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Combined flow cytometry and confocal laser scanning microscopy for evaluation of BR96 antibody cancer cell targeting
Amir H Iranpour Feridani1, Bo Holmqvist, Hans-Olov Sjögren
1Department of Oncology, Lund University, Sweden. Amir.Iranpour@med.lu.se
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.
Method:
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.
Conclusion:
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.
Insights
Monoclonal antibodies targeting cancer cells, like BR96, can internalize into tumor cells. This antibody-drug conjugate delivery mechanism shows potential for cancer therapy by inducing cytotoxic effects.
Area of Science:
- Oncology
- Immunology
- Biotechnology
Background:
- Monoclonal antibodies (mAb) are crucial for cancer management.
- Targeting specific cancer cells and achieving intracellular delivery are key to enhancing therapeutic outcomes.
Purpose of the Study:
- To evaluate the cancer cell targeting and internalization capabilities of a specific monoclonal antibody, BR96.
- To assess the potential of BR96 as a carrier for cytotoxic conjugates in cancer therapy.
Main Methods:
- Utilized flow cytometry (FCM) and confocal laser scanning microscopy (CLSM) for quantitative and qualitative analysis.
- Studied fluorescein-conjugated BR96 mAb targeting the Lewis Y (LeY) antigen on a rat tumor cell line (BN7005-H1D2).
Main Results:
- BR96 specifically bound to LeY on the cell membrane and was internalized into the cytoplasm as BR96/LeY immunocomplexes in approximately 15% of cells.
- Internalized BR96/LeY localized within the cytoplasm, sometimes near the nucleus.
- Evidence of BR96-induced cytotoxicity was observed, with affected cells showing pyknotic nuclei and disrupted DNA.
Conclusions:
- FCM and CLSM effectively demonstrated the spatial-temporal dynamics of BR96 targeting and internalization in LeY-expressing cancer cells.
- These combined techniques offer a valuable preclinical tool for analyzing antibody targeting and their efficacy as cytotoxic conjugate carriers in cancer treatment.
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