Studies on tumor-associated immunoglobulins
A direct and indirect radio-immune antiglobulin tests (RIAT) were developed and used 'to study tumor-associated immunoglobulin (TAIg) levels in transplanted freshly excised solid tumors. The studies were performed on 8-12 week old inbred CBA mice. The tumors investigated were grown from freshly excised and cultured CCH1 and T3 fibrosarcoma tumor cells. The RIATs developed were based on isotopic antiglobulin tests which had been used in this department to determine circulating anti¬ tumor antibodies in tumor-bearing mice. They were modified and improved in order to be used to detect in vivo bound TAIgs in solid tumors. The indirect RIAT developed was used in both quantitative and qualitative TAIg studies. The more sensitive direct RIAT was used solely in quantitative studies on TAIgs. In order to optimize the test a number of variableswere investigated. These included the method of tumor cell preparation and the influence of reagent concentration, tumor cell number, and the period of incubation. Furthermore, attempts were made to reduce nonspecific binding of reagents by precoating the plastic plates and tubes with various protein containing media. As a result of these investigations, techniques were developed which were sufficiently sensitive for semi-quantitative and semi-qualitative studies. In vivo bound TAIgs in solid tumors and its variation with age and size of tumors was assessed by both the direct and indirect RIATs. The more immunogenic CCH1 tumors were found to possSss higher levels of TAIgs than the less immunogenic T3 tumors. Moreover, tumors produced by the injection of tumor cells contaminated with host cells were found to possess higher levels of TAIgs than tumors produced by cultured tumor cells which are devoid of host cells. Similarly, tumor cells prepared in different ways(enzyme vs mechanical) were found to produce tumors of different TAIg content. In addition, the in vivo binding of TAIgs was found in general to vary positively with the age and size of the tumors. The injection of 1-5 million tumor cells was found to produce larger tumors ?of higher TAIg content than the injection of 0.1-0.5 million tumor cells. Similar effects were noted whether the tumors were compared in different mice or on opposite limbs of the same mouse. Comparative results were also obtained by a double radio—immunoassay test which was carried out in parallel with the RIATs. The amount of in vivo bound TAIg in solid tumors was thus found to be dependent on immunogenicity, method of maintenance (ie. in vivo or in vitro), and the method or preparation of the tumor cell inoculum. The cellular basis of TAIgs was studied in immuno-stimulated and immuno-deficient mice. Intraperitoneal G.parvum (CP) administration resulted in an increase of TAIgs in both CCK1 and T3 tumor models, though this effect was more marked in the former. Moreover, additional studies revealed that CP treated CCH1 tumors had higher levels of cell-surface bound IgA, IgG2b, and IgG3 than non-treated CCH1 tumors. Parallel qualitative studies on CCH1 tumor extracts showed that CP treated tumors also had more IgGl, IgA and IgM. Studies were also performed in thymectomized and nude mice in order to assess the effect of T-cell deprivation on the in vivo binding of TAIgs. The TAIg content in tumors grown in T-cell deficient mice was shown not to differ from tumors grown in normal mice. Preliminary experiments were also conducted on mice treated with gold salts in order to determine the influence of macrophages in TAIg binding. No significant difference was obtained between tumors grown in gold-salt treated mice and nontreated mice. Moreover, whole body irradiation (400 rads) 24 hrs, before the injection of tumor cells was not found to appreciably effect the in vivo binding of TAIg. The significance of these results and their relevance to tumor immunodiagnosis and immunotherapy are discussed.