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ls cancer a disease of self-seeding?Larry Norton & foan Massagu6

The high cell density, rapid growth rate and large population size of cancer are conventionally attributed to apathologically high ratio of cell production to cell death. Yet these features might also or instead result frominappropriate cell movement, already understood to underlie invasion and metastasis. This integrating concept couldinduce a broadening of our existing anticancer pharmacopoeia, which, with mitosis as its predominant target, is nowseldom curative.

Although epithelial cancers are diverse geno-tlpically and phenoryaically, several featuresare universall-5.One of these is the abnormalcapacity of cancer cells to migrate, which manifests in two ways. The first is invasion: the cells'ability to become dislodged and travel withinthe tissue of origin. The second, metastasis,involves travel beyond the tissue of origin.Aberrant cell mobility is thought to be distinctfrom (while sharing some molecular pathwayswith) another universal feature: an increasedratio ofproliferation to cell deathr,6,7. This lat-ter paired abnormality, rather than cell mobil-ity, is thought to underlie tumorigenesis, theexpanded generation of cancer cells that pro-duces large, destructive masses. So, the currentview of cancer incorporates a set of overlap-ping dichotomies: invasion versus metastaticspread, both relating to cell mobility; tumori-genesis versus metastatic behavior, the formerthought to result from cell proliferation, thelatter from cell movement.

Here we seek to unifir these dichotomies byconsidering the possibiliry that pathologic cellmobility, in addition to behg crucial to tumorinvasion and metastatic dissemination, canalso contribute significantly to primary tumorgrowth. Moreover, the mechanism by whichcell mobility can increase tumor size can alsoproduce high cell density and rapid grouth rate.

Lary \o'tor is in lre Depa'r'rerrs or Meoicire,Vemor al Sloan-Kenerirg Carcer Ce.ter aro tl-eWeill College of Med cine of Cornell University, Newvork, New Yor( 10021. USA ano Joa^ lvassagLes in the Cancer B ology and Genet cs Programand Howard Hughes Medlca Institute, MemorialSloan-Kettering Cancer Center, New York, New York1002i, usA.[-marl I nortonpub@mskcc.org

We call this proposed mechanism'self-seeding',a term used in botany to describe the abilityof plants to overgrow an ecosystem. Considerhow a mass of weeds dominates a field: not bythe massive increase in size of individual weedplants, but rather by the continuous propaga-tion of new weeds both within the weed bed(density) and at its periphery (invasion). Incancer, this concept would translate to escapeecells that constantly re-seed a tumor mass,making that mass a dense collection of con-tiguous small growths that inflltrate and poten-tially destroy its host organ. Furthermore, justas a self-seedingweed population might spreadbeyond its field of origin, so might cancers seeddistant metastases.

The logic of self-seedingMany of the faculties that permit distant seed-ing could logically enable self-seeding (Fig. I ).These include separating from an anatomicmooring,lysing a proteinaceous and carbohy-drate matrix, intravasating, circulating, adher-ing to an endothelium, extravasating, attachingin a target location, inducing angiogenesis andpropagating in the target environmentl. Seedsmight depart from the primary site of tumorformation or from a metastatic site. Returningto the site of origin would constitute self-seed-ing or re-seeding, whereas lodging in another(distant) site would constitute the process ofmetastasis.

Cancer invasion is symbolized in Figure Iby pathways A and D. Metastasis is syrnbolizedby pathway C. Here we hlpothesize the exis-tence of pathways B and E, through which adislodged, wandering cancer cell, having accessto the entire systemic circulation, has a finiteprobability of returning to its site of birth. Tobe sure, this return trip could well be faced with

an unfavorable circulation pattern, includingencounters with intricate, tortuous capillarybeds that may retain the wandering cells beforethey can return to their site of origin. Thosecells that do manage the return trip, however,would find themselves in thewelcoming micro-environment in which they first developed orin which they took rootl-s. The co-evolution ofcancer cells and their stroma that creates sucha microenvironment is often termed'field can-cerization', and maybe reflected in the presenceof tumor and stroma-specific gene expression

._ q l)Datrerns" -'.The molecular mediators of self-seeding

in the primary site (pathways A and/or B)and self-seeding in metastatic sites (pathwaysD and/or E) might be partially overlapping.Successful self-seeding might well require thecontin ual gene ration of sel l-renewi n g p rogeny.Therefore, we envision that some proportionof self-seeds may be acting the role of, or besynonpnous with, cancer'stem' cells, moreaccurately called tumor-initiating ce11s13,14.And, to the extent that normal stem cells mayundergo tumorigenic mutations, their intrinsicself-renewing and migratory properties mightturn such cells into higtrly effective self-seeds.

An appealing aspect of the concept of self-seeding is that it could explain diverse char-acteristics of cancer, extending the analogy toa weed-strewn garden. Dysplasia could be theconsequence of the tumor being a spatiallydisoriented conglomerate of functionallyindependent smaller masses. As self-seededloci continue to self-seed, the result wouldbe more and younger loci of new growthand hence more histologic disorganizationas well as a preponderance of immature cells.Furthermore, independent masses at theperiphery of a cancer would be de facto points

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of invasion whether they arose by pathways Aor B in the primary site, or pathways D or E ina metastatic site. Each new growing compo-nent of the conglomerate would tend to attractits own independentblood supply, promotinghypervascularityls. If self-seeds return to theprimary tumor's organ of origin but do notattach to the mass of the primary tumoq thiswould convey the appearance of multifocal-ity. (Of course, true multifocality-multiplepoints of primary carcinogenesis-may alsocoexist with this process.) As the cells estab-lishing these noncontiguous seeded loci aresamples of the heterogeneous collection ofcells already present, they may be sufficientlydistinct that they convey the appearance ofpolyclonality. Moreover, once established, sat-ellite lesions could further evolve and inter-mingle, consistent with the observation thatmultifocal lesions often seem to be polyclonal.Self-seeding might also be relevant to thehotly debated question of whether a primarytumor exhibits the expression signature of itsmetastatic descendents: self-seeding wouldtend to equalize the molecular profiles of anaggressive tumor and its metastases, theirdegree of similarity increasing proportionalto the degree to which self-seeding accountsfor the growing mass of the primary tumor.

The mathematics of self-seedingSelf-seeding could explain increased celldensity, high mitotic rate and large tumorsize by reference to two related biomath-ematical concepts: fractal geometry andGompertzian kinetics. Biological structuresare best described mathematically by fractalgeometry rather than.the Euclidian geom-etry of regular solid masses like spheresand cubesl6'17. By Euclidian geometry thevolume of a biologic mass increases by thecube of its diameter. In contrast, by fractalgeometry the number of cells in that massincreases by a power constant less thanthree. Hence, the ratio of cell number tovolume (termed 'cell density') falls as thevolume increases. Because of this, a fullydeveloped normal organ, or even a largesection of that adult organ, would in mostcases have a lower total cell density than thewhole organ in its primordial, smaller state.In contrast, if-as self-seeding would sug-gest-a cancerous mass arising from thatorgan is a conglomerate of small, incipi-ent, component masses, each would have asmall volume and hence a high cell density'creating a high cell density in the cumula-tive whole. (An important exception to thisgeneral rule is discussed below.)

The abnormally high cell density found inmost cancers is labeled'hyperproliferation'

4{

A*Tff;t n"X

Metaslatictumor

Systemic circulalion

Figure I The self seeding concept of cancer growth and metastasis. The primary tumor mass on thelower left is a composite of three smaller sub tumors: the central, larger one is a proliferating collectionof cells at the original site of carcinogenesis; the other two sub-tumors on the lower left are the progenyof self-seeds that spread from ihat original site. Because each of ihe three is relatively small comparedwith the volume of their sum, they are relatively both dense and rapidly growing (see text). A self-seedto the primary tumor may follow pathway A, comprised of dislodging (orange arrow) and proteolysisof the extracellular matrix (yellow arrow) followed by reattachment in or at the primary site, thenproliferation and angiogenesis. Or the self-seed might follow pathway B, which includes intravasation(green anow), circulation and extravasation (purple arrow), then return to the primary tumor bed,proliferation and angiogenesis. A cell following pathway C traces the same steps, but relocates to ametastatic siie, where growth can occur by proliferation, angiogenesis and self-seedtng at that site,either directly (pathway D) or via circulatory flow (pathway E). The expressed gene sets responsible forthese processes may be shared completely or partially. For example, pathways B and C may require thesame biochemical processes, and hence the same gene expression pattern, with the final destinaiion(primary or metastatic sites) determined stochastically. Or pathway C may use the same genes aspathway B, but with some additional properties. The genes responsible for paihways D or E may besignificantly overexpressed, whereas the genes associated with pathways A or B may not, which wouldexplain virulent growth in a metastatic but not the primary site. Many oiher patterns of local and distantgrowth are consistent with the model, as discussed in the text.

because its microscopic appearance-more to imperceptible further growth (Fig' 2).cells per unit of space-is regarded properly Gompertzian kinetics has proven useful asasevidenceofmorecelldivisionsinconjunc- well as applicable in the clinic, particularlytion with relatively fewer cell deaths. This in the design of improved cancer treatmentdoes not, however, imply that the regulation regimensls.of mitosis or apoptosis is necessarilyabnor- If a malignant tumol is a conglomeratemal. An alternative cause of high cell density of component Gompertzian masses, eachcould be the cells' normal response to the with a relatively high growth rate because it'start-up'nature of the small tumor seedlings is relatively small, then the whole conglom-comprising the malignant conglomerate. erate-being the sum of its parts-would

One of the consequences of high cell den- have a high growth rate as well. In addition, asity is a high density of proliferating cells. conglomerate of small Gompertzian tumorsThis might be one reason why small-vol- would grow large because it would sum theume masses grow mole rapidly relative to many individual Gompertzian plateaus oftheir sizes than masses of larger volume, as the components. Hence, the concept of self-first described mathematically by Benjamin seeding provides an explanation for both theGompertzl8. This phenomenon results in a increased growth rate and the increased totalsigmoid growth curve on an arithmetic plot volume of malignancy without needing toof size versus time, with population size even- hlpothesize deviant regulation of mitosis ortually approaching a plateau phase of slow cell death at the individual cellular level.

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The relationship between ptoliferationand self-seedingThe above analysis does not ignore the obvi-ous facts that abnormalities in mitotic regu-lation and an aberrant ability to survive suchstresses as cell-cycle checkpoint alterations,hypoxia, glucose deprivation or interstitialfluid pressure changes are commonly foundin cancer cel1sl,6. We merely indicate thatabnormal cell mobility could contribute tophenomena previously attributed entirely toother processes. Furthermore, it is possiblethat hlperactive mitogenic pathways and eva-sion of growth inhibitory constraints may benecessary but not sufficient for overt carcino-genesis. Hyperproliferation is also common inmany benign conditions such as dermatosesand premalignant lesions. Furthermore, micegenetically engineered to overexpress purelymitogenic oncogenes characteristically demon-strate benign hlperplasias in many organsT'1e.Tiue cancers arise from some, but only some, ofthe cells in these genetically altered organs. Tobecome malignant, therefore, l"umors requireadditional abilities, which maybe present fromthe outset (for example, in normal tissue stemcells that undergo transformation) or may beacquired later. We argue that one of these abili-ties is the capacity to self-seed. In this regard,the observation that some potent oncogenessimultaneously disrupt both cell adhesion(part ofthe seeding process) and mitotic-apop-totic regulation may explain their efficienry incausing virulent cancetT'19. That the expres-sion of matrix metalloproteases (also part ofthe seeding process) maybe sufficient to causemalignant transformation in transgenic mice issimilarly supportive of our concept2-1.

Increased proliferation is clearly an adverseprognostic marker in many types of epithe-lial cancer2o. Although this is usually thoughtto reflect the cancer cells' mitotic-apoptoticdysfunction, we may wish to consider that italso could reflect the aggressiveness of self-seeding. As more vigorous self-seeding wouldproduce more loci of growthwithin the tumorconglomerate, it would result in faster overallgrowth rate as well as larger potential totaltumor volume. Furthermore, and perhapsmost importantly, a self-seeding etiology ofhlperproliferation would correlate well witha tumor's metastatic ability, which is, after all,the prime cause of poor prognosis in clinicalcancer.

This line of reasoning may cast new light onone of the clinical truisms of epithelial can-cer, that large primary tumor size is a poorprognostic feature. The conventional view isthat cancers gain metastatic ability throughan accumulation of mutations as they grow tolarge size. Yet perhaps some tumors grow to be

large, and have a poor prognosis, because theyseed aggressively to self and to distant sites.Cancers may not be bad because they are big;they may be big because they are bad.

The molecular links between metastasisand self-seedingLending credence to the mathematical reason-ing above is recent molecular evidence that theability of a wandering cancer cell to re-seed itsoriginal mass may be linked to the ability toseed distant sites. Primary tumors frequentlyexpress genes whose products alter the micro-environment, such as extracellular matrix pro-teases, glycosylases, proangiogenesis factors,regulators of cell adhesion, and mediators ofinflammation and angiogenesisl s'7'e-14'1e.Gene expression signatures that include thesegenes predict metastatic behavior and hencepoor prognosis in breast cancers9-12. Indeed,so important is the abiliry of a cancer to alter itsenvi ron m ent that poor-prognosis gene-expres-sion signatures are emerging which largelyexclude genes that purely mediate proliferationor survivalll'12.

It is commonly assumed that these environ-ment-altering genes are associated with poorprognosis only because they are responsiblefor metastatic behavior. However, althoughhuman breast cancer xenografts with highmetastatic capacity in the laboratory do dem-onstrate a gene expression profile associatedwith metastatic behavior (and hence poorprognosis) in the clinic, so do cell clones withless prominent metastatic behaviorl9'21'22.We therefore consider that the genes in the'poor-prognosis' environment-altering setmay underlie the self-seeding that permitsgrowth in the primary site and also lay thefoundation for distant seeding. It should beexpected, therefore, that genes in addition tothose in the poor-prognosis set are neededto promote growth in metastatic sites, andin fact there is evidence that this is the case.In highly metastatic laboratory models ofhuman cancer, there are genes that are bothhighly expressed and responsible for site-specific metastases22. Some of these genes arealso found to be expressed in human primarytumors that show a high risk of developingmetastasis to the lung22.

Of particular importance in this regard isthe experimental evidence that a subset oflungmetastasis-associated genes also contribute totumor growth in the mammary gland22. Thisfinding may be interpreted as follows: theseparticular genes may act not only as metasta-sis-producing, distant-seeding genes (pathwaysC and possibly D and/or E in Fig. 1), but alsoas tumor self-seeding genes (pathways B andpossibly A). Furthermore, a different and dis-

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8x

2x

01020304050Arbitrary time scale

Figure 2 Gompertzian growth curves, This patternof growth is ubiquitous in nature. As the numberof cells increases with time the growth rate relativeto the number of cells decreases, eventuallyapproaching zero as the number of cells approachesa limiting plateau size (1010 in this example). lf atumor is comprised of many Gompertzian masses,each arising from a self-seed, rather than one mass,as would occur in the absence of self-seeding, theconglomerate's maximum size will be the sum ofmany plateaus, which could be very large, possibleeven lethal. Self seeding might cause Gompertziangrowth itself . Gompertz's original equation isshown as the solid line18. The dashed line, whichis almost indistinguishable from the solid line, is anew equation (below) in which proliferation occursin an anatomic region of the mass that has a lowerfractal dimension than the region where apoptosisoccurs. Self-seeding would create such a patternsince the seeds, approaching the mass from theoutside (pathway B in Fig. 1) or migrating outward(pathway A), would congregate on the periphery (orleading edge) of the growing mass rather ihan themass's volume. d/V/df = h1 . /Vt(a/c) - h2 . /Vt(b/c).The constants are h1 > h2 > 0, and 3 >c > b > a >2. (ln this example, a = 2.8, b = c = 2.9.)

tinct gene subset is associated with the growthof metastases in the lung but not in the pri-marysite22. Perhaps some of these genes,whichmight be associated with pathway C, are caus-ing self-seeding (pathways D and/or E) in themetastatic masses but not in the primarv site(pathways A and/or B).

Some natural history implications of self-seedingThe concept that many genes may be involvedin these processes could explain some of thecomplex and enigmatic phenotypes that areobserved in nature. For example, one couldenvision a cancer that expresses metastasis-permissive genes (pathway C in Fig. 1) butlacks prominent capability to colonize meta-static sites (pathways D and/or E). This wouldresult in the production of latent metastasesrestricted in their volume expansion. Perhapsthis is why some individuals with breast cancerwho have cells of epithelial origin in their bonemarrow presumed to be breast cancer metas-tases, never demonstrate clinically significantosseous disease.

Another eccentric phenotlpe would bethat of a cancer with such intense self-seedine

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power (pathways A and B) that it would neverform a discrete tumor mass in the primary site,but rather demonstrate a diffuse infiltrationof the organ of origin. This would provide anexception to the general rule cited above thatcancer cells form dense masses. As the genesthat underlie such behavior maywell be associ-ated also with pathways C, D and E, we shouldexpect to see virulent metastases in many dis-tant organs. Many adenocarcinomas of thepancreas may act in this way. In other cancert)?es, metastases in organs with especiallycancer-supporting stroma may also exhibitsuch behavior. If a cancer's pathways C andD and/or E are most prominent, the diseasecould become widely metastatic while neverdemonstrating a large or even a discernableprimary mass. Indeed, on rare occasions wedo observe breast and lung cancers that actlike this. Another possibility is that a primarycancer could spin offa cell qpe with the capac-ify to strongly self-seed (pathways D and/or E)onlyin one spot in one distant organ. In such acase the individual would benefit considerablyfrom resection or irradiation of that solitarymetastasis in addition to control of the pri-mary tumor. Hence, variations on the themesdepicted in Figure I could well encompassdiverse clinical presentations.

It must be emphasized, however, that all ofthe above examples ofdiscrepancies in growthcharacteristics between primary and metastaticsites are exceptions to the general ruie. In thevast preponderance of cases of clinical cancerthere is such a consistent association of largetumor size, anaplasia, rapid growth rate andmetastatic behavior that it leads one to hypoth-esize. as we have, that the molecular roots ofthese phenomena are likely to be the same orvery closely related.

A therapeutic implication of self-seedingBecause of our community's historic focus oncell proliferation as the core aberrancy in can-cer, almost al1 anticancer drugs now in com-mon use were developed as antiproliferativeinterventions. These drugs have unquestion-ably proven usefi-rl because they shrink tumors,which improves prognosis bypostponing deathor delaying recurrence after resectioni8'20. Cureof established epithelial cancers is uncommon,

however, and even the advances we have madeir the use ofpostsurgical adjuvant drug therapymay reflect time delays (as regrowth proceedsfrom residual cells) rather than eradicationof all cells in some individuals with cancerts.Moreover, whatever benefits are gained fromthe use of antimitotic drugs are often at thecost of considerable toxicity because cellularproliferation is so intrinsic to the viability andfunction of most normal tissues.

The fact that our current therapeutic strat-egies have been largely disappointing to dateshould not surprise us if further research con-firms that seeding as well as mitosis is at thecore of malignanry. Only recently have clinicaloncologists started to explore agents directedagainst targets other than those involved incell proliferation. Antiangiogenesis is onesuch example. In these clinical trials, however,our bias toward mitosis as a primary targetis evident in our tendency to combine newtherapeutics with antiproliferative agents,usually chemotherapy23 2s. This may notbe an ideal long-term strategy. In addition,neoangiogenesis is only one of many possibletargets along the paths to successful self-seed-ing, and may not be the best, as it is far downthe pathway. But as we develop antiseedingdrugs we must beware of new toxicities, aswe might be disrupting certain crucial physi-ological processes-wound healing and gam-ete production being examples.

ConclusionExperimental and theoretical results aresuggesting the possibility that the processof self-seeding, a putative close relative ofthe process of metastasis, may influence orunderlie many important features of epithe-lial cancer. These include high ceil density,invasion, multifocality, amplified growthrate, enlarged cell-population size, and thecapacity to spread to and reproduce thesefeatures in distant organ sites. Testing thevalidity of this hypothesis will require acombination of approaches. For example, itwould be of interest to see whether tumorsize is causally linked to the effect of metas-tasis-gene expression signatures. We alsoneed to determine whether metastatic tumormasses have the ability to attract circulating

cells released from the same tumor, andwhether this self-seeding ability, contribut-ing to tumor growth, depends on pro-meta-static genes. Yet ifvalidated by these or othermeans, this new concept would help us tobetter understand malignant diseases andperhaps to discover how to manage themmore effectively.

ACKNOWLEDGMENTSThe authors express their appreciation to A.B. Olshenfor his review of mathematical details, to T.J. Rileyfor drawing the figures, and to C. Pearce for editingthe manuscript. We also acknowledge with gratitudediscussions with our principal P0 1 -CA94060-04colleagues R. Benezra, M. Iasin, S. Larson,M. Moynahan, N. Rosen and H. Varmus and withJ. Bromberg, G. Gupta, C. Hudis, R. Kurtz,A. Minn,D. Spriggs and C. Van Poznak.

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