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A molecular mechanism of complement resistance of human melanoma cells
Abstract:
The susceptibility of human melanoma cells to lysis by human complement after sensitization with the R24 murine IgG3 monoclonal antibody to the GD3 ganglioside antigen was investigated. It was found that the melanoma cell lines were either susceptible (greater than or equal to 70% cytotoxicity) or resistant (less than or equal to 30% cytotoxicity) to complement-mediated killing. We determined the kinetics of binding of C3 to and its subsequent fate on the melanoma cells. We found that on susceptible cell lines, maximal binding of C3 occurred within 10 min of incubation. At that time, approximately 90% of the bound C3 was in the form of C3b. During the subsequent incubation, the C3b was slowly inactivated, apparently generating the physiologic degradation products iC3b, C3dg, and C3d. However, this degradation of C3b could be inhibited without affecting the final degree of cytotoxicity, indicating that it is of no apparent consequence for the killing of susceptible melanoma cells. Very different results were obtained with resistant melanoma cells. Bound C3b was rapidly inactivated, and C3d was the predominant form of C3 on resistant cells throughout the incubation. Therefore, rapid inactivation of C3b was identified as a protective mechanism of human melanoma cells against complement attack. In addition, we found that resistance to complement is not an inherent property of the cells but depends on the antibody used for sensitization, because the resistant cell lines could be lysed after sensitization with polyclonal antiserum.
Insights
Human melanoma cells show varied susceptibility to complement-mediated lysis. Rapid inactivation of C3b protects resistant melanoma cells from complement attack, a mechanism dependent on the sensitizing antibody.
Area of Science:
- Immunology
- Oncology
- Complement System
Background:
- The complement system is crucial for innate immunity, mediating pathogen lysis and inflammation.
- Melanoma, a significant skin cancer, presents challenges in therapeutic targeting.
- Monoclonal antibodies targeting tumor antigens are investigated for cancer immunotherapy.
Purpose of the Study:
- To investigate the susceptibility of human melanoma cells to complement-mediated lysis after sensitization with a specific monoclonal antibody (R24) targeting the GD3 ganglioside antigen.
- To elucidate the mechanisms underlying complement resistance in melanoma cells.
- To determine the role of C3 deposition and degradation in complement-mediated killing of melanoma cells.
Main Methods:
- Sensitization of human melanoma cell lines with R24 murine IgG3 monoclonal antibody.
- Incubation with human complement to assess complement-mediated cytotoxicity.
- Kinetics analysis of C3 binding and degradation on melanoma cell surfaces using techniques like flow cytometry.
- Comparison of C3 deposition and inactivation patterns on susceptible versus resistant cell lines.
Main Results:
- Melanoma cell lines exhibited differential susceptibility to complement-mediated killing, categorized as susceptible (≥70% cytotoxicity) or resistant (≤30% cytotoxicity).
- On susceptible cells, maximal C3 binding occurred within 10 minutes, with C3b being the predominant form, followed by slow degradation.
- Resistant melanoma cells showed rapid inactivation of bound C3b, with C3d being the predominant C3 fragment, indicating a protective mechanism.
- Resistance was antibody-dependent, as resistant cell lines became susceptible to lysis when sensitized with polyclonal antiserum.
Conclusions:
- Rapid inactivation of C3b serves as a key protective mechanism for human melanoma cells against complement-mediated attack.
- Melanoma cell resistance to complement is not an intrinsic property but is influenced by the specific antibody used for sensitization.
- Understanding these mechanisms can inform the development of more effective antibody-based immunotherapies for melanoma.