- Email: [email protected]
Comparison of Tissue Culture Cells and Histological Sections for Use in Screening Monoclonal Antibodies
0022-534 7/85/1345-0982$02.00 /0 Vol. 134, November
THE JOURNAL OF UROLOGY
Printed in U.S.A.
Copyright© 1985 by The Williams & Wilkins Co.
COMPARISON OF TISSUE CULTURE CELLS AND HISTOLOGICAL SECTIONS FOR USE IN SCREENING MONOCLONAL ANTIBODIES FRANK P. BEGUN
H. BARTON GROSSMAN*
AND
From the Section of Urology, Department of Surgery, University of Michigan, Ann Arbor, Michigan
ABSTRACT
Monoclonal antibodies have been screened using either tissue culture cells or histologic sections as a source of antigen. We evaluated and compared these two methods using six murine monoclonal antibodies on human kidney cultures and histologic sections of human normal kidney and renal carcinoma. Antibody binding demonstrated shared antigens on kidney culture cells, renal tubular cells and renal carcinoma cells. The patterns of antibody binding were generally similar with either cultured cells or histologic sections. However, certain antibodies demonstrated significant differences in antibody binding depending on the method used for screening. An essential part of the evaluation of a new monoclonal antibody is the determination of the specificity of the antibody. This may be operationally defined as the range (number of types) of cells that contain the antigen of interest. The accurate characterization of an antibody's specificity is important because it helps define the purposes for which an antibody can be used. Antibody specificity has been determined by two fundamentally different approaches, tissue culture cell lines and non-cultured tissues. 1• 2 To evaluate these two approaches we studied six murine monoclonal antibodies from om laboratory, using serologic analysis of tissue culture cell lines and immunoperoxidase assays on histological tissue sections. Normal kidney tissue was used for this evaluation because some of our antibodies were found to react with cultured normal kidney cells. The two approaches yielded similar data. However, significant differences were observed. MATERIALS AND METHODS
Monoclonal antibody production. Hybridoma cells were produced as previously described.a Antibodies A2, A68.7 and A80 were the result of the fusion of the murine myeloma cell line NS1 with spleen cells from a Balb/c mouse immunized with the human transitional cell carcinoma line RT4. Hybridomas E58 and E76 were produced by the fusion of NSl with spleen cells from a Balb/c mouse immunized with the bladder cancer cell line 253J. The spleen cells that yielded F31 were from a Balb/c mouse immunized with the bladder cancer cell line 5637. Screening was done as previously described using a mixed hemadsorption (MHA) assay and subcloning was performed using limiting dilution.a Hybridoma cells were grown in a serum free medium (RPMI with 0.2 mM hypoxanthine, 32 µM thymidine, 5 µg./ml. insulin and 5 µg./ml. transferrin) 4 or medium containing serum.a Supernatants from serum free medium hybridoma cultures were concentrated 10 times using CX-30 ultrafiltration units (Millipore Corporation). All supernatants were aliquoted and frozen at -20C. The antibody was thawed just prior to use. Si~ilar results were obtained with both antibody preparations. Cell lines. Cell cultures were maintained in Eagle's Minimum Essential Medium with 1 per cent non-essential amino acids, 2 mM glutamine, 100 u/ml. penicillin, 100 µg./ml. streptomycin and 15 per cent fetal bovine serum. Normal kidney cell cultures were established from non-neoplastic portions of nephrectomy Accepted for publication June 4, 1985. * Requests for reprints: Urology Research Library, 1405 E. Ann St., Box 03 C5116, Ann Arbor, MI 48109. Supported by PHS grant CA36933, awarded by the National Cancer Institute, DHHS.
specimens. 5 •6 UM-UC-3 is a new human cell line derived from a bladder transitional cell carcinoma. It produces tumors in athymic nude mice (Balb/c background). Immunoperoxidase assay. The immunoperoxidase (IP) assay utilized a biotin-avidin system (Vector Labs). 7 Four micron thick sections of normal kidney and renal cell carcinomas were mounted on slides using Soho's glue. The tissue sections, obtained from nephrectomies of patients with renal cell carcinomas, frequently contained areas of both normal kidney and malignant tissue. The slides were deparaffinized using graded xylene and ethanol baths. All the reactions were carried out at room temperature. The sections were treated with 0.3 per cent hydrogen peroxide in methanol for 20 minutes, washed for 20 minutes in Dulbecco's phosphate buffered saline (PBS) solution and transferred to a moist chamber to prevent desiccation. A 2 per cent solution of normal horse serum was added to reduce nonspecific background staining. Excess serum was blotted off and the sections were covered with the monoclonal antibody or PBS for one hour and then washed for 10 minutes in PBS. Biotin labeled horse anti-mouse IgG was placed upon the slides for 30 minutes. Excess antibody was removed by washing with PBS followed by the addition of an avidin-biotin peroxidase complex for one hour. The slides were again washed using PBS and then transferred to a Copley jar containing PBS solution. Staining was produced by incubating the sections in a 0.01 per cent hydrogen peroxide and 0.05 per cent diaminobenzidine tetrahydrochloride solution in PBS for 2.5 minutes. The slides were then washed for 10 minutes and counterstained with hematoxylin. The slides were again passed through graded ethanol and xylene baths and mounted with Permount (Fisher Scientific Co.). They were then examined under a light microscope for the presence of brown staining. Frozen sections. Areas of normal kidney for frozen section were obtained from nephrectomy specimens of patients with renal carcinoma. The kidney was frozen at -70C and placed on a copper sectioning disc using OCT embedding medium. Six micron thick sections were cut, mounted and fixed in acetone for 10 minutes followed by a distilled water wash. They were then treated as described above for formalin fixed tissues. A further modification of this staining method was to eliminate the acetone fixation step and incubate the frozen sections for an equal period of time in PBS. The reminder of the steps were the same as described above. MHA assay. The MHA assay was carried out in NUNC 163118 microtest plates (Roskilde) as previously described.a Target cells (normal kidney or UM-UC-3 cultures) were trypsinized, washed and pipetted into microtest plates at a density of 200 cells/well (10 µl.). The plates were incubated at 37C until used. On the day of the assay, the plates were washed
982
ME?½GDS
7
~R SCkEENI~G norxDc:._i hurnan
T'en
with mouse anti-mouse immunoglobuwere added in 10 to each well. The were incubated at room temperature for 45 minutes and washed with PBS-FBS. Positive as indicated rosettes of erythrocytes around the target cells, were determined microscopically. Immunoperoxidase assay on UA!f.-UC-3 cells. The IP assay was performed on UM-UC-3 cells in suspension. UM-UC-3 cells growing in tissue culture flasks were harvested with 0.25 per cent trypsin and resuspended in PBS. Formalin treated cells were resuspended in 10 per cent buffered formalin and incubated for 30 minutes. UM-UC-3 cells and UM-UC-3 formalin treated cells were then stained with the same technique used for histologic sections except that all reactions were carried out in suspension. Centrifugation was used to facilitate washing the cells between reagents. After staining was complete, the cells were placed on a microscope slide and permanent slides were made using a method similar to that described above for formalin fixed histological sections except that counterstaining with hematoxylin was not performed. Formalin fixed nude mouse tumor and kidney. A tumor was injecting 7 x grown in a nude mouse, Balb/c background, 105 UM-UC-3 cells subcutaneously into the flank. The tumor and nude mouse were fixed in formalin and prepared for mo,,CI,JHloH!e using a standard paraffin embedding procedure. Four micron thick sections were made and stained the method described above for formaiin fixed human h11,to,101~1ca1 sections. -H'-j[~-"~
RESULTS
Serological tests. Four of the six monoclonal antibodies tested bound to cultured normal kidney cells a mixed hemadsorption assayo Antibody A2 did not bind to any of the seven normal kidney cultureso "''""°,v"" A68.7 bound to one of seven cultures (table Immunoperoxidase assays on formalin normal and renal carcinoma. Antibodies A80, E58, E76 and F31 to renal tubules when tested on formalirr fixed sections of TABLE
983
1. Monoclonai antibody binding to cultured normal kidney cells
'These antibodies also stained ~reas of
vvas nonreactive •»ith norrr1al and renal A2 exhibited diffuse of all renal structures including cwJC"'°"' glomernli and connective tissue elements. Antibody A2 also demonstrated diffuse binding to all renal carcinoma elements (table Imniunoperoxidase assays sections of normal kidney. Frozen sections of normal kidney were used to attempt to clarify the differences of antibody reactivity that were noted between formalin fixed sections and tissue culture cells. Although histologic preservation with frozen sections was poor, an assessment of immunoperoxidase staining could be made (table 2). Antibody reactivity was similar in acetone and non-acetone fixed frozen sections. Four of the antibodies (A2, A68.7, ABO and E76) demonstrated identical patterns of staining with formalin fixed and frozen sections. Antibodies E58 and F31 exhibited additional binding sites (glomeruli) on frozen sections. Immunoperoxidase staining of UM-UC-3. Antibodies A2, A80, E58 and F31 were tested with the IP assay on non-fixed and formalin fixed cell suspenions of the human transitional cancer cell line UM-UC-3 (table 3). Only antibody F31 was reactive with this cell line using the MHA assay (table 4). Antibodies A80, E58 and F31 showed complete correlation between the MHA and IP assays. Antibody A2 was non-reactive MHA assay and IP assay of non-fixed UM-UC-3 cell suspensions but stained formalin fixed UM-UC-3 cell suspensions (IP). staining of formalin nude mouse E58 and F31 reactivity with a UMtissues. Antibodies UC-3 nude mouse tumor was identical to that obtained using UM-UC-3 tissue culture suspensions (table 4). Antibody A2 demonstrated diffuse reactivity with all cellular components. Antibody A2 was also tested against formalin fixed nude mouse kidney. It also stained this tissue with a diffuse, non-specific pattern. DISCUSSION
The potential applications of monoclonal antibodies, altheoretically boundless, are governed by the distribution of the antigen (specificity) to which the monoclonal antibody binds. 1 Characterization of the specificity has been accomplished with both cultured and non-cultured cells. 1 · 2 Tissue
(mixed hemadsorption assay) Normal Kidney
A2
A68.7
A80
E58
E76
F31
+
+ + +
+ NT NT
+
+
DE
+ +
+ NT NT + +
DO
CB CT
cu DB
DQ
NT
NT +
3. Representative experiment demonstrating monoclonal antibody binding to UM-UC-3 cells (immunoperoxidase assay)
TABLE
Non-fixed
PBS
+
+ +
+
+
A80
+
+
+
+
E58 F31
A2
+=binding.
- = no bi'1ding. NT = not tested.
PBS
Formalin Fixed
0%''
2%
5% 0% 0% 80%
90% 0% 0% 100%
= phosphate buffered saline.
* = results are per cent positive (stained) celiso
TABLE 2.
----------~
Summary of monoclonal antibody binding to normal kidney and renal carcinoma
Antibody
MHA Assay
Normal Kidney Cultures
A2 A68.7 A80
E58 E76 F31 MBA assay = mixed hemadsorption assay. +=binding. - = no binding.
+ + + +
!mmunopemxidase Assay Formalin Fixed Normal Kidney
Renal Cell Carcinoma
diffuse
+
diffuse
tubules tubules tubules tubules
+ + + +
tubules tubules, glomeruli tubules tubules, glomeruli
Frozen Section Normal Kidney
984
BEGUN AND GROSSMAN
TABLE 4.
Summary of monoclonal antibody binding to UM-UC-3 cells Immunoperoxidase
Antibody
MHA Cell Suspension
A2 A80 E58 F31
+
+
Formalin Fixed Cell Suspension
Formalin Fixed Nude Mouse Tumor
+
+
+
+
MHA = mixed hemadsorption assay. +=binding. - = no binding.
culture cells are used because established lines have extended cell lives and are therefore easily studied at various intervals. However, cultured cells undergo enormous selection pressures and do not necessarily possess the cell surface antigens found on the original tissue. The immunoperoxidase technique permits the analysis of histologic sections that have not been exposed to the bias induced by cell culture. However, the tissues are manipulated and antigens may be altered particularly when chemical treatment (fixed tissues as opposed to frozen sections) is used. Our monoclonal antibodies were producing using human transitional cell carcinoma lines as the immunizing material. It was noted during early characterization of these antibodies that one antibody, A80, bound to cultured normal kidney cells using the MHA assay. 3 We therefore chose normal kidney as our experimental model to compare the tissue culture data to that derived from histologic sections. Initial testing was done on formalin fixed specimens of normal kidney from eight patients that had undergone nephrectomy for renal cell carcinoma. Seven of the eight specimens also had areas of renal cell carcinoma in the tissue sections. Five of the six antibodies demonstrated similar binding with the two testing methods. Antibodies A80, E58, E76 and F31 were reactive with normal kidney grown in tissue culture and with renal tubules in normal kidney sections (IP). Antibody A68.7 was negative with both assay methods. One antibody, however, yielded different results with the two techniques. Antibody A2 was unreactive with cultured normal kidney cells, but diffuse staining of all renal components was seen on immunoperoxidase testing. Immunoperoxidase analysis of frozen sections of normal kidney was used to evaluate possible alteration of cell surface antigens by formalin fixation. Although the architecture of frozen sectioned material was poorly preserved, sufficient detail remained to allow determination of the staining pattern of the antibodies. Four of the six antibodies exhibited the same patterns of staining using both frozen and formalin fixed sections of normal kidney. Antibodies E58 and F31 showed additional binding to glomeruli on frozen sections that was not seen when formalin fixed material was used. This also reflects antigen alteration by chemical processing. The finding that those antibodies that bound to cultured normal kidney cells also bound to renal tubules (IP) suggests that the cultured cells are derived from renal tubular epithelial cells. The antibodies that bound to renal tubules also stained renal cell carcinoma (IP). This offers further evidence that renal carcinomas are derived from renal tubular cells.&---10 The binding of antibody A2 in a diffuse fashion to all cells (tubules, glomeruli and connective tissue) in both formalin fixed and frozen sectioned material suggests a novel cause for the lack of staining by MHA. The MHA assay detects antibody binding to surface membranes of living cells. However, the
interior of cells becomes the dominant area of antibody exposure in histologic sections. The results could be accounted for if the antigen defined by A2 is commonly found intracellularly and only rarely on the cell surface, e.g., as an alteration accompaning neoplastic transformation. This hypothesis is strongly supported by the observed data. First, A2 does not bind to cultured normal kidney cells by MHA, but diffusely stains both frozen and formalin fixed histologic sections of normal kidney. Second, A2 only stains UM-UC-3 cells in suspension after they have been treated with formalin (destroying the integrity of the cell membrane). Third, although radiolabeled A2 does not image UM-UC-3 tumors in nude mice (unpublished data) or nude mouse kidneys, 11 A2 binds to formalin fixed sections of UM-UC-3 tumors and mouse kidney in a diffuse pattern. Thus in this instance, the use of living target cells is a better indicator of antibody binding in vivo. Both tissue culture cells and histologic sections may be used to obtain information about antibody binding to human cells. In general, there is good correlation between these techniques. 12 However, each method has its own strengths and weaknesses and differences do occur with some antibodies. Tissue culture cells permit the use of sensitive assays on intact cells that may not accurately reflect their origins. Histologic sections preserve cellular detail but may alter antigen presentation by physical or chemical means. A thorough understanding of the strengths and limitations of these assays is essential for the evaluation of experimental data and the determination of the potential clinical usefulness of new antibodies. REFERENCES
1. Cairncross, J. G., Mattes, M. J., Beresford, H. R., Albino, A. P., Houghton, A. N., Lloyd, K. 0. and Old, L. J.: Cell surface antigens of human astrocytoma defined by mouse monoclonal antibodies: identification of astrocytoma subsets. Proc. Natl. Acad. Sci. USA, 79: 5641, 1982. 2. McGee, J. O'D., Woods, J. C., Ashall, F., Bramwell, M. E. and Harris, H.: A new marker for human cancer cells. 2, Immunohistochemical detection of the Ca antigen in human tissues with the Cal antibody. Lancet, 2: 7, 1982. 3. Grossman, H. B.: Hybridoma antibodies reactive with human bladder carcinoma cell surface antigens. J. Urol., 130: 610, 1983. 4. Chang, T. H., Steplewski, Z. and Koprowski, H.: Production of monoclonal antibodies in serum free medium. J. Immunol. Methods, 39: 369, 1980. 5. Ueda, R., Shiku, H., Pfreundschuh, M., Takahashi, T., Li, L. T. C., Whitmore, W. F., Oettgen, H. F. and Old, L. J.: Cell surface antigens of human renal cancer defined by autologous typing. J. Exp. Med., 150: 564, 1979. 6. Grossman, H. B., Wedemeyer, G. and Ren, L.: Human renal carcinoma: characterization of five new cell lines. J. Surg. One., 28: 237, 1985. 7. Hsu, S. M., Raine, L. and Farzer, H.: A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am. J. Clin. Pathol., 75: 734, 1981. 8. Seljelid, R. and Ericsson, J. L. E.: Electron microscopic observations on specializations of the cell surface in renal clear cell carcinoma. Lab. Invest., 14: 435, 1965. 9. Wallace, A. C. and Nairn, R. C.: Renal tubular antigens in kidney tumors. Cancer, 29: 977, 1972. 10. Reznik, G., Ward, J. M., Hardisty, J. F. and Russfield, A.: Renal carcinogenic and nephrotoxic effects of the flame retardant tris (2,3-dibromopropyl) phosphate in F344 rats and (C57BL/6N x C3H/HeN)F 1 mice. JNCI, 63: 205, 1979. 11. Gross, M. D., Skinner, R. W. S. and Grossman, H. B.: Radioimmunodetection of a transplantable human bladder carcinoma in a nude mouse. Invest. Radiol., 19: 530, 1984. 12. Bander, N.: Comparison of antigen expression of human renal cancers in vivo and in vitro. Cancer, 53: 1235, 1984.
THE JOURNAL OF UROLOGY
Printed in U.S.A.
Copyright© 1985 by The Williams & Wilkins Co.
COMPARISON OF TISSUE CULTURE CELLS AND HISTOLOGICAL SECTIONS FOR USE IN SCREENING MONOCLONAL ANTIBODIES FRANK P. BEGUN
H. BARTON GROSSMAN*
AND
From the Section of Urology, Department of Surgery, University of Michigan, Ann Arbor, Michigan
ABSTRACT
Monoclonal antibodies have been screened using either tissue culture cells or histologic sections as a source of antigen. We evaluated and compared these two methods using six murine monoclonal antibodies on human kidney cultures and histologic sections of human normal kidney and renal carcinoma. Antibody binding demonstrated shared antigens on kidney culture cells, renal tubular cells and renal carcinoma cells. The patterns of antibody binding were generally similar with either cultured cells or histologic sections. However, certain antibodies demonstrated significant differences in antibody binding depending on the method used for screening. An essential part of the evaluation of a new monoclonal antibody is the determination of the specificity of the antibody. This may be operationally defined as the range (number of types) of cells that contain the antigen of interest. The accurate characterization of an antibody's specificity is important because it helps define the purposes for which an antibody can be used. Antibody specificity has been determined by two fundamentally different approaches, tissue culture cell lines and non-cultured tissues. 1• 2 To evaluate these two approaches we studied six murine monoclonal antibodies from om laboratory, using serologic analysis of tissue culture cell lines and immunoperoxidase assays on histological tissue sections. Normal kidney tissue was used for this evaluation because some of our antibodies were found to react with cultured normal kidney cells. The two approaches yielded similar data. However, significant differences were observed. MATERIALS AND METHODS
Monoclonal antibody production. Hybridoma cells were produced as previously described.a Antibodies A2, A68.7 and A80 were the result of the fusion of the murine myeloma cell line NS1 with spleen cells from a Balb/c mouse immunized with the human transitional cell carcinoma line RT4. Hybridomas E58 and E76 were produced by the fusion of NSl with spleen cells from a Balb/c mouse immunized with the bladder cancer cell line 253J. The spleen cells that yielded F31 were from a Balb/c mouse immunized with the bladder cancer cell line 5637. Screening was done as previously described using a mixed hemadsorption (MHA) assay and subcloning was performed using limiting dilution.a Hybridoma cells were grown in a serum free medium (RPMI with 0.2 mM hypoxanthine, 32 µM thymidine, 5 µg./ml. insulin and 5 µg./ml. transferrin) 4 or medium containing serum.a Supernatants from serum free medium hybridoma cultures were concentrated 10 times using CX-30 ultrafiltration units (Millipore Corporation). All supernatants were aliquoted and frozen at -20C. The antibody was thawed just prior to use. Si~ilar results were obtained with both antibody preparations. Cell lines. Cell cultures were maintained in Eagle's Minimum Essential Medium with 1 per cent non-essential amino acids, 2 mM glutamine, 100 u/ml. penicillin, 100 µg./ml. streptomycin and 15 per cent fetal bovine serum. Normal kidney cell cultures were established from non-neoplastic portions of nephrectomy Accepted for publication June 4, 1985. * Requests for reprints: Urology Research Library, 1405 E. Ann St., Box 03 C5116, Ann Arbor, MI 48109. Supported by PHS grant CA36933, awarded by the National Cancer Institute, DHHS.
specimens. 5 •6 UM-UC-3 is a new human cell line derived from a bladder transitional cell carcinoma. It produces tumors in athymic nude mice (Balb/c background). Immunoperoxidase assay. The immunoperoxidase (IP) assay utilized a biotin-avidin system (Vector Labs). 7 Four micron thick sections of normal kidney and renal cell carcinomas were mounted on slides using Soho's glue. The tissue sections, obtained from nephrectomies of patients with renal cell carcinomas, frequently contained areas of both normal kidney and malignant tissue. The slides were deparaffinized using graded xylene and ethanol baths. All the reactions were carried out at room temperature. The sections were treated with 0.3 per cent hydrogen peroxide in methanol for 20 minutes, washed for 20 minutes in Dulbecco's phosphate buffered saline (PBS) solution and transferred to a moist chamber to prevent desiccation. A 2 per cent solution of normal horse serum was added to reduce nonspecific background staining. Excess serum was blotted off and the sections were covered with the monoclonal antibody or PBS for one hour and then washed for 10 minutes in PBS. Biotin labeled horse anti-mouse IgG was placed upon the slides for 30 minutes. Excess antibody was removed by washing with PBS followed by the addition of an avidin-biotin peroxidase complex for one hour. The slides were again washed using PBS and then transferred to a Copley jar containing PBS solution. Staining was produced by incubating the sections in a 0.01 per cent hydrogen peroxide and 0.05 per cent diaminobenzidine tetrahydrochloride solution in PBS for 2.5 minutes. The slides were then washed for 10 minutes and counterstained with hematoxylin. The slides were again passed through graded ethanol and xylene baths and mounted with Permount (Fisher Scientific Co.). They were then examined under a light microscope for the presence of brown staining. Frozen sections. Areas of normal kidney for frozen section were obtained from nephrectomy specimens of patients with renal carcinoma. The kidney was frozen at -70C and placed on a copper sectioning disc using OCT embedding medium. Six micron thick sections were cut, mounted and fixed in acetone for 10 minutes followed by a distilled water wash. They were then treated as described above for formalin fixed tissues. A further modification of this staining method was to eliminate the acetone fixation step and incubate the frozen sections for an equal period of time in PBS. The reminder of the steps were the same as described above. MHA assay. The MHA assay was carried out in NUNC 163118 microtest plates (Roskilde) as previously described.a Target cells (normal kidney or UM-UC-3 cultures) were trypsinized, washed and pipetted into microtest plates at a density of 200 cells/well (10 µl.). The plates were incubated at 37C until used. On the day of the assay, the plates were washed
982
ME?½GDS
7
~R SCkEENI~G norxDc:._i hurnan
T'en
with mouse anti-mouse immunoglobuwere added in 10 to each well. The were incubated at room temperature for 45 minutes and washed with PBS-FBS. Positive as indicated rosettes of erythrocytes around the target cells, were determined microscopically. Immunoperoxidase assay on UA!f.-UC-3 cells. The IP assay was performed on UM-UC-3 cells in suspension. UM-UC-3 cells growing in tissue culture flasks were harvested with 0.25 per cent trypsin and resuspended in PBS. Formalin treated cells were resuspended in 10 per cent buffered formalin and incubated for 30 minutes. UM-UC-3 cells and UM-UC-3 formalin treated cells were then stained with the same technique used for histologic sections except that all reactions were carried out in suspension. Centrifugation was used to facilitate washing the cells between reagents. After staining was complete, the cells were placed on a microscope slide and permanent slides were made using a method similar to that described above for formalin fixed histological sections except that counterstaining with hematoxylin was not performed. Formalin fixed nude mouse tumor and kidney. A tumor was injecting 7 x grown in a nude mouse, Balb/c background, 105 UM-UC-3 cells subcutaneously into the flank. The tumor and nude mouse were fixed in formalin and prepared for mo,,CI,JHloH!e using a standard paraffin embedding procedure. Four micron thick sections were made and stained the method described above for formaiin fixed human h11,to,101~1ca1 sections. -H'-j[~-"~
RESULTS
Serological tests. Four of the six monoclonal antibodies tested bound to cultured normal kidney cells a mixed hemadsorption assayo Antibody A2 did not bind to any of the seven normal kidney cultureso "''""°,v"" A68.7 bound to one of seven cultures (table Immunoperoxidase assays on formalin normal and renal carcinoma. Antibodies A80, E58, E76 and F31 to renal tubules when tested on formalirr fixed sections of TABLE
983
1. Monoclonai antibody binding to cultured normal kidney cells
'These antibodies also stained ~reas of
vvas nonreactive •»ith norrr1al and renal A2 exhibited diffuse of all renal structures including cwJC"'°"' glomernli and connective tissue elements. Antibody A2 also demonstrated diffuse binding to all renal carcinoma elements (table Imniunoperoxidase assays sections of normal kidney. Frozen sections of normal kidney were used to attempt to clarify the differences of antibody reactivity that were noted between formalin fixed sections and tissue culture cells. Although histologic preservation with frozen sections was poor, an assessment of immunoperoxidase staining could be made (table 2). Antibody reactivity was similar in acetone and non-acetone fixed frozen sections. Four of the antibodies (A2, A68.7, ABO and E76) demonstrated identical patterns of staining with formalin fixed and frozen sections. Antibodies E58 and F31 exhibited additional binding sites (glomeruli) on frozen sections. Immunoperoxidase staining of UM-UC-3. Antibodies A2, A80, E58 and F31 were tested with the IP assay on non-fixed and formalin fixed cell suspenions of the human transitional cancer cell line UM-UC-3 (table 3). Only antibody F31 was reactive with this cell line using the MHA assay (table 4). Antibodies A80, E58 and F31 showed complete correlation between the MHA and IP assays. Antibody A2 was non-reactive MHA assay and IP assay of non-fixed UM-UC-3 cell suspensions but stained formalin fixed UM-UC-3 cell suspensions (IP). staining of formalin nude mouse E58 and F31 reactivity with a UMtissues. Antibodies UC-3 nude mouse tumor was identical to that obtained using UM-UC-3 tissue culture suspensions (table 4). Antibody A2 demonstrated diffuse reactivity with all cellular components. Antibody A2 was also tested against formalin fixed nude mouse kidney. It also stained this tissue with a diffuse, non-specific pattern. DISCUSSION
The potential applications of monoclonal antibodies, altheoretically boundless, are governed by the distribution of the antigen (specificity) to which the monoclonal antibody binds. 1 Characterization of the specificity has been accomplished with both cultured and non-cultured cells. 1 · 2 Tissue
(mixed hemadsorption assay) Normal Kidney
A2
A68.7
A80
E58
E76
F31
+
+ + +
+ NT NT
+
+
DE
+ +
+ NT NT + +
DO
CB CT
cu DB
DQ
NT
NT +
3. Representative experiment demonstrating monoclonal antibody binding to UM-UC-3 cells (immunoperoxidase assay)
TABLE
Non-fixed
PBS
+
+ +
+
+
A80
+
+
+
+
E58 F31
A2
+=binding.
- = no bi'1ding. NT = not tested.
PBS
Formalin Fixed
0%''
2%
5% 0% 0% 80%
90% 0% 0% 100%
= phosphate buffered saline.
* = results are per cent positive (stained) celiso
TABLE 2.
----------~
Summary of monoclonal antibody binding to normal kidney and renal carcinoma
Antibody
MHA Assay
Normal Kidney Cultures
A2 A68.7 A80
E58 E76 F31 MBA assay = mixed hemadsorption assay. +=binding. - = no binding.
+ + + +
!mmunopemxidase Assay Formalin Fixed Normal Kidney
Renal Cell Carcinoma
diffuse
+
diffuse
tubules tubules tubules tubules
+ + + +
tubules tubules, glomeruli tubules tubules, glomeruli
Frozen Section Normal Kidney
984
BEGUN AND GROSSMAN
TABLE 4.
Summary of monoclonal antibody binding to UM-UC-3 cells Immunoperoxidase
Antibody
MHA Cell Suspension
A2 A80 E58 F31
+
+
Formalin Fixed Cell Suspension
Formalin Fixed Nude Mouse Tumor
+
+
+
+
MHA = mixed hemadsorption assay. +=binding. - = no binding.
culture cells are used because established lines have extended cell lives and are therefore easily studied at various intervals. However, cultured cells undergo enormous selection pressures and do not necessarily possess the cell surface antigens found on the original tissue. The immunoperoxidase technique permits the analysis of histologic sections that have not been exposed to the bias induced by cell culture. However, the tissues are manipulated and antigens may be altered particularly when chemical treatment (fixed tissues as opposed to frozen sections) is used. Our monoclonal antibodies were producing using human transitional cell carcinoma lines as the immunizing material. It was noted during early characterization of these antibodies that one antibody, A80, bound to cultured normal kidney cells using the MHA assay. 3 We therefore chose normal kidney as our experimental model to compare the tissue culture data to that derived from histologic sections. Initial testing was done on formalin fixed specimens of normal kidney from eight patients that had undergone nephrectomy for renal cell carcinoma. Seven of the eight specimens also had areas of renal cell carcinoma in the tissue sections. Five of the six antibodies demonstrated similar binding with the two testing methods. Antibodies A80, E58, E76 and F31 were reactive with normal kidney grown in tissue culture and with renal tubules in normal kidney sections (IP). Antibody A68.7 was negative with both assay methods. One antibody, however, yielded different results with the two techniques. Antibody A2 was unreactive with cultured normal kidney cells, but diffuse staining of all renal components was seen on immunoperoxidase testing. Immunoperoxidase analysis of frozen sections of normal kidney was used to evaluate possible alteration of cell surface antigens by formalin fixation. Although the architecture of frozen sectioned material was poorly preserved, sufficient detail remained to allow determination of the staining pattern of the antibodies. Four of the six antibodies exhibited the same patterns of staining using both frozen and formalin fixed sections of normal kidney. Antibodies E58 and F31 showed additional binding to glomeruli on frozen sections that was not seen when formalin fixed material was used. This also reflects antigen alteration by chemical processing. The finding that those antibodies that bound to cultured normal kidney cells also bound to renal tubules (IP) suggests that the cultured cells are derived from renal tubular epithelial cells. The antibodies that bound to renal tubules also stained renal cell carcinoma (IP). This offers further evidence that renal carcinomas are derived from renal tubular cells.&---10 The binding of antibody A2 in a diffuse fashion to all cells (tubules, glomeruli and connective tissue) in both formalin fixed and frozen sectioned material suggests a novel cause for the lack of staining by MHA. The MHA assay detects antibody binding to surface membranes of living cells. However, the
interior of cells becomes the dominant area of antibody exposure in histologic sections. The results could be accounted for if the antigen defined by A2 is commonly found intracellularly and only rarely on the cell surface, e.g., as an alteration accompaning neoplastic transformation. This hypothesis is strongly supported by the observed data. First, A2 does not bind to cultured normal kidney cells by MHA, but diffusely stains both frozen and formalin fixed histologic sections of normal kidney. Second, A2 only stains UM-UC-3 cells in suspension after they have been treated with formalin (destroying the integrity of the cell membrane). Third, although radiolabeled A2 does not image UM-UC-3 tumors in nude mice (unpublished data) or nude mouse kidneys, 11 A2 binds to formalin fixed sections of UM-UC-3 tumors and mouse kidney in a diffuse pattern. Thus in this instance, the use of living target cells is a better indicator of antibody binding in vivo. Both tissue culture cells and histologic sections may be used to obtain information about antibody binding to human cells. In general, there is good correlation between these techniques. 12 However, each method has its own strengths and weaknesses and differences do occur with some antibodies. Tissue culture cells permit the use of sensitive assays on intact cells that may not accurately reflect their origins. Histologic sections preserve cellular detail but may alter antigen presentation by physical or chemical means. A thorough understanding of the strengths and limitations of these assays is essential for the evaluation of experimental data and the determination of the potential clinical usefulness of new antibodies. REFERENCES
1. Cairncross, J. G., Mattes, M. J., Beresford, H. R., Albino, A. P., Houghton, A. N., Lloyd, K. 0. and Old, L. J.: Cell surface antigens of human astrocytoma defined by mouse monoclonal antibodies: identification of astrocytoma subsets. Proc. Natl. Acad. Sci. USA, 79: 5641, 1982. 2. McGee, J. O'D., Woods, J. C., Ashall, F., Bramwell, M. E. and Harris, H.: A new marker for human cancer cells. 2, Immunohistochemical detection of the Ca antigen in human tissues with the Cal antibody. Lancet, 2: 7, 1982. 3. Grossman, H. B.: Hybridoma antibodies reactive with human bladder carcinoma cell surface antigens. J. Urol., 130: 610, 1983. 4. Chang, T. H., Steplewski, Z. and Koprowski, H.: Production of monoclonal antibodies in serum free medium. J. Immunol. Methods, 39: 369, 1980. 5. Ueda, R., Shiku, H., Pfreundschuh, M., Takahashi, T., Li, L. T. C., Whitmore, W. F., Oettgen, H. F. and Old, L. J.: Cell surface antigens of human renal cancer defined by autologous typing. J. Exp. Med., 150: 564, 1979. 6. Grossman, H. B., Wedemeyer, G. and Ren, L.: Human renal carcinoma: characterization of five new cell lines. J. Surg. One., 28: 237, 1985. 7. Hsu, S. M., Raine, L. and Farzer, H.: A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am. J. Clin. Pathol., 75: 734, 1981. 8. Seljelid, R. and Ericsson, J. L. E.: Electron microscopic observations on specializations of the cell surface in renal clear cell carcinoma. Lab. Invest., 14: 435, 1965. 9. Wallace, A. C. and Nairn, R. C.: Renal tubular antigens in kidney tumors. Cancer, 29: 977, 1972. 10. Reznik, G., Ward, J. M., Hardisty, J. F. and Russfield, A.: Renal carcinogenic and nephrotoxic effects of the flame retardant tris (2,3-dibromopropyl) phosphate in F344 rats and (C57BL/6N x C3H/HeN)F 1 mice. JNCI, 63: 205, 1979. 11. Gross, M. D., Skinner, R. W. S. and Grossman, H. B.: Radioimmunodetection of a transplantable human bladder carcinoma in a nude mouse. Invest. Radiol., 19: 530, 1984. 12. Bander, N.: Comparison of antigen expression of human renal cancers in vivo and in vitro. Cancer, 53: 1235, 1984.