Combined flow cytometry and confocal laser scanning microscopy for evaluation of BR96 antibody cancer cell targeting and internalization.
BACKGROUND
Monoclonal antibodies (mAb) are important tools in the management of tumor disease, and the discovery of antibodies with both specific cancer cell targeting and capacity to enter the cells by internalization are critical to improve the therapeutic efficacy.


METHODS
Antibody cancer cell targeting and internalization properties of fluoroscein-conjugated mAb made against Lewis Y (BR96) were evaluated quantitatively and qualitatively by means of flow cytometry (FCM) and confocal laser scanning microscopy (CLSM), respectively, on cells from a rat tumor cell line (BN7005-H1D2).


RESULTS
The study demonstrated a specific binding of BR96 to LewisY (LeY) located in the cell membrane and as BR96/LeY immunocomplexes (BR96/LeY) internalized into the cytoplasm. BR96/LeY was internalized into about 15% of the cells, usually distributed throughout the cytoplasm, but also located close to the nuclei. Cytotoxic effects by BR96 were indicated, and CLSM visualized subpopulations containing cells with bound or internalized BR96/LeY that possessed morphologically pyknotic nuclei and disrupted DNA.


CONCLUSIONS
The spatial-temporal pattern by BR96 cell targeting and internalization processes of BR96/LeY into the cancer cells expressing LeY was demonstrated by FCM and CLSM. Used together, the FCM and CLSM techniques provide a valuable tool for preclinical analyses of antibody targeting and their capacities as carriers of cytotoxic conjugates for the use in cancer therapy.
Feridani
Amir H Iranpour
AH
Department of Oncology, Lund University, Sweden. Amir.Iranpour@med.lu.se
Holmqvist
Bo
B
Sjögren
Hans-Olov
HO
Strand
Sven-Erik
SE
Tennvall
Jan
J
Baldetorp
Bo
B
D021581:Active Transport, Cell Nucleus; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism* / Q000633:toxicity; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003110:Colonic Neoplasms / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D003593:Cytoplasm / Q000378:metabolism; D004249:DNA Damage; D004337:Drug Carriers; D005434:Flow Cytometry / Q000379:methods*; D007983:Lewis Blood Group Antigens / Q000276:immunology*; D018613:Microscopy, Confocal / Q000379:methods*; D051381:Rats; D013997:Time Factors
D000911:Antibodies, Monoclonal; C507851:BR96 monoclonal antibody; D004337:Drug Carriers; D007983:Lewis Blood Group Antigens; C094162:Lewis Y antigen