? Most?of?our?mechanistic?understanding?of?how?human?cancer?cells?migrate?and?invade?has?been?obtained?by? observing?cell?behavior?in?an?artificial?2?D?environment.??Although?progress?has?been?made?using?this? approach,?important?new?evidence?indicates?that?cell?migration?in?2?D?systems?does?not?completely? recapitulate?events?associated?with?locomotion?in?a?more?physiological?environment?using?reconstituted?3?D? matrices?and?tissue?explants.??Work?by?others?and?novel?evidence?provided?in?this?research?proposal? demonstrate??invasive?cells?can?utilize?either?a?mesenchymal?type?of?cell?invasion?that?involves?formation?of?an? elongated?invadapodia?and?a?spindle?shaped?morphology?or?a?primitive?amoeboid??movement?that?involves? membrane?blebbing?though?small?holes?in?the?extracellular?matrix.??These?breakthrough?findings?prompted? the?hypothesis?that?cells?are?armed?with?different?invasive?programs?that?allow?them?to?traverse?complex? tissues?and?colonize?foreign?sites?in?the?body.??Most?importantly?though?these?findings?indicate?that? therapeutic?prevention?of?this?process?in?patients?will?require?a?multifaceted?approach?that?targets?both?modes? of?cell?invasion.??It?is?crucial?then?that?we?identify?invasive?mechanisms?utilized?by?disseminating?tumor?cells? in?vivo?so?that?the?appropriate?therapeutic?agent(s)?can?be?designed?to?completely?eradicate?the?spread?of? cancer?in?patients.??However,?tumor?cell?invasion?is?a?complex?and?dynamic?process?that?involves?the?intricate? interplay?between?the?tumor?cells?and?the?remodeling?vasculature?and?stroma.??Understanding?this?process?in? vivo?has?been?difficult?because?it?has?not?been?possible?to?visualize?this?process?in?high?resolution?in?live? animals.??To?address?this?problem,?we?have?developed?a?novel?model?of?cancer?progression?that?utilizes? human?cancer?cells?growing?in?optical?clear?zebrafish?genetically?engineered?to?express?green?fluorescent? protein?in?all?blood?vessels.??Using?this?model?and?dual?color?high?resolution?confocal?microscopy,?we? discovered?that?the?metastatic?gene?RhoC?induces?a?rapid?cell?invasion?process?that?facilitates?cell?intravasation? through?vascular?openings?induced?by?VEGF?secretion.??In?contrast,?mesenchymal?cell?invasion?involves? formation?of?elongated?invadopodia?and?membrane?integration?into?the?vascular?wall,?but?not?cell? intravasation.??Our?goal?in?the?proposed?work?is?to?understand?the?signaling?mechanism?that?control?amoeboid? and?mesenchymal?invasion?as?cells?intravasate?and?how?the?vascular?pores?form?in?response?to?VEGF? secretion.??Based?on?our?preliminary?findings?and?the?work?of?others,?we?hypothesize?that?the?metastatic?gene? RhoC?mediates?amoeboid?invasion?through?Rho?kinase?activity?(ROCK)?and?myosin?II?mediated?contractility.?? We?also?hypothesize?that?PI3K?harboring?activating?mutations?found?in?human?cancers?induces?mesenchymal? cell?invasion?through?activation?of?the?FAK?Src?CAS?Crk?Rac?signaling?module,?which?facilitates?actin? mediated?invadopodial?protrusion.??We?hypothesize?that?the?vascular?pores?form?through?disruption?of?cell? cell?junctions,?which?is?regulated?by?src?phosphorylation?of?VE?cadherin.??Therefore,?our?overall?goal?is?to? examine?in?detail?how?RhoC?and?mutated?PI3K?signaling?pathways?regulate?cancer?cell?invasion?and? intravasation?and?the?molecular?signaling?mechanisms?that?control?vascular?pore?formation.?? ?? Cancer?cells?spread?throughout?the?body?by?invading?into?blood?vessels?where?they?are?carried?to? distant?organs?and?form?secondary?tumors.??Work?in?this?proposal?will?determine?the?mechanism?of? how? cancer? cells? invade? through? the? vessel? wall? utilizing? high? resolution? confocal? imaging? of? optically?transparent?zebrafish?harboring?metastatic?human?cancer?cells.???
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