Oncology 34 : 53-57 (1977)
Immunological Studies of Induced Tumors of the Rat Submandibular Gland S. E l-Mo fit Facility of Dentistry, Cairo University, Cairo
Key Words. Salivary gland tumors - Tumor-specific antigens — Chemical carcinogenesis - Salivary glands
Introduction One of the basic concepts in the immunology of cancer is that in a normal animal there is a ’’serveillance system” responsible for the inhibition or elimination of aberrant cells protecting the host against the outgrowth of potentially neoplastic cells. This concept is based on the assumption that tumor cells are different antigenically from normal cells and that under proper conditions the immunologic mechanism of the host is capable of recognizing the difference. The presence of antigenic differ ences between normal and tumor cells has been the first criterion in demonstrating the validity of this assumption. Studies on different tissues indicated that induction of a variety of tumors resulted in loss or deletion of tissue-specific antigens [1—4], and it was postulated that loss of tissue-specific antigens resulted in the cells escaping the tissue homeostatic control and regulatory mechanisms, and thus undergoing malignant transformation. Furthermore, the presence of additional antigens associated with malignant transformation was observ ed in many tumors. The appearance of these tumor-specific antigens has been demonstrated by different techniques in a wide variety of experimentally-induced as well as in spon taneous malignant tumors [5-7]. Thus, malignant transforma tion is characterized by morphologic alteration in the tissues as well as alterations of their antigenic profile. These alter ations do not only involve changes in concentration of existing antigens and disappearance of some of them but also the appearance of new ones.
Only few studies have dealt with oral structures and only in direct evidence suggests the importance of immunologic mechanisms in tumorigenesis of these structures. The import ance of the role played by the immunologic mechanism in the development of oral tumors has been suggested primarily as a result of studies dealing with effect of different agents capable of non-specifically depressing the immune response on the development of chemically-induced tumors. It has been demonstrated that chemical carcinogenesis of the submandi bular gland [8] and buccal pouch [9, 10] has been accelerated by the systemic use of cortisone. Although the tumors induced in animals injected with cortisone presented the same histo logic characteristics as those in the control animals, invasion of the underlying tissue was more extensive and the tumors developed to a larger size. Administration of the antimeta bolite, methotrexate, in animals treated with 9,10 dimethyl1,2 benzanthracene (DMBA) resulted in the development of a more anaplastic carcinoma in the cheek pouch which developed more rapidly and invaded deeper than DMBA con trol animals [11]. Administration of the antimetabolite, azathioprine, enhanced the carcinogenic action of DMBA upon the buccal pouch mucosa of the hamster. The carcinoma developed more rapidly and tended to be of the anaplastic variety rather than the well-differentiated carcinoma usually found in DMBA treated animals [12]. These observations are similar to those reported in animals injected with specific antilymphocyte serum [13, 14|. It has been postulated that suppression of the immunologic response allows the chemically-induced neoplasm to develop more rapidly and to develop into a more anaplastic carcinoma [15]. The predominance of anaplastic cells led G iunta and S hklar [14] to raise the possibility of an antigenic difference between the anaplastic and well-differentiated types of tumors. In recent studies, the presence of antigenic differences be tween normal and tumor cells has been shown in oral epitheli um. C h av ez and T o to [16] noted an alteration in cell surface antigens associated with DMBA-induced carcinoma of the hamster cheek pouch. These alterations were detected by decrease of the intensity of immunofluorescent staining of the tumor cells in comparison with normal cells, when they were evaluated with rabbit antiserum to normal cheek pouch. Furthermore, it has been shown, using immunofluorescence, that blood group antigens which are normally present on the cell membranes of human oral epithelial cells, disappear in most oral carcinomas [17] and premalignant lesions [18]. Previous studies have demonstrated that saline extracts of
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Abstract. The antigenic activity of rat submandibular gland carcinoma induced by 9,10 dimethyl-1,2 benzanthracene (DMBA) has been evaluated using rabbit antisera to normal submandibular gland extract and to extracts of the induced tumors. Extracts of all tumors and premalignant lesions show reduction in the concentration of several of the intrinsic antigens of the normal submandibular gland when they were evaluated by imunodiffusion and immunoelectrophoresis. Although all the induced tumors, including those used for antisera preparation, are well-differentiated epidermoid carcinoma, a consid erable variation is present in the antigenic pattern of each tumor. When antisera to several tumors, absorbed with rat serum and kidney extract, were used in studying extracts of all induced tumors, antigenic bands were observed. The number of these bands varies from three to five. All the induced carcinomas share the presence of two of these bands in the a and (f-globulin region, the remaining bands differ from one tumor to another.
El-Mofty: Immunology of Submandibular Gland Tumors
54
Materials and Methods Tumors of the submandibular gland were induced in four month old, inbred male Fischer rats. The animals were an esthetized with diethyl ether and a midline incision was made on the ventral surface of the neck. The right submandibular gland was exposed and a 15 gauge syringe needle was used to insert a pellet (5 mg) of the carcinogen 9,10 dimethyl-1,2 benazanthracene (DMBA), (Calbiochem, Los Angeles, Cali fornia) in powder form in the body of the gland according to the technique described by C a ia l d o and Shklar [21], The incision was sutured and the animals were maintained on Lab Chow and water ad libitum. Starting at four weeks, groups of four animals together with two sham operated controls were sacrificed every two weeks up to and including 20 weeks. The animal was anesthetized with diethyl ether and blood was collected from the aorta. The salivary glands were removed and weighed individually. The pellet of DMBA was removed from the submandibular gland and the lesion was cut in half. One half was fixed and prepared for histological and histochemical examination. The remaining portion of the tissue was quick-frozen and used for studying the antigenic characteristics of these tumors. Autopsy was performed on all the animals and lymph nodes, lung, liver, spleen and pancreas were fixed and prepared for histological examination. Histologic examinations revealed that the chemical carcinogen induced a constant sequence of events consisting of degener ation, metaplasia and neoplasia. The submandibular glands of the rats sacrificed at four-week intervals showed degene ration, hyperplasia and metaplasia. At six-week intervals, two rats developed squamous cell carcinoma while the glands of the other two animals showed hyperplastic and metaplastic changes. At eight-week intervals, three rats out of four devel oped squamous cell carcinoma, the remaining rat showed only hyperplasia and metaplasia. All animals in which the carcinogen was implanted for ten weeks or more developed carcinoma. The induced tumors were all well differentiated squamous cell carcinoma [22].
The submandibular glands as well as other tissues and blood of normal adult male rats were collected. The tumors and nor mal glands were homogenized in saline (1 mg/g of tissue), using a Sorvall Omnimixer at 0°C. The homogenates were centrifuged at 10,000 rpm for 10 min and supernatant was collected. Extracts of other rat tissue, e. g., kidney, liver and spleen were prepared in the same manner. The protein con centration of the saline extracts was determined by the method of L o w ry etal. [23]. Eight adult male New Zealand rabbits, 2-3 kg in weight, were used for preparing the antisera. Antisera to normal sub mandibular gland and to the induced tumors were prepared by injecting the rabbits intracutaneously and subcutaneously into sites on the back and shoulders. Four rabbits were injected with extract of pooled normal submandibular glands, and the remaining four rabbits were injected with tumor extracts, each was inoculated with extract of a different tumor. The extracts were emulsified with an equal volume of Freund’s complete adjuvant (Difco Laboratories, Detroit, Michigan) for the first injection and into incomplete adjuvant for sub sequent injections. Each rabbit received a weekly injection of about 10 mg of protein of the extract. When satisfactory titers were obtained, the animals were exsanguinated, a week to ten days after the last injection. Also, blood of unimunized rabbits was collected to be used as a control. Blood was allow ed to clot for one hour then the serum was separated by centri fugation and stored at -20 °C. Antisera were absorbed with lyophilized rat serum in a concentration of 80 mg/ml, which was found to be a proper concentration for neutralization pur poses by a preliminary gel diffusion test. The absorption mix tures were incubated for 30 min at 37 °C and then centrifuged to remove the precipitates. When necessary, further absorp tion was carried out using lyophilized extracts of rat kidney. Two-dimensional immunodiffusion as described by O u c h t e r LONY [24] was carried out using disposable Petri dishes (Falcon Plastics, Culver City, California) and the reactions were read two to three days later. Immunoelectrophoresis was carried out according to the method of G ra bar and B urtin [25] as modified by ScHElDEGGER [26] using Agafor immunoelectrophoresis apparatus (National Instruments Laboratories, Rockville, Maryland). One per cent agarose in 0.05 m barbital buffer at pH 8.6 was used. After the antigens were placed in the wells, a constant current of 4 mA milliamperes per slide was applied for two hours. Diffusion of the antisera was allowed to proceed for two to three days.
Results Preliminary screening by immunodiffusion of sera of all rabbits inoculated with extracts of normal submandibular gland and of induced squamous cell carcinoma of the gland exhibited several lines of precipitation when reacted with ex tracts of normal submandibular gland, of the induced tumors, as well as of glands showing premalignant changes. Absorption of the antisera with lyophilized rat serum and extract of rat kidney abolished the reaction with the majority of rat tissue extracts and demonstrated that rat submandibular gland contained at least five intrinsic antigenic components
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normal rat submandibular gland tissue contain at least five instrinsic antigenic components as well as other antigens, shared with blood serum and tissues of the rat. Of these anti gens, two to three were specific for the submandibular gland, and the remaining antigens were shared to varying degrees with extracts of related exocrine glands: parotid, sublingual, pancreas, extraorbital and intraorbital lacrimal [19, 20). These findings encouraged us to investigate the effect of malignant transformation on the tissue-specific antigens of the rat submandibular gland, particularly since it appears that no previous studies have evaluated changes in the salivary gland antigenic profile associated with such transformation and the possible appearance of tumor-specific antigens. The primary objective of the present study was to investigate the antigenic relationship between saline extractable antigens of induced tumors in comparison with those of the normal rat submandibular gland. It is aimed to monitor any change in the tissue-specific antigens in association with malignant trans formation, as well as to evaluate the possibility of appearance of new antigenic components in these tumors.
El-Mofty: Immunology of Submandibular Gland Tumors
as well as other antigens shared with blood serum and tissues of the rat [20], No difference was found between normal submandibular gland from normal rats, glands from shamoperated rats and normal mates to submandibular glands in which the tumors were induced. For the purpose of compari son, extracts of 33 induced carcinomas and seven submandi bular glands with premalignant alterations, i. e., hyperplasia and metaplasia, were evaluated with rabbit antisera to normal rat submandibular gland absorbed with rat serum and kidney. Extracts of all the tumors showed decrease of concentration of some of the submandibular gland intrinsic antigens with considerable variation noted in the antigenic pattern of each tumor (figure 1). Evaluation of premalignant lesions also revealed decrease of concentration of some antigenic com ponents with the antigenic pattern, varying from one lesion to another. Immunodiffusion and immunoelectrophoretic studies were carried out to monitor the appearance of tumor-specific antigens, using antisera to induced tumors. The antisera pre pared in four rabbits by injection of four different tumors, were all tested versus extracts of all the induced tumors and premalignant lesions. All DMBA-induced tumors gave preci pitation bands with the different antisera absorbed with nor mal rat serum and kidney extract. Immunoelectrophoretic evaluation of extracts of the tumors demonstrated the pres ence of three to five precipitation bands. While the immuniz ing antigens were from well-differentiated squamous cell carcinoma and the examined tumors were all well-differen tiated squamous cell carcinoma, the immunoelectrophoretic
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patterns varied from one tumor to another. However, all shared the presence of two distinct precipitation bands in the a and (5-globulin region. Two precipitation bands could be seen on evaluating extracts of normal rat submandibular gland and extracts of premalignant lesions with antisera to tumor extract. Figure 2 depicts the pattern obtained on eval uating normal submandibular gland and tumor extracts from different rats with absorbed antisera. Absorption of the antisera with normal submandibular gland extract did not exhaust the antigenic capacity of the antiserum against extracts of the tumors. After absorption with lyophilized normal rat serum, the antisera showed no reaction with normal sera, nor with sera from tumorbearing animals. Ab sorption of an antiserum with a tumor extract does not exhaust its antigenic capacity against all other tumors, particularly the one used in preparing the antiserum. On the other hand, absorption of an antiserum with the same tumor used for its preparation exhausted its antigenic capacity against all other tumors. No reaction was found between sera from tumor bearing animals and different tumors, including tumors of the same animals.
Discussion Implantation of powdered DMBA in the submandibular glands of inbred Fischer rats resulted in degeneration, prolifer ation, metaplasia and neoplasia. All animals in which the carcinogen was implanted for ten weeks or more, developed
Figure 2. Immunoelectrophoresis in agar gel. Troughs contain rabbit antiserum to carcinoma of the rat submandibular gland absorbed with rat scrum and kid ney extract. The first well from the left (N) contains normal sub mandibular gland extract. The remaining wells contain extracts of induced carcinomas.
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Figure 1. Immunoelectrophoresis in agar gel. Troughs contain rabbit anti-submandibular gland extract absorbed with rat serum and kidney extract. The first well from the left (N) contains normal rat submandibular gland extract. The remaining wells contain extracts of induced carcinomas.
El-Mofty: Immunology of Submandibular Gland Tumors
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host cannot be dismissed, particularly since these tumors arc characterized by their localized invasiveness, lack of metastasis and failure to be transplanted to syngeneic hosts [22, 34], Since the morphology and biological behavior of these induced tumors has been shown to be similar to human oral car cinoma [34], further studies with this system of chemical car cinogenesis and immunologic alterations might provide an ef fective model system for investigation of oral tumor.
References 1
2
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W eii .f.r , E.: Loss of specific cell antigen in relation to carcino genesis. In Wolstenholme and O ’Connor Ciba Foundation Symposium on Carcinogenesis: Mechanisms of Action: p. 165 (Churchill, London 1959). Nairn , R.; R ichmond, H. G.; Mc E ntegart , M. G., and T othergill , J. E.: Immunological differences between normal and malignant cells. Brit. med. J. ii: 1335-1340 (1960). A bei.ev , G. I.: Antigenic structure of chemically-induced hepatomas. Prog. exp. Tumor Res. 7:104—108 (1965). A lbin , R. J.: Immunologic studies of normal, benign, and malignant human prostatic tissue. Cancer 29: 1570-1574 (1972). P rehn , R. T.: Cancer antigens in tumors induced by chemicals. Fed. Proc. 2 4 :1018-1022 (1965). Baldwin, R. W. and Barker, C. R.: Tumor-specific antigeni city of aminazo-dyc-induced rat hepatomas. Int. J. Cancer 2: 355-364(1967). Keller , J. A.; Lo, A. C., and Kolos, T. O.: Immunoelectrophoretic analysis of DMBA-induced rat mammary tumors. Oncology 25:401-409 (1971). A nbari, N.; Shki.ar , G., and Cataldo , E.: The effect of systemically administered cortisone on salivary gland carcino genesis in the rat. J. dent. Res. 44:1056 (1965). Shklar, G.: Cortisone and hamster buccal pouch carcino genesis. Cancer Res. 36:2461-2463 (1966). Shklar, G.: The effect of cortisone on the induction and development of hamster buccal pouch carcinomas. Oral Surg. 23: 241-248 (1967). Shklar, G.; Cataldo , E., and F itzgerald , A. L.: The effect of methotrexate on chemical carcinogenesis of hamster buccal pouch. Cancer Res. 26:2218-2224 (1966). Sheehan , R.; Shklar, G., and T ennenbaum , R.: Azathioprine effects on the development of hamster pouch carcinomas. Arch. Path. 91:264-270 (1971). Woods , D. A.: Influence of antilymphocyte serum on DMBA induction of oral carcinomas. Nature 224:276-277 (1967). G iunta , J. L. and Shklar, G.: The effect of antilymphocyte serum on experimental hamster buccal pouch carcinogenesis. Oral Surg. 31:344-353 (1971). Shki.ar , G.: Recent advances in experimental oral and salivary gland tumors. J. oral Surg. 28:495-500 (1970). Chavez , R. F. and Toto , P. D.: Staining differences between normal and carcinogen induced carcinomas of the hamster pouch. J. dent. Res. 49:721-724 (1970). D abelsteen, E. and P indborg, J. J.: Loss of epithelial blood group substance A in oral carcinomas. Acta path, microbiol. scand. Sect. A, 81:435-444 (1973). D abei^stf.en , E.; R oed -P etersen, B.. and P indborg, J. J.: Loss of epithelial blood group antigens A and B in oral premalig nant lesions. Acta path, microbiol. scand. Sect. A, 83: 292-300 (1975). E l -M ofty. S.: Immunologic tissue- and species-specificity of the rat submaxillary gland. Fed. Proc. 29:304(1971).
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squamous cell carcinoma. The induced squamous cell carcin oma lesions were all well-differentiated, did not metastasize, and could not be transplanted to syngeneic hosts [22]. Histological studies revealed that the induced tumors were very similar to one another morphologically. However, evaluation of their antigenic profile demonstrated consider able difference from one tumor to another, whether they were studied by antiserum to normal submandibular gland or to tumor extract. In the present investigation, it was noted that malignant transformation was associated with decrease of concentration of several of the tissue-specific antigens pre viously identified to be characteristic of the normal rat sub mandibular gland [19, 20], These findings are in general agreement with results obtained from tumors induced in other organs [2, 4], However, it should be emphasized that alteration of antigenic characteristics of the cells and neo plastic transformation are not necessarily interdependent and that such alteration has been noted in epithelial cells taking part in wound healing [27] and in parotid gland following denervation [28]. Thus, it is more probable that these antigenic changes are associated with other factors such as growth rate, rather than malignancy per se. It is of interest to notice that these antigenic alterations were noted in lesions representing preneoplastic changes; before malignant transformation has taken place and that the anti genic properties of the preneoplastic cells are carried over into the resulting tumors. The present findings are in agreement with previous studies on premalignant lesions of other tissues [29, 30]. The value of the presence of antigenic alteration in premalignant lesions in predicting the prognosis of the condition is still to be evaluated. The presence of additional antigens associated with malignant transformation has also been observed in these tumors. Of the three to five tumorspecific precipitation bands observed in each tumor, two were present in all of them, and the re maining bands were specific for each tumor. The tumorspecific antigens were different from those present in normal gland; and traceable after absorption with normal rat serum and organs. The possibility that some of these tumor-specific antigens are of embryonic origin cannot be ruled out [31] but remains to be proven. This is particularly of interest since it has been shown that postnatal morphologic development of rat submandibular gland is associated with parallel antigenic alteration [32], The appearance of immunogenic proteins undetectable in the organs of normal rats can be the expression of protein neo synthesis due to an altered genetic information as a result of the carcinogenic effect of DMBA. The observation that although the induced tumors were very similar morphologic ally, yet each tumor has its own characteristic antigenic pro file, agrees with previous studies on other tissues that demon strated that chemically induced tumors in contrast to virus induced tumors, are characterized by their highly specific antigenicity [33], The absence in tumor-bearing rats of autoimmunoantisera directed at their own neoplasm can be explained either by insufficient sensitivity of the method used or by continuous absorption of the antibodies formed by the tumor mass. How ever, the possibility that the antigenic alterations noted in these tumors play a role in the immunologic response of the
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El-Mofty: Immunology of Submandibular Gland Tumors
21 22 23
24
25 26 27
28
E l-M ofty, S.: Tissue- and species-specific antigens of the rat submandibular gland. Experientia 30:301-302 (1974). Cataldo, E. and Shklar, G.: Chemical carcinogenesis in the hamster submaxillary, gland. J. dent. Res. 43:568-579(1964). E l-Mofty, S.: Chemical carcinogenesis in the rat submandi bular gland (in Press). Lowry, O. H.; Rosf.brough , N. J.; Farr , A. L. and R andall , R. J.: Protein measurement with the folin phenol reagent. J. biol. Chem. 193:265-275 (1951). O uchterlony, O.: In vitro method for testing the toxinproducing capacity of diphtheria bacteria. Acta path, microbiol. scand. Sect. A, 2 5 :186-191 (1948). G rabar, P. et B urtin, P.: Analyse immuno-électrophorétique (Masson, Paris 1960). Schneidegger , J. J.: Une micro-méthode de l’immuno électrophorèse Int. Arch. Allergy 7: 103-110 (1955). D abelsteen, E. and F ejerskov, O.: Loss of epithelial blood group antigen A during wound healing in oral mucous mem brane. Acta path, microbiol. scand. Sect. A, 82: 431-434 (1974). E l-M ofty, S. and Schneyer, C. A.: Alteration of antigenic components of rat parotid gland following denervation. Ex perientia 30:950-951 (1974).
29
B urtin, P.; Martin , E.; Sabine , M. C., and Von Kleist, S.:
Immunological study of polyps of the colon. J. nat. Cancer Inst. 48:25-29 (1972). 30
31
32
33 34
H alpin, T. Z .; Vaage , J., and B lair, P. B.: Lack of anti genicity of mammary tumors induced by carcinogens in a nonantigenic preneoplastic lesion. Cancer Res. 32: 2197-2200 (1972). G old , P. and F reeman , S.: Specific carcinoembryonic anti
gens of the human digestive system. J. exp. Med. 122: 467-481 (1965). E l-M ofty, S.: Changes in antigenic profile of the rat sub maxillary gland during postnatal development. Int. Ass. dent. Res. 50:800(1972). Klein, G.: Experimental studies in tumor immunology. Fed. Proc. 28:1739-1753 (1969). B rown, A. M.: A comparative assay of the in vitro and in vivo behavior of three chemically induced neoplasmas of rat sub mandibular glands. J. oral Path. 2:355-364 (1973).
Request reprints from: S. E l-Mofty , BchD, PhD, 28 Abd El-Azim Rashed Str., Agouza, Cairo (Egypt)
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Immunological Studies of Induced Tumors of the Rat Submandibular Gland S. E l-Mo fit Facility of Dentistry, Cairo University, Cairo
Key Words. Salivary gland tumors - Tumor-specific antigens — Chemical carcinogenesis - Salivary glands
Introduction One of the basic concepts in the immunology of cancer is that in a normal animal there is a ’’serveillance system” responsible for the inhibition or elimination of aberrant cells protecting the host against the outgrowth of potentially neoplastic cells. This concept is based on the assumption that tumor cells are different antigenically from normal cells and that under proper conditions the immunologic mechanism of the host is capable of recognizing the difference. The presence of antigenic differ ences between normal and tumor cells has been the first criterion in demonstrating the validity of this assumption. Studies on different tissues indicated that induction of a variety of tumors resulted in loss or deletion of tissue-specific antigens [1—4], and it was postulated that loss of tissue-specific antigens resulted in the cells escaping the tissue homeostatic control and regulatory mechanisms, and thus undergoing malignant transformation. Furthermore, the presence of additional antigens associated with malignant transformation was observ ed in many tumors. The appearance of these tumor-specific antigens has been demonstrated by different techniques in a wide variety of experimentally-induced as well as in spon taneous malignant tumors [5-7]. Thus, malignant transforma tion is characterized by morphologic alteration in the tissues as well as alterations of their antigenic profile. These alter ations do not only involve changes in concentration of existing antigens and disappearance of some of them but also the appearance of new ones.
Only few studies have dealt with oral structures and only in direct evidence suggests the importance of immunologic mechanisms in tumorigenesis of these structures. The import ance of the role played by the immunologic mechanism in the development of oral tumors has been suggested primarily as a result of studies dealing with effect of different agents capable of non-specifically depressing the immune response on the development of chemically-induced tumors. It has been demonstrated that chemical carcinogenesis of the submandi bular gland [8] and buccal pouch [9, 10] has been accelerated by the systemic use of cortisone. Although the tumors induced in animals injected with cortisone presented the same histo logic characteristics as those in the control animals, invasion of the underlying tissue was more extensive and the tumors developed to a larger size. Administration of the antimeta bolite, methotrexate, in animals treated with 9,10 dimethyl1,2 benzanthracene (DMBA) resulted in the development of a more anaplastic carcinoma in the cheek pouch which developed more rapidly and invaded deeper than DMBA con trol animals [11]. Administration of the antimetabolite, azathioprine, enhanced the carcinogenic action of DMBA upon the buccal pouch mucosa of the hamster. The carcinoma developed more rapidly and tended to be of the anaplastic variety rather than the well-differentiated carcinoma usually found in DMBA treated animals [12]. These observations are similar to those reported in animals injected with specific antilymphocyte serum [13, 14|. It has been postulated that suppression of the immunologic response allows the chemically-induced neoplasm to develop more rapidly and to develop into a more anaplastic carcinoma [15]. The predominance of anaplastic cells led G iunta and S hklar [14] to raise the possibility of an antigenic difference between the anaplastic and well-differentiated types of tumors. In recent studies, the presence of antigenic differences be tween normal and tumor cells has been shown in oral epitheli um. C h av ez and T o to [16] noted an alteration in cell surface antigens associated with DMBA-induced carcinoma of the hamster cheek pouch. These alterations were detected by decrease of the intensity of immunofluorescent staining of the tumor cells in comparison with normal cells, when they were evaluated with rabbit antiserum to normal cheek pouch. Furthermore, it has been shown, using immunofluorescence, that blood group antigens which are normally present on the cell membranes of human oral epithelial cells, disappear in most oral carcinomas [17] and premalignant lesions [18]. Previous studies have demonstrated that saline extracts of
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Abstract. The antigenic activity of rat submandibular gland carcinoma induced by 9,10 dimethyl-1,2 benzanthracene (DMBA) has been evaluated using rabbit antisera to normal submandibular gland extract and to extracts of the induced tumors. Extracts of all tumors and premalignant lesions show reduction in the concentration of several of the intrinsic antigens of the normal submandibular gland when they were evaluated by imunodiffusion and immunoelectrophoresis. Although all the induced tumors, including those used for antisera preparation, are well-differentiated epidermoid carcinoma, a consid erable variation is present in the antigenic pattern of each tumor. When antisera to several tumors, absorbed with rat serum and kidney extract, were used in studying extracts of all induced tumors, antigenic bands were observed. The number of these bands varies from three to five. All the induced carcinomas share the presence of two of these bands in the a and (f-globulin region, the remaining bands differ from one tumor to another.
El-Mofty: Immunology of Submandibular Gland Tumors
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Materials and Methods Tumors of the submandibular gland were induced in four month old, inbred male Fischer rats. The animals were an esthetized with diethyl ether and a midline incision was made on the ventral surface of the neck. The right submandibular gland was exposed and a 15 gauge syringe needle was used to insert a pellet (5 mg) of the carcinogen 9,10 dimethyl-1,2 benazanthracene (DMBA), (Calbiochem, Los Angeles, Cali fornia) in powder form in the body of the gland according to the technique described by C a ia l d o and Shklar [21], The incision was sutured and the animals were maintained on Lab Chow and water ad libitum. Starting at four weeks, groups of four animals together with two sham operated controls were sacrificed every two weeks up to and including 20 weeks. The animal was anesthetized with diethyl ether and blood was collected from the aorta. The salivary glands were removed and weighed individually. The pellet of DMBA was removed from the submandibular gland and the lesion was cut in half. One half was fixed and prepared for histological and histochemical examination. The remaining portion of the tissue was quick-frozen and used for studying the antigenic characteristics of these tumors. Autopsy was performed on all the animals and lymph nodes, lung, liver, spleen and pancreas were fixed and prepared for histological examination. Histologic examinations revealed that the chemical carcinogen induced a constant sequence of events consisting of degener ation, metaplasia and neoplasia. The submandibular glands of the rats sacrificed at four-week intervals showed degene ration, hyperplasia and metaplasia. At six-week intervals, two rats developed squamous cell carcinoma while the glands of the other two animals showed hyperplastic and metaplastic changes. At eight-week intervals, three rats out of four devel oped squamous cell carcinoma, the remaining rat showed only hyperplasia and metaplasia. All animals in which the carcinogen was implanted for ten weeks or more developed carcinoma. The induced tumors were all well differentiated squamous cell carcinoma [22].
The submandibular glands as well as other tissues and blood of normal adult male rats were collected. The tumors and nor mal glands were homogenized in saline (1 mg/g of tissue), using a Sorvall Omnimixer at 0°C. The homogenates were centrifuged at 10,000 rpm for 10 min and supernatant was collected. Extracts of other rat tissue, e. g., kidney, liver and spleen were prepared in the same manner. The protein con centration of the saline extracts was determined by the method of L o w ry etal. [23]. Eight adult male New Zealand rabbits, 2-3 kg in weight, were used for preparing the antisera. Antisera to normal sub mandibular gland and to the induced tumors were prepared by injecting the rabbits intracutaneously and subcutaneously into sites on the back and shoulders. Four rabbits were injected with extract of pooled normal submandibular glands, and the remaining four rabbits were injected with tumor extracts, each was inoculated with extract of a different tumor. The extracts were emulsified with an equal volume of Freund’s complete adjuvant (Difco Laboratories, Detroit, Michigan) for the first injection and into incomplete adjuvant for sub sequent injections. Each rabbit received a weekly injection of about 10 mg of protein of the extract. When satisfactory titers were obtained, the animals were exsanguinated, a week to ten days after the last injection. Also, blood of unimunized rabbits was collected to be used as a control. Blood was allow ed to clot for one hour then the serum was separated by centri fugation and stored at -20 °C. Antisera were absorbed with lyophilized rat serum in a concentration of 80 mg/ml, which was found to be a proper concentration for neutralization pur poses by a preliminary gel diffusion test. The absorption mix tures were incubated for 30 min at 37 °C and then centrifuged to remove the precipitates. When necessary, further absorp tion was carried out using lyophilized extracts of rat kidney. Two-dimensional immunodiffusion as described by O u c h t e r LONY [24] was carried out using disposable Petri dishes (Falcon Plastics, Culver City, California) and the reactions were read two to three days later. Immunoelectrophoresis was carried out according to the method of G ra bar and B urtin [25] as modified by ScHElDEGGER [26] using Agafor immunoelectrophoresis apparatus (National Instruments Laboratories, Rockville, Maryland). One per cent agarose in 0.05 m barbital buffer at pH 8.6 was used. After the antigens were placed in the wells, a constant current of 4 mA milliamperes per slide was applied for two hours. Diffusion of the antisera was allowed to proceed for two to three days.
Results Preliminary screening by immunodiffusion of sera of all rabbits inoculated with extracts of normal submandibular gland and of induced squamous cell carcinoma of the gland exhibited several lines of precipitation when reacted with ex tracts of normal submandibular gland, of the induced tumors, as well as of glands showing premalignant changes. Absorption of the antisera with lyophilized rat serum and extract of rat kidney abolished the reaction with the majority of rat tissue extracts and demonstrated that rat submandibular gland contained at least five intrinsic antigenic components
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normal rat submandibular gland tissue contain at least five instrinsic antigenic components as well as other antigens, shared with blood serum and tissues of the rat. Of these anti gens, two to three were specific for the submandibular gland, and the remaining antigens were shared to varying degrees with extracts of related exocrine glands: parotid, sublingual, pancreas, extraorbital and intraorbital lacrimal [19, 20). These findings encouraged us to investigate the effect of malignant transformation on the tissue-specific antigens of the rat submandibular gland, particularly since it appears that no previous studies have evaluated changes in the salivary gland antigenic profile associated with such transformation and the possible appearance of tumor-specific antigens. The primary objective of the present study was to investigate the antigenic relationship between saline extractable antigens of induced tumors in comparison with those of the normal rat submandibular gland. It is aimed to monitor any change in the tissue-specific antigens in association with malignant trans formation, as well as to evaluate the possibility of appearance of new antigenic components in these tumors.
El-Mofty: Immunology of Submandibular Gland Tumors
as well as other antigens shared with blood serum and tissues of the rat [20], No difference was found between normal submandibular gland from normal rats, glands from shamoperated rats and normal mates to submandibular glands in which the tumors were induced. For the purpose of compari son, extracts of 33 induced carcinomas and seven submandi bular glands with premalignant alterations, i. e., hyperplasia and metaplasia, were evaluated with rabbit antisera to normal rat submandibular gland absorbed with rat serum and kidney. Extracts of all the tumors showed decrease of concentration of some of the submandibular gland intrinsic antigens with considerable variation noted in the antigenic pattern of each tumor (figure 1). Evaluation of premalignant lesions also revealed decrease of concentration of some antigenic com ponents with the antigenic pattern, varying from one lesion to another. Immunodiffusion and immunoelectrophoretic studies were carried out to monitor the appearance of tumor-specific antigens, using antisera to induced tumors. The antisera pre pared in four rabbits by injection of four different tumors, were all tested versus extracts of all the induced tumors and premalignant lesions. All DMBA-induced tumors gave preci pitation bands with the different antisera absorbed with nor mal rat serum and kidney extract. Immunoelectrophoretic evaluation of extracts of the tumors demonstrated the pres ence of three to five precipitation bands. While the immuniz ing antigens were from well-differentiated squamous cell carcinoma and the examined tumors were all well-differen tiated squamous cell carcinoma, the immunoelectrophoretic
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patterns varied from one tumor to another. However, all shared the presence of two distinct precipitation bands in the a and (5-globulin region. Two precipitation bands could be seen on evaluating extracts of normal rat submandibular gland and extracts of premalignant lesions with antisera to tumor extract. Figure 2 depicts the pattern obtained on eval uating normal submandibular gland and tumor extracts from different rats with absorbed antisera. Absorption of the antisera with normal submandibular gland extract did not exhaust the antigenic capacity of the antiserum against extracts of the tumors. After absorption with lyophilized normal rat serum, the antisera showed no reaction with normal sera, nor with sera from tumorbearing animals. Ab sorption of an antiserum with a tumor extract does not exhaust its antigenic capacity against all other tumors, particularly the one used in preparing the antiserum. On the other hand, absorption of an antiserum with the same tumor used for its preparation exhausted its antigenic capacity against all other tumors. No reaction was found between sera from tumor bearing animals and different tumors, including tumors of the same animals.
Discussion Implantation of powdered DMBA in the submandibular glands of inbred Fischer rats resulted in degeneration, prolifer ation, metaplasia and neoplasia. All animals in which the carcinogen was implanted for ten weeks or more, developed
Figure 2. Immunoelectrophoresis in agar gel. Troughs contain rabbit antiserum to carcinoma of the rat submandibular gland absorbed with rat scrum and kid ney extract. The first well from the left (N) contains normal sub mandibular gland extract. The remaining wells contain extracts of induced carcinomas.
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Figure 1. Immunoelectrophoresis in agar gel. Troughs contain rabbit anti-submandibular gland extract absorbed with rat serum and kidney extract. The first well from the left (N) contains normal rat submandibular gland extract. The remaining wells contain extracts of induced carcinomas.
El-Mofty: Immunology of Submandibular Gland Tumors
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host cannot be dismissed, particularly since these tumors arc characterized by their localized invasiveness, lack of metastasis and failure to be transplanted to syngeneic hosts [22, 34], Since the morphology and biological behavior of these induced tumors has been shown to be similar to human oral car cinoma [34], further studies with this system of chemical car cinogenesis and immunologic alterations might provide an ef fective model system for investigation of oral tumor.
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W eii .f.r , E.: Loss of specific cell antigen in relation to carcino genesis. In Wolstenholme and O ’Connor Ciba Foundation Symposium on Carcinogenesis: Mechanisms of Action: p. 165 (Churchill, London 1959). Nairn , R.; R ichmond, H. G.; Mc E ntegart , M. G., and T othergill , J. E.: Immunological differences between normal and malignant cells. Brit. med. J. ii: 1335-1340 (1960). A bei.ev , G. I.: Antigenic structure of chemically-induced hepatomas. Prog. exp. Tumor Res. 7:104—108 (1965). A lbin , R. J.: Immunologic studies of normal, benign, and malignant human prostatic tissue. Cancer 29: 1570-1574 (1972). P rehn , R. T.: Cancer antigens in tumors induced by chemicals. Fed. Proc. 2 4 :1018-1022 (1965). Baldwin, R. W. and Barker, C. R.: Tumor-specific antigeni city of aminazo-dyc-induced rat hepatomas. Int. J. Cancer 2: 355-364(1967). Keller , J. A.; Lo, A. C., and Kolos, T. O.: Immunoelectrophoretic analysis of DMBA-induced rat mammary tumors. Oncology 25:401-409 (1971). A nbari, N.; Shki.ar , G., and Cataldo , E.: The effect of systemically administered cortisone on salivary gland carcino genesis in the rat. J. dent. Res. 44:1056 (1965). Shklar, G.: Cortisone and hamster buccal pouch carcino genesis. Cancer Res. 36:2461-2463 (1966). Shklar, G.: The effect of cortisone on the induction and development of hamster buccal pouch carcinomas. Oral Surg. 23: 241-248 (1967). Shklar, G.; Cataldo , E., and F itzgerald , A. L.: The effect of methotrexate on chemical carcinogenesis of hamster buccal pouch. Cancer Res. 26:2218-2224 (1966). Sheehan , R.; Shklar, G., and T ennenbaum , R.: Azathioprine effects on the development of hamster pouch carcinomas. Arch. Path. 91:264-270 (1971). Woods , D. A.: Influence of antilymphocyte serum on DMBA induction of oral carcinomas. Nature 224:276-277 (1967). G iunta , J. L. and Shklar, G.: The effect of antilymphocyte serum on experimental hamster buccal pouch carcinogenesis. Oral Surg. 31:344-353 (1971). Shki.ar , G.: Recent advances in experimental oral and salivary gland tumors. J. oral Surg. 28:495-500 (1970). Chavez , R. F. and Toto , P. D.: Staining differences between normal and carcinogen induced carcinomas of the hamster pouch. J. dent. Res. 49:721-724 (1970). D abelsteen, E. and P indborg, J. J.: Loss of epithelial blood group substance A in oral carcinomas. Acta path, microbiol. scand. Sect. A, 81:435-444 (1973). D abei^stf.en , E.; R oed -P etersen, B.. and P indborg, J. J.: Loss of epithelial blood group antigens A and B in oral premalig nant lesions. Acta path, microbiol. scand. Sect. A, 83: 292-300 (1975). E l -M ofty. S.: Immunologic tissue- and species-specificity of the rat submaxillary gland. Fed. Proc. 29:304(1971).
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squamous cell carcinoma. The induced squamous cell carcin oma lesions were all well-differentiated, did not metastasize, and could not be transplanted to syngeneic hosts [22]. Histological studies revealed that the induced tumors were very similar to one another morphologically. However, evaluation of their antigenic profile demonstrated consider able difference from one tumor to another, whether they were studied by antiserum to normal submandibular gland or to tumor extract. In the present investigation, it was noted that malignant transformation was associated with decrease of concentration of several of the tissue-specific antigens pre viously identified to be characteristic of the normal rat sub mandibular gland [19, 20], These findings are in general agreement with results obtained from tumors induced in other organs [2, 4], However, it should be emphasized that alteration of antigenic characteristics of the cells and neo plastic transformation are not necessarily interdependent and that such alteration has been noted in epithelial cells taking part in wound healing [27] and in parotid gland following denervation [28]. Thus, it is more probable that these antigenic changes are associated with other factors such as growth rate, rather than malignancy per se. It is of interest to notice that these antigenic alterations were noted in lesions representing preneoplastic changes; before malignant transformation has taken place and that the anti genic properties of the preneoplastic cells are carried over into the resulting tumors. The present findings are in agreement with previous studies on premalignant lesions of other tissues [29, 30]. The value of the presence of antigenic alteration in premalignant lesions in predicting the prognosis of the condition is still to be evaluated. The presence of additional antigens associated with malignant transformation has also been observed in these tumors. Of the three to five tumorspecific precipitation bands observed in each tumor, two were present in all of them, and the re maining bands were specific for each tumor. The tumorspecific antigens were different from those present in normal gland; and traceable after absorption with normal rat serum and organs. The possibility that some of these tumor-specific antigens are of embryonic origin cannot be ruled out [31] but remains to be proven. This is particularly of interest since it has been shown that postnatal morphologic development of rat submandibular gland is associated with parallel antigenic alteration [32], The appearance of immunogenic proteins undetectable in the organs of normal rats can be the expression of protein neo synthesis due to an altered genetic information as a result of the carcinogenic effect of DMBA. The observation that although the induced tumors were very similar morphologic ally, yet each tumor has its own characteristic antigenic pro file, agrees with previous studies on other tissues that demon strated that chemically induced tumors in contrast to virus induced tumors, are characterized by their highly specific antigenicity [33], The absence in tumor-bearing rats of autoimmunoantisera directed at their own neoplasm can be explained either by insufficient sensitivity of the method used or by continuous absorption of the antibodies formed by the tumor mass. How ever, the possibility that the antigenic alterations noted in these tumors play a role in the immunologic response of the
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El-Mofty: Immunology of Submandibular Gland Tumors
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E l-M ofty, S.: Tissue- and species-specific antigens of the rat submandibular gland. Experientia 30:301-302 (1974). Cataldo, E. and Shklar, G.: Chemical carcinogenesis in the hamster submaxillary, gland. J. dent. Res. 43:568-579(1964). E l-Mofty, S.: Chemical carcinogenesis in the rat submandi bular gland (in Press). Lowry, O. H.; Rosf.brough , N. J.; Farr , A. L. and R andall , R. J.: Protein measurement with the folin phenol reagent. J. biol. Chem. 193:265-275 (1951). O uchterlony, O.: In vitro method for testing the toxinproducing capacity of diphtheria bacteria. Acta path, microbiol. scand. Sect. A, 2 5 :186-191 (1948). G rabar, P. et B urtin, P.: Analyse immuno-électrophorétique (Masson, Paris 1960). Schneidegger , J. J.: Une micro-méthode de l’immuno électrophorèse Int. Arch. Allergy 7: 103-110 (1955). D abelsteen, E. and F ejerskov, O.: Loss of epithelial blood group antigen A during wound healing in oral mucous mem brane. Acta path, microbiol. scand. Sect. A, 82: 431-434 (1974). E l-M ofty, S. and Schneyer, C. A.: Alteration of antigenic components of rat parotid gland following denervation. Ex perientia 30:950-951 (1974).
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Immunological study of polyps of the colon. J. nat. Cancer Inst. 48:25-29 (1972). 30
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H alpin, T. Z .; Vaage , J., and B lair, P. B.: Lack of anti genicity of mammary tumors induced by carcinogens in a nonantigenic preneoplastic lesion. Cancer Res. 32: 2197-2200 (1972). G old , P. and F reeman , S.: Specific carcinoembryonic anti
gens of the human digestive system. J. exp. Med. 122: 467-481 (1965). E l-M ofty, S.: Changes in antigenic profile of the rat sub maxillary gland during postnatal development. Int. Ass. dent. Res. 50:800(1972). Klein, G.: Experimental studies in tumor immunology. Fed. Proc. 28:1739-1753 (1969). B rown, A. M.: A comparative assay of the in vitro and in vivo behavior of three chemically induced neoplasmas of rat sub mandibular glands. J. oral Path. 2:355-364 (1973).
Request reprints from: S. E l-Mofty , BchD, PhD, 28 Abd El-Azim Rashed Str., Agouza, Cairo (Egypt)
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