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Biphasic effect of a primary tumor on the growth of secondary tumor implants

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Biphasic effect of a primary tumor on the growth of secondary tumor implants
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  J Cancer Res Clin Oncol (2010) 136:1605–1615DOI 10.1007/s00432-010-0818-7  1 3 ORIGINAL PAPER Biphasic e V  ect of a primary tumor on the growth of secondary tumor implants Juan Bruzzo · Paula Chiarella · Roberto P. Meiss · Raúl A. Ruggiero Received: 5 October 2009 / Accepted: 1 February 2010 / Published online: 21 February 2010 󰂩  Springer-Verlag 2010 Abstract  Background  The phenomenon of hormesis is character-ized by a biphasic dose–response, exhibiting oppositee V  ects in the low- and high-dose zones. In this study, weexplored the possibility that the hormesis concept maydescribe the interactions between two tumors implanted ina single mouse, such that the resulting tumors are of di V  er-ent sizes.  Materials and methods We used two murine tumors of spontaneous srcin and undetectable immunogenicity grow-ing in BALB/c mice. A measure of cell proliferation wasobtained by immunostaining for Ki-67 protein and by usingthe [ 3 H] thymidine uptake assay. For serum fractionation, weutilized dialysis and chromatography on Sephadex G-15.  Results The larger primary tumor induced inhibitory orstimulatory e V  ects on the growth of the smaller secondaryone, depending on the ratio between the mass of the largertumor relative to that of the smaller one, with high ratiosrendering inhibition and low ratios inducing stimulation of the secondary tumor. Conclusion Since metastases can be considered as naturalsecondary tumor implants in a tumor-bearing host and thatthey constitute the main problem in cancer pathology, theuse of the concept of hormesis to describe those biphasice V  ects might have signi W cant clinical implications. Ine V  ect, if the tumor-bearing host were placed in the inhibi-tory window, tumor extirpation could enhance the growthof distant metastases and, reciprocally, if placed in the stim-ulatory window, tumor extirpation would result not only ina reduction or elimination of primary tumor load but also ina slower growth or inhibition of metastases. Keywords Murine tumors · Hormesis · Biphasic dose–response · Secondary tumor implants · Metastases Introduction The phenomenon of hormesis is characterized by anon-monotonic dose–response that is biphasic, exhibitingopposite e V  ects at low and high doses (Thong and Maibath2007). Although biomedical sciences have been dominatedduring the past century by the assumed sigmoidal nature of dose–responses, evidence accumulated over the years sug-gests that the hormetic dose–response model may be morecommon and fundamental than previously thought. This evi-dence was stressed over the past decade as a consequence of more importance being given to low-dose e V  ects and the useof more powerful tools aimed to detect those e V  ects.In e V  ect, hormetic dose–responses have been reportedfor many agents designed for the treatment of numerouspathological conditions such as anxiety, memory disorders,seizure, stroke, dermatological, ocular and cardiac diseases,osteoporosis, cancer, etc. (Calabrese 2008a, b; Eichler etal. 2002; Gao etal. 2002; Honar etal. 2004; Wise and Lichtman 2007). J. Bruzzo · P. Chiarella · R. A. Ruggiero ( & )División Medicina Experimental (ILEX-CONICET), Academia Nacional de Medicina de Buenos Aires, Pacheco de Melo 3081, 1425 Buenos Aires, Argentinae-mail: ruloruggiero@yahoo.com.arR. P. MeissInstituto de Estudios Oncológicos (IEO), Academia Nacional de Medicina de Buenos Aires, Pacheco de Melo 3081, 1425 Buenos Aires, ArgentinaJ. Bruzzo · P. Chiarella · R. A. RuggieroCONICET (Consejo Nacional de Investigaciones Cientí  W cas y Técnicas), Buenos Aires, Argentina  1606J Cancer Res Clin Oncol (2010) 136:1605–1615  1 3 Regarding cancer research, substantial evidence showsthat low doses of di V  erent anti-tumor agents commonlystimulate the in vitro proliferation of many di V  erent typesof tumor cells, while high doses inhibit their proliferationor kill them. This is consistent with the hormetic dose–response relationship (Calabrese 2007). Similarly, agentsthat a V  ect capillary development, and in consequence con-sidered potentially signi W cant in the treatment of neoplasticdiseases that depend on angiogenesis, also exhibit a hor-metic dose–response, with high doses inhibiting and lowdoses enhancing the in vitro proliferation of microvascularendothelial cells (Folkman 2007).In the same way, the immunostimulatory theory, pro-posed by Prehn many years ago, states that the immunereaction may be either stimulatory or inhibitory to tumorgrowth depending on the local ratio of immune reactants totumor cells, with high ratios rendering tumor inhibition,intermediate and very low rendering null e V  ects and lowratios (between intermediate and very low ones) renderingtumor stimulation (Prehn 1972, 2006, 2007). This theory, which is an application of the hormesis concept for immu-nological reactions, has been supported by numerous datafrom his and other laboratories (Chiarella etal. 2008a, b; Norbury 1977; Prehn 1976; Shearer and Parker 1978). Implications of having a stimulatory zone may be clini-cally signi W cant. In e V  ect, chemotherapeutic, anti-angiogenicand immunological agents designed to kill tumor cells orsuppress their proliferation in patients may eventuallyenhance tumor growth when the concentration of the anti-tumor agent is low (i.e., stimulatory) or reaches a low valuein the body some days after treatment is initiated. This couldbe an important problem for agents with a long half life.In this study, to know if the hormesis concept can beextended to other W elds of cancer biology, we studied thee V  ect of a primary tumor on the growth of secondary tumorimplants. This phenomenon has received little attentiondespite the fact that it has been detected in association withhuman cancers (Beecken etal. 2009; Lange etal. 1980; Southam 1968; Sugarbaker etal. 1977) and despite its pos- sible relevance to predicting the fate of metastatic foci intumor-bearing hosts on the basis that metastasis can appro-priately be considered as natural secondary tumor implantsin a tumor-bearing host (Beecken etal. 2009). Further, therelatively scanty number of papers addressing this phenom-enon has rendered controversial results, since inhibitoryand stimulatory e V  ects have been reported, depending onthe tumor models utilized (McAllister etal. 2008; O’Reillyetal. 1994; Ruggiero etal. 1985). We have used herein, two murine tumors of spontaneoussrcin and non-detectable immunogenicity because wewished to explore the phenomenon without confusion fromthe immune reaction and because murine spontaneoustumors could presumably best be compared with humantumors. We are going to demonstrate that a primary tumorcan exert both inhibitory and stimulatory e V  ects on thegrowth of secondary implants of the same tumor dependingon the relative masses of both tumors. Materials and methods AnimalsBALB/c mice, 3–5months old were used throughout. Theywere raised in our own colony and maintained on Coopera-tion pellets (San Nicolás, Buenos Aires, Argentina) andwater adlibitum. The care of animals was according to thepolicies of Academia Nacional de Medicina of Buenos Aires,Argentina (NIH Guide and Use of Laboratory Animals).TumorsLB: It is a non-immunogenic T-lymphoid leukemia-lym-phoma that arose spontaneously in a 6-month-old BALB/cmale. It was maintained by subcutaneous (s.c.) serial pas-sages in syngeneic mice. A more detailed description of this tumor is given elsewhere (Ruggiero etal. 1985; Zahalkaetal. 1993).CEI: It is a non-immunogenic undi V  erentiated epider-moid carcinoma, which arose spontaneously in a 12-month-old BALB/c female. It was maintained by syngeneic s.c.serial passages. More details of this tumor are given else-where (Meiss etal. 1986).Tumor volume was expressed according to the formulaof Attia and Weiss: volume=0.4 ( ab 2 ) where a  and b  rep-resent the larger and the smaller diameters, respectively(Meiss etal. 1986). Male mice were used in the experi-ments for LB tumor and female mice were utilized in theexperiments for CEI tumor.Histological studiesSkin with or without macroscopic tumor was removed and W xed in 15% formaldehyde, 5% acetic acid and 80% methanol.The tumor was sliced along the largest diameter and embeddedwith the overlying skin. Serial sections (3–5   m) wereobtained, stained with hematoxylin and eosin and studied.Mitotic number was evaluated per high power W eld(HPF) at £ 1,000 magni W cation in well-preserved areas(non-necrotic) with similar cell densities, while taking intoaccount mainly metaphase and anaphase. A measure of cellproliferation was obtained by immunostaining for Ki-67(Goat polyclonal M19; Santa Cruz), a nuclear protein thatis expressed in proliferating cells in the G1, S, G2 and Mphases and absent in the G0 phase. Staining was developedunder microscope with diaminobenzidine peroxidase  J Cancer Res Clin Oncol (2010) 136:1605–16151607  1 3 substrate. The number of cells expressing Ki-67 wasevaluated with HPF at £ 400. In each case, 50 W elds (HPF)were counted.MediumThe medium used was RPMI-1640 (Gibco, Grand Island,NY, USA), with penicillin G sodium (10   g/ml), strepto-mycin sulfate (25   g/ml) and amphotericin B as fungizone(25   g/ml). The medium was supplemented with 5% fetalcalf serum.SerumNormal and tumor-bearing mice were bled and the bloodwas kept at room temperature for 1h for clotting. Serumobtained after centrifugation was stored at ¡ 20°C until use.For [ 3 H] thymidine uptake assays, serum was decomple-mented at 56°C for 30min.[ 3 H] Thymidine uptake assayProliferation of tumor cells in 0.1ml of medium was deter-mined in 96-well microtiter plates (NUNC, Denmark) inthe presence of several twofold dilutions of serum fromnormal or tumor-bearing mice. Immediately afterward, thecultures were pulsed with [ 3 H] thymidine (Dupont, NENResearch Products, Boston, MA, USA) at a W nal concentra-tion of 1   Ci/ml, the mixture was incubated at 37°C for 18–24h in a 5% carbon dioxide humidi W ed atmosphere andharvested with an automated cell harvester. The radioactiv-ity incorporated into the cells was counted in a liquid scin-tillation beta counter (Beckman). The assays were carriedout in quadruplicate or sextuplicate.Cell-mediated cytotoxicity assayThis test was carried out as previously described (Francoetal. 1996). Brie X y, 0.1ml of 51 Cr-labeled tumor cells wasincubated with the same volume of di V  erent spleen cell sus-pension at an e V  ector ratio of 100:1, for 4h at 37°C in a 5%carbon dioxide humidi W ed atmosphere. Afterward, cells werecentrifuged and radioactivity in the supernatant was mea-sured in a gamma counter (Beckman). The percentage of spe-ci W c lysis was calculated as [(experimental c.p.m. ¡ normalc.p.m.)/(maximal c.p.m. ¡ normal c.p.m.)] £ 100.Serum fractionation  Dialysis Serum from normal and tumor-bearing mice was subjectedto dialysis (12,500 molecular weight cut-o V  ). Chromatography on Sephadex G-15 The dialyzable fraction of serum was concentrated bylyophilization, resuspended in 0.5ml of water and appliedto 66 £ 0.7cm chromatographic column of SephadexG-15; elution was performed with water with a 0.44mlmin ¡ 1 X ow rate. Each fraction obtained was assayed on in vitroproliferation of tumor cells using the [ 3 H] thymidine uptakeassay.Statistical analysisValues were expressed as mean § standard error (SE). TheStudent’s t   test was used and di V  erences were consideredsigni W cant when  p · 0.05. Results Kinetics of a primary LB tumorThe growth of LB tumor initiated with an s.c. inoculum of 10 6  LB cells is plotted in Fig.1. The tumor becomes ini-tially detectable on day 3 and then its size increases pro-gressively killing the host on, approximately, day 22.Biphasic e V  ect of a primary LB tumor on the growth of secondary LB tumor implantsA total of 42 mice received an s.c. implant of 1 £ 10 6  LBtumor cells in the right X ank on day 0 (primary tumor) andon days 0, 1, 2, 3, 4, 6 and 9, when the primary tumor vol-ume was 0, 0, 0, 10, 50, 250 and 700mm 3 , respectively.They received a secondary s.c. implant of 1 £ 10 5  LB cellsin the left X ank ( n =6 mice per group). Six mice that onlyreceived the s.c. implant of 1 £ 10 5  LB cells in the left Fig.1 Growth of s.c. LB tumor initiated with an inoculum of 1 £ 10 6 LB cells in the right X ank. Each point represents the mean § SE of 12mice 051015200100020003000 Days of tumor growth    T  u  m  o  r  v  o   l  u  m  e   (  m  m    3    )  1608J Cancer Res Clin Oncol (2010) 136:1605–1615  1 3 X ank (that is, not bearing the primary tumor) served ascontrols.As shown in Fig.2, the primary tumor produced di V  er-ent e V  ects on the second tumor growth, depending on theday on which the second implant was carried out. In e V  ect,when a second tumor was implanted on days 0 or 2, nodi V  erence as compared with the control was observed.When the second tumor was implanted on day 1, a signi W -cant enhancement was detected. Finally, when the secondtumor was implanted on day 3 onwards, a signi W cant inhi-bition was observed. The most profound inhibition wasdetected on days 6 and 9, when the primary tumor was thelargest at the time of the secondary tumor implant.Although on days 0, 1 and 2 no primary tumor mass wasmacroscopically detected, the mass of the primary tumor onthe day when the second tumor began to be perceptible Fig.2 Growth of a secondary LB tumor implant in primary LB tumor-bearing mice. The primary LB tumor was initiated with 1 £ 10 6  tumor cells inoculated in the right X ank on day 0. A sec-ondary tumor was implanted (1 £ 10 5  LB tumor cells) in the left X ank on days 0 ( a ), 1 ( b ), 2 ( c ), 3 ( d ), 4 ( e ), 6 ( f  ) or 9 ( g ) of primary tumor growth ( n =6 mice per group). Controls were six mice receiving only the inoc-ulum of 1 £ 10 5  LB cells in the left X ank. *  p <0.05, **  p <0.01, ***  p <0.001 2° Tumor at Day 0of 1° tumor growth 051015200100020003000 ControlPrimary tumor bearing A Days of second tumor growth    S  e  c  o  n   d   t  u  m  o  r  v  o   l  u  m  e   (  m  m    3    ) 2°Tumor at Day 1of 1° tumor growth 051015200100020003000 ControlPrimary tumor bearing B ** Days of second tumor growth    S  e  c  o  n   d   t  u  m  o  r  v  o   l  u  m  e   (  m  m    3    ) 2° Tumor at Day 2of 1° tumor growth 051015200100020003000 ControlPrimary tumor bearing C Days of second tumor growth    S  e  c  o  n   d   t  u  m  o  r  v  o   l  u  m  e   (  m  m    3    ) 2°Tumor at Day 3of 1° tumor growth 051015200100020003000 ControlPrimary tumor bearing D * Days of second tumor growth    S  e  c  o  n   d   t  u  m  o  r  v  o   l  u  m  e   (  m  m    3    ) 2° Tumor at Day 4of 1° tumor growth 051015200100020003000 ControlPrimary tumor bearing E *** Days of second tumor growth    S  e  c  o  n   d   t  u  m  o  r  v  o   l  u  m  e   (  m  m    3    ) 2° Tumor at Day 6of 1° tumor growth 051015200100020003000 ControlPrimary tumor bearing F ***** Days of second tumor growth    S  e  c  o  n   d   t  u  m  o  r  v  o   l  u  m  e   (  m  m    3    ) 2° Tumor at Day 9of 1° tumor growth 051015200100020003000 ControlPrimary tumor bearing G ***** Days of second tumor growth    S  e  c  o  n   d   t  u  m  o  r  v  o   l  u  m  e   (  m  m    3    )  J Cancer Res Clin Oncol (2010) 136:1605–16151609  1 3 (day 7 of second tumor growth, tumor volume 11mm 3 )was di V  erent in the cases in which the second tumor wasimplanted on day 0 (Fig.2a), 1 (Fig.2b) or 2 (Fig.2c), because the primary tumor had been inoculated 7, 8 or9days, respectively, before that day (Fig.1 shows thattumor volume on day 9 was larger than that on day 8 andthis was in turn larger than that on day 7 of tumor growth).That is, the secondary tumor growth could be eitherstimulated or inhibited, apparently depending on the ratiobetween the mass of the primary tumor relative to that of the second tumor implant, with high ratios (secondarytumor implanted on day 3 onwards) rendering inhibition,low ratios (secondary tumor implanted on day 1) inducingstimulation and intermediate or very low ratios (secondarytumor implanted on day 2 or 0, respectively) producing noe V  ect on secondary tumor growth.A hormetic or biphasic dose–response curve describingthese observations is depicted in Fig.3.Although the above-mentioned experiments suggest thehypothesis that inhibition or stimulation of secondarytumor implants depend on the relative tumor sizes betweenprimary and secondary tumors, it remains only an assump-tion, since other changes might well take place with thepassage of time other than or in addition to changes intumor size. To further support that hypothesis, mice bearinga primary tumor in the right X ank (initiated with an s.c.inoculum of 1 £ 10 6  LB tumor cells) received, at a constanttime (6days) after the primary tumor inoculation, a vari-able number of secondary inoculations in the left X ank (1 £ 10 5 , 1 £ 10 6  or 1 £ 10 7  LB tumor cells) and thegrowth of the secondary tumor was registered. As shown inFig.4, while secondary tumor implants of 1 £ 10 5  and1 £ 10 6  LB tumor cells were completely and partiallyinhibited, respectively, as compared with the correspondingcontrols, the growth of 1 £ 10 7  LB tumor cells wasenhanced, especially during the W rst days of growth. Fourdays after the secondary implant with 1 £ 10 7  LB tumor cells,the histological examination of the enhanced tumors revealed ahigher number of both mitosis (mean number per HPF § SE=2.02 § 0.13; n =50 W elds, £ 1,000 magni W cation) and Fig.3 Hormetic curve summarizing the stimulatory and inhibitorye V  ects induced by a primary LB tumor implanted in the right X ank (1 £ 10 6  tumor cells) on the growth of secondary LB tumor implanted(1 £ 10 5  tumor cells) in the left X ank, on selected days of primarytumor growth.  Dotted line  represents the tumor volume in control miceonly receiving the tumor implant in the left X ank. *  p <0.05,**  p <0.01, ***  p <0.001 02468100100200 ********** Days of LB primary tumor growth at whichsecondary tumor implants were carried out    2   °   L   B   t  u  m  o  r  v  o   l  u  m  e  a   t   d  a  y   1   5  a   f   t  e  r  s  e  c  o  n   d  a  r  y   t  u  m  o  r   i  m  p   l  a  n   t  a  s  a   %   o   f  c  o  n   t  r  o   l Fig.4 A total of 23 mice received an s.c. implant of 1 £ 10 6  LB cellsin the right X ank on day 0. Six days later (day 6), mice were s.c. chal-lenged with 1 £ 10 5  ( n =6) ( a ); 1 £ 10 6  ( n =6) ( b ) or 1 £ 10 7  ( n =11)( c ) LB cells in the left X ank. Controls only received 1 £ 10 5  ( n =6),1 £ 10 6  ( n =6) or 1 £ 10 7  ( n =15) LB cells in the left X ank. *  p <0.05,**  p <0.01, ***  p <0.001 10 5 024681012140100200300400500600700 ControlExperimetantal Group ****** A Days of second tumor growth    S  e  c  o  n   d   t  u  m  o  r  v  o   l  u  m  e   (  m  m    3    ) 10 6 02468101214050010001500 ControlExperimental Group ******** B Days of second tumor growth    S  e  c  o  n   d   t  u  m  o  r  v  o   l  u  m  e   (  m  m    3    ) 10 7 024681002505007501000 Experimental GroupControl * C Days of second tumor growth    S  e  c  o  n   d   t  u  m  o  r  v  o   l  u  m  e   (  m  m    3    )
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