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Primary SeedingTesting the self-seeding hypothesiswith a mathematical modelJacob G Scott1,2, David Basanta1, Philip Gerlee...
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Selfseedposter mss2013

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Selfseedposter mss2013

  1. 1. Primary SeedingTesting the self-seeding hypothesiswith a mathematical modelJacob G Scott1,2, David Basanta1, Philip Gerlee3,4 & Alexander RA Anderson11. Integrated Mathematical Oncology, H. Lee Moffitt Cancer Center, USA 2. Centre for Mathematical Biology, Oxford University, UK3. Sahlgrenska Cancer Center, University of Gothenburg, Sweden 4.Mathematical Sciences, Chalmers University of Technology, SwedenLungs and heartBrainLiverGutBladder and prostateBoneβηγ, 20130011, published 20 February 2013102013J. R. Soc. InterfaceJacob G. Scott, David Basanta, Alexander R. A. Anderson and Philip Ggrowthsecondary metastatic deposits as drivers ofA mathematical model of tumour self-seedinon March 11, 2013rsif.royalsocietypublishing.orgDownloaded fromUnifying metastasis — integratingintravasation, circulation andend-organ colonizationJacob Scott1,2, Peter Kuhn3and Alexander R. A. Anderson1Abstract|Recenttechnologicaladvancesthathaveenabledthemeasurementofcirculatingtumourcells(CTCs)inpatientshavespurredinterestinthecirculatoryphaseofmetastasis.TechniquesthatdonotsolelyrelyonabloodsampleallowsubstantialbiologicalinterrogationbeyondsimplycountingCTCs.1Integrated MathematicalOncology, Moffitt CancerCenter, Tampa,Florida 33612, USA.2Oxford University Centre forMathematical Biology,Mathematical Institute,Oxford OX1 3LB, UK.3Department of Cell Biology,The Scripps ResearchInstitute, La Jolla,California 92037, USA.Correspondence to A.R.A.A.and J.S.e-mails: alexander.anderson@moffitt.org;jacob.scott@moffitt.orgdoi:10.1038/nrc3287Published online 24 May 2012In patients with advanced primary cancer, circulatingtumour cells (CTCs)1can be found throughout the entirevascular system2. When and where these CTCs formmetastasis is not fully understood, and is currently thesubject of intensive biological study. Paget’s well-knownseed–soil hypothesis3suggests that the ‘soil’ (the site ofa metastasis) is as important as the ‘seed’ (the metastaticcells) in the determination of successful metastasis. Themechanism by which seeds are disseminated to specificsoil has, to date, been a ‘known unknown’. We think thatit is during this poorly understood phase of metastasisthat we stand to answer important questions4.We hypothesize that the rich variety of possible meta-static disease patterns not only stems from the physicalaspects of the circulation but also from CTC hetero-geneity (FIG. 1). These seeds represent many differentpopulations that are derived from a diverse populationof competing phenotypes within the primary tumour5.Because such seeds need to pass through a system ofphysical and biological filters in the form of specificorgans, the circulatory phase of metastasis could bemodelled as a complex deterministic filter. In theory,until the evolution of a suitable seed, any number ofCTCs could flow through the circulation and arrestat end organs without metastases forming. As tumourheterogeneity is thought to expand as the tumour pro-gresses, it follows that at some point a seed will comeinto existence that is suited to a specific soil within thatpatient’s body. If this seed is to propagate it must findits soil, a process that we hypothesize is governed bysolvable physical rules that relate to the dynamics ofthe circulatory flow between different organs and howthese organs filter (not only by size, but probably also byother biological mechanisms). Although these biologi-cal mechanisms are not yet known, we might be able toinfer their existence by finding out which measurementsdo not fit a model that is defined only by physical flowand filtration.To begin the process of physical interrogation, wepropose a model that represents the human circulatorysystem as a directed and weighted network, with nodesrepresenting organs and edges representing arteries andveins.The novelty is only fully realized when combinedwith a heterogeneous CTC population (driven by primarytumour heterogeneity) modulated by the complex organfilter system (with physiologically relevant connections)under dynamic flow. Four important biological processesemerge from this representation. First, the shedding rate,which is defined as the rate at which the tumour shedsCTCs into the vasculature. Second, CTC heterogeneity,which is defined as the distribution of CTC phenotypespresent in the circulation. Third, the filtration fraction,which is defined as the proportion (and type) of CTCsthat arrest in a given organ. Fourth, the clearance rate,which is defined as the rate at which cancer cells arecleared from the blood and/or organ after arrest. Each ofthese biological processes is probably disease- and evenpatient-specific, and each is extremely poorly understood.Using this representation to motivate the develop-ment of a mathematical model we can define both theconcentration of CTCs and their phenotypic distribu-tion at any given point in the network, as well as organ-specific filtration values. To parameterize this model,characterization and enumeration of CTCs taken froma single patient at different time points and from differ-ent points in this network will need to be undertaken.A complete understanding of the model will also pro-vide information about the behaviour of the system asa whole. Specifically, the average lifespan of a CTC in apatient’s circulation will be able to be calculated with onlya minimum of measurements. Although this seems tobe a simple calculation, the scientific literature on thisNATURE REVIEWS | CANCER VOLUME 12 | JULY 2012 | 445astasis — integratingn, circulation andolonizationand Alexander R. A. Anderson1ladvancesthathaveenabledthemeasurementofcirculatinghavespurredinterestinthecirculatoryphaseofmetastasis.elyonabloodsampleallowsubstantialbiologicalinterrogationncer, circulatingughout the entirehese CTCs formd is currently theget’s well-known‘soil’ (the site ofd’ (the metastaticl metastasis. Thenated to specificwn’. We think thatase of metastasisstions4.of possible meta-rom the physicalm CTC hetero-many differentverse populationrimary tumour5.ugh a system ofform of specificastasis could befilter. In theory,any number ofation and arrestming. As tumourthe tumour pro-a seed will comec soil within thatgate it must finde is governed byhe dynamics oforgans and howprobably also bygh these biologi-might be able toh measurementsdo not fit a model that is defined only by physical flowand filtration.To begin the process of physical interrogation, wepropose a model that represents the human circulatorysystem as a directed and weighted network, with nodesrepresenting organs and edges representing arteries andveins.The novelty is only fully realized when combinedwith a heterogeneous CTC population (driven by primarytumour heterogeneity) modulated by the complex organfilter system (with physiologically relevant connections)under dynamic flow. Four important biological processesemerge from this representation. First, the shedding rate,which is defined as the rate at which the tumour shedsCTCs into the vasculature. Second, CTC heterogeneity,which is defined as the distribution of CTC phenotypespresent in the circulation. Third, the filtration fraction,which is defined as the proportion (and type) of CTCsthat arrest in a given organ. Fourth, the clearance rate,which is defined as the rate at which cancer cells arecleared from the blood and/or organ after arrest. Each ofthese biological processes is probably disease- and evenpatient-specific, and each is extremely poorly understood.Using this representation to motivate the develop-ment of a mathematical model we can define both theconcentration of CTCs and their phenotypic distribu-tion at any given point in the network, as well as organ-specific filtration values. To parameterize this model,characterization and enumeration of CTCs taken froma single patient at different time points and from differ-ent points in this network will need to be undertaken.A complete understanding of the model will also pro-vide information about the behaviour of the system asa whole. Specifically, the average lifespan of a CTC in apatient’s circulation will be able to be calculated with onlya minimum of measurements. Although this seems tobe a simple calculation, the scientific literature on thisVOLUME 12 | JULY 2012 | 445d into one mammary gland in mice to formor mass. Unlabeled MDA231-LM2 cells were inoc-ontralateral mammary gland to form a ‘‘recipient’’ame tumor (Figure 1A). After 60 days, the recipientxcised and examined for the presence of seedingns of ex vivo bioluminescence imaging (BLI).%) of the recipient tumors showed extensive seed-31-LM2 cells (Figure 1B and Table 1). Tumorsmore indolent MDA231 parental population weres MDA231-LM2 tumors at capturing seed cellsTable 1). No seeding was observed in mock-inoc-ary glands within the same time period (Figure 1C).ce microscopy analysis of MDA231 recipientmed the presence of numerous GFP+ MDA231-cells as distinct patches typically encompassinguarter of a tumor section (Figure 1D and data notn recipient tumors were generated using red-otein (RFP)-labeled cells, the infiltrating GFP+ cellswere observed intermingling with resident RFP+ cancer cells andwith unlabeled areas of presumptive tumor stroma (Figure 1E).Quantitative RT-PCR analysis of firefly-luciferase mRNA levelin seeded recipient tumors revealed that seeder cells accountedfor 5%–30% of the recipient tumor mass (data not shown).To establish the generality of this seeding phenomenon, weperformed similar experiments with different cancer cell lines.Recipient mammary tumors became seeded with high frequency(53% to 100% of mice) by donor tumors that were formed withbone-metastatic (MCF7-BoM2), lung-metastatic (MDA231-LM2), or brain-metastatic (CN34-BrM2) cells from different sub-types of breast cancer (basal, estrogen receptor-negativeMDA231 cells versus luminal, estrogen receptor-positive MCF7cells) or patient-derived malignant cell cultures (CN34 cells)(Figure 1B and Table 1). Seeding of a recipient tumor by its ownaggressive progeny was also observed between subcutaneoustumors formed by the human colon carcinoma line SW620 andits lung-metastatic derivative SW620-LM1, and between theing of Established Tumors by CTCsontralateral seeding experiment. Unlabeled and GFP/luciferase-expressing breast cancer cells were injected into contralateral No. 2 mammarypient tumor’’ and a ‘‘donor tumor,’’ respectively.t tumors extracted from mice bearing the indicated GFP/luciferase-expressing donor tumors. Color-range bars: photon flux. LM2: a lung-meta-of MDA231. MCF7-BoM2: a bone-metastatic derivative of MCF7. CN34-BrM2: a brain-metastatic derivative of pleural effusion CN34. PyMT:m mammary tumors developed in MMTV-PyMT transgenic mice.ree and tumor-bearing mammary glands from mice bearing GFP/luciferase-expressing donor tumors. n = 9–18. Error bars represent SEM.ns of seeded MDA231-LM2 tumors were visualized by fluorescence microscopy. An entire tumor section and a higher-magnification image (310)d are shown.al seeding experiment was performed with RFP- and GFP-expressing MDA231-LM2 cells. Frozen sections from RFP-labeled tumors wereconfocal microscopy at 320.test mammary tumor seeding from lung metastases. GFP/luciferase-expressing MDA231-LM2 cells were injected intravenously. Once lungestablished, unlabeled MDA231 cells were injected into a mammary gland No. 2.f CTCs derived from lung metastases in mice described in (F). Relative levels of CTC were plotted against the luminescent signals of recipientLI of three representative recipient tumors (i, ii, and iii) identified in the graph., 1315–1326, December 24, 2009 ª2009 Elsevier Inc.CTCs to infiltrate tumors in response to this attraction (Fig-ure 3E).To gain further insight into these attraction and infiltrationfunctions, we performed a trans-endothelial migration assay inwhich tumor cell-conditioned media were placed in the bottomwell of the chamber (Figure 4A). Media conditioned by MDA231breast carcinoma or A375 melanoma cells were several-foldmore active at stimulating the trans-endotheilal migration ofMDA231-LM2 cells than were media conditioned by MCF10Acells, a human breast epithelial cell line derived from untrans-formed tissue (Figure 4B). Similarly, A375-BoM2 melanoma cellsmigrated through endothelial cell layers more actively inresponseto these cancer cell-conditioned media than to media condi-tioned by HaCat cells (Figure 4B), a human keratinocyte cell linerepresenting the most abundant cell type in skin epidermis.Media from MDA231 and MDA231-LM2 cultures were equivalentas a source of attraction in these experiments (Figure 4C), whichis consistent with the equivalent ability of these two cell lines toact as recipient tumors in self-seeding assays (refer to Figure 1Band Table 1).MDA231 cells further stimulated the trans-endothelial migrationof MDA231-LM2 cells (Figure 4C). Parental MDA231 cellsshowed low trans-endothelial migration activity even in the pres-ence of media conditioned by tumor cells (Figure 4C). Similarly,the migration of A375-BoM2 cells through endothelial layerswas several-fold more efficient than that of the parental A375cells in the presence of conditioned media from A375 or A375-BoM2 (Figure 4C). These results demonstrated that cancer cellsrelease signals that attract their progeny across endotheliallayers. In addition, these results suggest that aggressive cancercells are superior to their more indolent counterparts in theirability to migrate in response to these signals.Tumor-Derived Mediators of Cancer Cell AttractionTo identify candidate tumor-derived attractants for CTCs, wecompared the secreted levels of 180 cytokines in conditionedmedia. This analysis uncovered several cytokines whoseproduction was higher (IL-6, IL-8, oncostatin M, and vascularendothelial growth factor [VEGF]) or lower (CCL2) in MDA231and its derivatives than in MCF10A cells (Figures 5A, S2A, andFigure 3. Tumor Attraction and Infiltration Functions(A) Unlabeled MDA231 cells were injected into a mammary gland No. 2. When tumors became palpable, LacZ/GFP/luciferase-expressing MDA231-LM2 cellswere introduced into the circulation by intracardiac injection.(B) BLI of mice with seeded and unseeded tumors. Arrow, recipient tumor.(C) Comparative tumor-seeding ability of MDA231 and MDA231-LM2 cells from the circulation. Luminescent signals from recipient tumors at the indicated timepoints are shown.(D) Luminescent signals of recipient tumors from mice injected with indicated cell lines were quantified 10 (MDA-231) and 5 (A375) days after injection. n = 6–10.(E) A diagram summarizing two functions involved in tumor self-seeding.Error bars in all cases represent SEM and p values were based on two-tailed Mann-Whitney test.Kim et al. (2009) Celland inoculated into one mammary gland in mice to forma ‘‘donor’’ tumor mass. Unlabeled MDA231-LM2 cells were inoc-ulated into a contralateral mammary gland to form a ‘‘recipient’’mass of the same tumor (Figure 1A). After 60 days, the recipienttumors were excised and examined for the presence of seedingcells by means of ex vivo bioluminescence imaging (BLI).A majority (85%) of the recipient tumors showed extensive seed-ing by MDA231-LM2 cells (Figure 1B and Table 1). Tumorsformed by the more indolent MDA231 parental population wereas effective as MDA231-LM2 tumors at capturing seed cells(Figure 1B and Table 1). No seeding was observed in mock-inoc-ulated mammary glands within the same time period (Figure 1C).Fluorescence microscopy analysis of MDA231 recipienttumors confirmed the presence of numerous GFP+ MDA231-LM2 seeding cells as distinct patches typically encompassingless than a quarter of a tumor section (Figure 1D and data notshown). When recipient tumors were generated using red-fluorescent protein (RFP)-labeled cells, the infiltrating GFP+ cellswere observed intermingling with resident RFP+ cancer cells andwith unlabeled areas of presumptive tumor stroma (Figure 1E).Quantitative RT-PCR analysis of firefly-luciferase mRNA levelin seeded recipient tumors revealed that seeder cells accountedfor 5%–30% of the recipient tumor mass (data not shown).To establish the generality of this seeding phenomenon, weperformed similar experiments with different cancer cell lines.Recipient mammary tumors became seeded with high frequency(53% to 100% of mice) by donor tumors that were formed withbone-metastatic (MCF7-BoM2), lung-metastatic (MDA231-LM2), or brain-metastatic (CN34-BrM2) cells from different sub-types of breast cancer (basal, estrogen receptor-negativeMDA231 cells versus luminal, estrogen receptor-positive MCF7cells) or patient-derived malignant cell cultures (CN34 cells)(Figure 1B and Table 1). Seeding of a recipient tumor by its ownaggressive progeny was also observed between subcutaneoustumors formed by the human colon carcinoma line SW620 andits lung-metastatic derivative SW620-LM1, and between theFigure 1. Seeding of Established Tumors by CTCs(A) A diagram of contralateral seeding experiment. Unlabeled and GFP/luciferase-expressing breast cancer cells were injected into contralateral No. 2 mammaryglands as a ‘‘recipient tumor’’ and a ‘‘donor tumor,’’ respectively.(B) BLI of recipient tumors extracted from mice bearing the indicated GFP/luciferase-expressing donor tumors. Color-range bars: photon flux. LM2: a lung-meta-static derivative of MDA231. MCF7-BoM2: a bone-metastatic derivative of MCF7. CN34-BrM2: a brain-metastatic derivative of pleural effusion CN34. PyMT:cells derived from mammary tumors developed in MMTV-PyMT transgenic mice.(C) BLI of tumor-free and tumor-bearing mammary glands from mice bearing GFP/luciferase-expressing donor tumors. n = 9–18. Error bars represent SEM.(D) Frozen sections of seeded MDA231-LM2 tumors were visualized by fluorescence microscopy. An entire tumor section and a higher-magnification image (310)of a selected field are shown.(E) A contralateral seeding experiment was performed with RFP- and GFP-expressing MDA231-LM2 cells. Frozen sections from RFP-labeled tumors werevisualized under confocal microscopy at 320.(F) A diagram to test mammary tumor seeding from lung metastases. GFP/luciferase-expressing MDA231-LM2 cells were injected intravenously. Once lungmetastases were established, unlabeled MDA231 cells were injected into a mammary gland No. 2.(G) Left: burden of CTCs derived from lung metastases in mice described in (F). Relative levels of CTC were plotted against the luminescent signals of recipienttumors. Right: BLI of three representative recipient tumors (i, ii, and iii) identified in the graph.1316 Cell 139, 1315–1326, December 24, 2009 ª2009 Elsevier Inc.Metastatic disease accounts for thelion’s share of cancer deaths, yet it isa process that remains poorlyunderstood. Many theories ofmetastasis have been posited in‘cartoon’ form. These include thewell known ‘seed soil’ hypothesis, theidea that removal of the primarytumor somehow increases thegrowth of metastasis and mostrecently, the ‘self-seeding’ hypothesis(right). In this work, we aim to testthe ‘self-seeding’ hypothesis with atheoretical construct we recentlyposited (below).Evidence for ‘self-seeding’?Models of metastasis should not ignore known vascular connectivityScott et al.Scott et al.A simple model derived fromNorton et al. (Nature Med 2006)iterated on the vascular networkData on CTC prevalence in vascularnetwork taken from literature: anopportunity for future personalization?Results suggest Secondary Seeding ismore likely the mechanism behind ‘self-seeding’. This suggests that treatmentof subclinical micromets in specificorgans (organ directed therapy) couldbe predicted to have clinical utility givenpatient specific parameters.Tumor simulation dynamicspp λλshedding rateλreturn ratep

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