title: Real-time intravital imaging establishes tumor-associated macrophages as the extraskeletal target of bisphosphonate action in cancer.
abstract: Recent clinical trials have shown that bisphosphonate drugs improve breast cancer patient survival independent of their antiresorptive effects on the skeleton. However, because bisphosphonates bind rapidly to bone mineral, the exact mechanisms of their antitumor action, particularly on cells outside of bone, remain unknown. Here, we used real-time intravital two-photon microscopy to show extensive leakage of fluorescent bisphosphonate from the vasculature in 4T1 mouse mammary tumors, where it initially binds to areas of small, granular microcalcifications that are engulfed by tumor-associated macrophages (TAM), but not tumor cells. Importantly, we also observed uptake of radiolabeled bisphosphonate in the primary breast tumor of a patient and showed the resected tumor to be infiltrated with TAMs and to contain similar granular microcalcifications. These data represent the first compelling in vivo evidence that bisphosphonates can target cells in tumors outside the skeleton and that their antitumor activity is likely to be mediated via TAMs.
  
  
  CONCLUSIONS
  Bisphosphonates are assumed to act solely in bone. However, mouse models and clinical trials show that they have surprising antitumor effects outside bone. We provide unequivocal evidence that bisphosphonates target TAMs, but not tumor cells, to exert their extraskeletal effects, offering a rationale for use in patients with early disease.
authors.affiliation: Garvan Institute of Medical Research and St. Vincent's Clinical School, Faculty of Medicine, UNSW Australia, Sydney, Australia.
authors.forename: Simon
authors.initials: S
authors.lastname: Junankar
authors.affiliation: Division of Applied Medicine, University of Aberdeen, Aberdeen, United Kingdom.
authors.forename: Gemma
authors.initials: G
authors.lastname: Shay
authors.affiliation: Garvan Institute of Medical Research and St. Vincent's Clinical School, Faculty of Medicine, UNSW Australia, Sydney, Australia.
authors.forename: Julie
authors.initials: J
authors.lastname: Jurczyluk
authors.affiliation: Garvan Institute of Medical Research and St. Vincent's Clinical School, Faculty of Medicine, UNSW Australia, Sydney, Australia.
authors.forename: Naveid
authors.initials: N
authors.lastname: Ali
authors.affiliation: Garvan Institute of Medical Research and St. Vincent's Clinical School, Faculty of Medicine, UNSW Australia, Sydney, Australia.
authors.forename: Jenny
authors.initials: J
authors.lastname: Down
authors.affiliation: Garvan Institute of Medical Research and St. Vincent's Clinical School, Faculty of Medicine, UNSW Australia, Sydney, Australia. Department of Nuclear Medicine, St. Vincent's Hospital, Sydney, Australia.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Pocock
authors.affiliation: Department of Pathology, St. Vincent's Hospital, Sydney, Australia.
authors.forename: Andrew
authors.initials: A
authors.lastname: Parker
authors.affiliation: Garvan Institute of Medical Research and St. Vincent's Clinical School, Faculty of Medicine, UNSW Australia, Sydney, Australia.
authors.forename: Akira
authors.initials: A
authors.lastname: Nguyen
authors.affiliation: University of Southern California, Los Angeles, California.
authors.forename: Shuting
authors.initials: S
authors.lastname: Sun
authors.affiliation: University of Southern California, Los Angeles, California.
authors.forename: Boris
authors.initials: B
authors.lastname: Kashemirov
authors.affiliation: University of Southern California, Los Angeles, California.
authors.forename: Charles E
authors.initials: CE
authors.lastname: McKenna
authors.affiliation: Garvan Institute of Medical Research and St. Vincent's Clinical School, Faculty of Medicine, UNSW Australia, Sydney, Australia.
authors.forename: Peter I
authors.initials: PI
authors.lastname: Croucher
authors.affiliation: Garvan Institute of Medical Research and St. Vincent's Clinical School, Faculty of Medicine, UNSW Australia, Sydney, Australia.
authors.forename: Alexander
authors.initials: A
authors.lastname: Swarbrick
authors.affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Katherine
authors.initials: K
authors.lastname: Weilbaecher
authors.affiliation: Garvan Institute of Medical Research and St. Vincent's Clinical School, Faculty of Medicine, UNSW Australia, Sydney, Australia.
authors.forename: Tri Giang
authors.initials: TG
authors.lastname: Phan
authors.affiliation: Garvan Institute of Medical Research and St. Vincent's Clinical School, Faculty of Medicine, UNSW Australia, Sydney, Australia. m.rogers@garvan.org.au.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Rogers
mesh_terms: D000818:Animals; D050071:Bone Density Conservation Agents / Q000378:metabolism* / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000175:diagnosis / Q000188:drug therapy / Q000276:immunology; D002114:Calcinosis; D002232:Carbocyanines; D004164:Diphosphonates / Q000378:metabolism* / Q000627:therapeutic use; D004195:Disease Models, Animal; D005260:Female; D006801:Humans; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008875:Middle Aged; D060787:Neoplasm Grading; D009361:Neoplasm Invasiveness; D009369:Neoplasms / Q000175:diagnosis* / Q000188:drug therapy; D010587:Phagocytosis / Q000276:immunology; D015899:Tomography, Emission-Computed, Single-Photon; D014057:Tomography, X-Ray Computed; D023041:Xenograft Model Antitumor Assays
chemical_list: C569686:Alexa Fluor 647; D050071:Bone Density Conservation Agents; D002232:Carbocyanines; D004164:Diphosphonates