsthe198Au mesentenic vein injection was delayed for 2 weeks following tumor introduction. All animals either died of tumor after 2 weeks or were sacrificed at 28 days. At autopsy all livers were examined grossly and microscopically for tumor growths. A summary of our results is recorded in Table 3. In 149 control rats that received “cold― colloidal gold through a mesentenic vein 5 mm after tumor injection, 83% devel oped bilateral massive liven tumors within 28 days. This significantly decreased to 15% or 19 of 123 animals in the treatment group that received intraportal 198Au5 mm after tumor administration. In the 30 control animals that re ceived “cold― colloidal gold through a systemic (femoral vein) injection 5 mm after tumor injection, there was a 93% liver tumor “take― as compared to 8% in the systemically treated group of rats that received 198Auvia a peripheral vein 5 mm after the tumor cells. When 198Auadministration via the portal route was delayed by 2 weeks after tumor 2742 infusion, the tumor “take― was 54% as compared to 83% in the comparable control group. DISCUSSION Previous experimental and limited clinical experiences with the use of fJ-emitting 90Y tagged to ceramic micro spheres have demonstrated that localized interstitial he patic radiation can be successfully produced with minimal systemic spillover and result in effective growth prevention of virulent tumors without significant systemic or liver toxicity (6, 7, 10, 12). Localization of organ radioactivity, however, relies on the trapping of microspheres in the size of 50 to 100 within the pnecapillary vessels by a process of microembolism and requires operative placement of a he patic artery catheter. Other radioactive colloidal compounds that are known to be selectively concentrated by the reticuloendothelial cells of the liver after i.v. administration have been studied. Grady et a!. (5) and Ackerman et al. (1) have separately reported on encouraging results with the use of radiocolloid suspensions of 32P-tagged chnomic phosphate in prevent ing liver “takes― of Walker 256 carcinoma and the Novikoff hepatoma injected into the rat portal vein to simulate liver metastases. A disadvantage of the use of Cr32PO4has been its reported high bone marrow uptake after systemic i.v. injection. A radiocolloid suspension of ‘“Au was chosen as the particulate matter for our present study. The isotope was currently available, it is easy to measure quantitatively, CANCER RESEARCHVOL. 38 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1978 American Association for Cancer Research. @ Prevention of Hepatic Metastases by i.v. 98AU and much has been known about its chemical preparation to small particles of approximately known size (22). A uniform small particle size (20 to 40 nm) is essential to avoid capillary aggregation and blockage and to allow effective reticuloendothelial phagocytosis. Unlike chromic [32Plphosphate, cumbersome mixing procedures are not necessary for its preparation. Radioactive gold has a half life of 2.73 days, emits negative /3 particles with an average energy of 0.315 MeV, and an a-ray with an energy of 0.411 MeV. Its tissue distribution and radiation effects have been extensively studied in rats, dogs, guinea pigs, and humans. The greatest part of its i .v. administered dose (80 to 90%) is concentrated by the liver. Only 5 to 15% is taken up by the spleen, which is relatively radionesistant, and very small quantities reach other tissues of the body. The compound is cleared from the blood within minutes inasmuch as most of the gold given becomes firmly lodged within Kupffer cells and distribution is in a fairly homogeneous pattern through out the liver parenchyma (16, 18, 19, 21). The administration of ‘98Au in larger i.v. doses has not been associated with untoward effects in humans (16). There is also virtually no problem of bone marrow depression following injection of the material. No immediate or long-term hepatic damage has been described following radiation of the organ with 2500 R. After massive experimental doses in rats, liver necrosis had occurred but the doses were considerably higher (50 times) than were those used in our present study. The smallest amount of 198Aureported in the literature that has caused liver damage is 12,000 equivalent R, this sug gests that the threshold for necroses in rat livers is between to 19 and 8% after mesentenic or femoral vein ‘98Au treat ment, respectively, is significant (p < 10@). These results have paralleled other studies with respect to optimal time of isotope administration, which was within 48 hr after tumor injection (1, 5). Treatment results were poorer when 198Auadministration was delayed for 2 weeks but signifi cantly different from control values. This observation is correlated with the fact that clinically obvious liver tumor growths were usually established after this time interval (2 weeks) in our experimental rat livers. Root (16) has similarly achieved poor therapeutic results in the treatment of estab lished liver tumors after i.v. administered colloidal radi ogold. In the areas of discrete “takes― where reticuloendo thelial tissues have been replaced, the radionuclide is not concentrated in the hepatic tissue immediately adjacent to the tumor nodules. This is in striking contrast to the very high levels of radioactivity in the remaining grossly normal liver panenchyma. While showers of viable malignant cells have been iso lated from the portal venous effluent area of the primary tumor after surgical manipulations, the cause of liver me tastases may be in the presence of already disseminated occult tumor microfoci at the time of initial resection. Current knowledge of tumor population growth kinetics suggests that micrometastases are rendered cytotoxically more sensitive after removal of all gross disease as the fraction of remaining actively dividing viable tumor cells increases in an inverse relationship to total tumor popula tion size (17). It is therefore logical to treat the disease in its earlier stages rather than wait until a point at which, to 25,000 and 12,000 A (11).Four hundred @Ciof 198Aucolloid date, only palliative results may be expected at best. Unfor with t112physical assumed equal to t112effective were calcu tunately, external irradiation and infusion or systemic lated to deliver 2944 rads to the liver (2840 from f3-radiation, chemotherapy leave much to be desired in an adjuvent 104 from a-radiation) and in our study appeared to be the setting because of inherent accompanying systemic and minimal effective dose in preventing liven “metastases.― local toxicity. The human threshold for hepatic damage that is secondary It is apparent that this radionuclide offers a means of to total fractionated hepatic radiation is approximately 3000 achieving localized radiation distribution to the liver follow rads (9, 20), although dramatic recovery of liver morphology ing i.v. administration with minimal systemic and local and function has been noted in humans who have received untoward effects. The chief therapeutic use reported for 198Auhas been in the treatment of leukemia and lymphoma 3000 to 5900 rads after fractionated hepatic radiotherapy (15). Dosage increments of 198Aucolloid from 400 to 2000 (16). While therapeutic possibilities of i.v. administered @.tCi did not cause significant changes in several parameters radiogold colloid in the treatment of established hepatic of nat liver function that were monitored at 7 and 21 days: metastases has appeared exceedingly limited, the unique serum bilinubin, alkaline phosphatase, SGOT and SGPT. characteristics of colloidal radionuclides and their prefer Bile acid secretion rates in the isolated hepatocyte after ential concentration in the reticuloendothelial tissues of the exposure to 400 @tCiwere unaltered . Morphological liver liver suggest that they may prove advantageous in the damage was produced only at significantly higher doses treatment of disseminated micnometastatic hepatic tumor (3000 MCi) at 6 months. The nadiocolloid tissue distribution foci. also remained similarto previously described patterns (16, 18, 19). In the main experiment ‘9Auwas injected either into a REFERENCES mesentenic or peripheral vein. 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The Preparation and Particle Size Measurement of Monodisperse Radioactive Gold Colloids. J. Lab. Clin. Med.,40: 261-266,1952. CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1978 American Association for Cancer Research. Prevention of Hepatic Metastases by Intravenous Radioactive Gold Antonio E. Alfonso, Ali Hassan, Bernard Gardner, et al. Cancer Res 1978;38:2740-2744. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/38/9/2740 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. 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