Kinetics of metastatic breast cancer cell trafficking in bone.
OBJECTIVE
In vivo studies have focused on the latter stages of the bone metastatic process (osteolysis), whereas little is known about earlier events, e.g., arrival, localization, and initial colonization. Defining these initial steps may potentially identify the critical points susceptible to therapeutic intervention.


METHODS
MDA-MB-435 human breast cancer cells engineered with green fluorescent protein were injected into the cardiac left ventricle of athymic mice. Femurs were analyzed by fluorescence microscopy, immunohistochemistry, real-time PCR, flow cytometry, and histomorphometry at times ranging from 1 hour to 6 weeks.


RESULTS
Single cells were found in distal metaphyses at 1 hour postinjection and remained as single cells up to 72 hours. Diaphyseal arrest occurred rarely and few cells remained there after 24 hours. At 1 week, numerous foci (2-10 cells) were observed, mostly adjacent to osteoblast-like cells. By 2 weeks, fewer but larger foci (> or =50 cells) were seen. Most bones had a single large mass at 4 weeks (originating from a colony or coalescing foci) which extended into the diaphysis by 4 to 6 weeks. Little change (<20%) in osteoblast or osteoclast numbers was observed at 2 weeks, but at 4 to 6 weeks, osteoblasts were dramatically reduced (8% of control), whereas osteoclasts were reduced modestly (to approximately 60% of control).


CONCLUSIONS
Early arrest in metaphysis and minimal retention in diaphysis highlight the importance of the local milieu in determining metastatic potential. These results extend the Seed and Soil hypothesis by demonstrating both intertissue and intratissue differences governing metastatic location. Ours is the first in vivo evidence that tumor cells influence not only osteoclasts, as widely believed, but also eliminate functional osteoblasts, thereby restructuring the bone microenvironment to favor osteolysis. The data may also explain why patients receiving bisphosphonates fail to heal bone despite inhibiting resorption, implying that concurrent strategies that restore osteoblast function are needed to effectively treat osteolytic bone metastases.
Department of Pathology, Comprehensive Cancer Center, Center for Metabolic Bone Disease, National Foundation for Cancer Research, Center for Metastasis Research, University of Alabama at Birmingham, Birmingham, Alabama 35294-0019, USA.
Pushkar A
PA
Phadke
Robyn R
RR
Mercer
John F
JF
Harms
Yujiang
Y
Jia
Andra R
AR
Frost
Jennifer L
JL
Jewell
Karen M
KM
Bussard
Shakira
S
Nelson
Cynthia
C
Moore
John C
JC
Kappes
Carol V
CV
Gay
Andrea M
AM
Mastro
Danny R
DR
Welch
D000818:Animals; D001859:Bone Neoplasms / Q000556:secondary*; D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor / Q000637:transplantation; D005260:Female; D005269:Femur / Q000473:pathology; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D051379:Mice; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D010006:Osteoblasts / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins