Removal of Breast Cancer Cells by Soybean Agglutinin in an Experimental Model for Purging Human Marrow1

(CANCER RESEARCH 48. 4573-4577, August 15. 1988] Removal of Breast Cancer Cells by Soybean Agglutinin in an Experimental Model for Purging Human Marrow1

Shoshana Morecki, Shlomo Margel, and Shimon Slavin2

Department of Bone Marrow Transplantation and Cancer Immunobiology Research Laboratory, Hadassah University Hospital, 91120 Jerusalem Â¡S.M., S. S.J, and Department of Chemistry, Bar-Han University, 52100 Kamat-Gan fS. Ma.], Israel

ABSTRACT (Â¿>)hematopoieticstem cells are SBA negative (15). SBA-treated marrow is used routinely in many centers as part of the T-cell Soybean agglutinin (SBA) was used as a differential reagent to achieve depletion procedure in allogeneic BMT following marrow abla selective elimination of human breast cancer cells (I -471) cell line) from tive doses of chemoradiotherapy (16-18). human marrow contaminated with tumor cells. Two successive cycles of direct agglutination by soluble SBA resulted in depletion of 3.5 logs of In the present study we have evaluated the use of SBA for tumor cells as determined by radiolabeling, whereas removal of more purging tumor cells using various types of magnetic beads as than 4 logs of tumor cells was demonstrated by a clonogenic bioassay. A SBA carriers, as well as by direct agglutination with soluble more convenient procedure for tumor purge involved the use of SBA SBA. Assessment of the degree of tumor cell elimination was bound to either polyglutaraldehyde magnetic beads or to commercial accomplished by a clonogenic bioassay. polystyrene magnetic beads. After one cycle of magnetic separation, 2 to 3.5 logs of tumor cells were removed. A second separation cycle using fresh magnetic beads improved depletion to more than 4 logs. Neither of MATERIALS AND METHODS these purging procedures affected the hematopoietic potential of granu- Breast Cancer Cell Lines. Breast cancer cell lines T-47D (19) (a gift loid-macrophage colony-forming unit cells. We suggest the use of SBA from Dr. I. Kedar, University of Tel Aviv, Israel), MDA-MB 231 (20), bound to magnetic beads as a convenient tool for effective ex vivo purging and MCF/7 (21) (a gift from Dr. D. Sulizeanu, Hebrew University, of marrow aspirates contaminated with metastatic breast cancer cells in Jerusalem, Israel) were grown in RPMI medium, as detailed in our patients with advanced disease. A similar procedure is applicable for all previous paper (12). Insulin (0.2 units/ml) was added only to the SBA-positive neoplasms. medium for culturing the T-47D cell line. Flow cytometric analysis, labeling of T-47D, preparation of tumor/ INTRODUCTION normal bone marrow cell mixtures, and depletion of tumor cells by agglutination were carried out, as previously described by us (12). High-dose chemotherapy or chemoradiotherapy followed by Binding of SBA to Magnetic Beads. PG magnetic beads were prepared rescue with cryopreserved autologous BM3 is a potentially by Dr. S. Margel (Weizmann Institute of Science, Israel) as previously curative therapeutic modality for an increasing number of pa described in detail (22). Binding of SBA was carried out by mixing 5 tients with various neoplastic disease (1-4). mg of magnetic beads with 0.5 mg of SBA in a total volume of 3 ml of distilled water for 3 h with continuous gentle agitation at room tem A variety of immunological and pharmacological procedures perature. Unbound SBA was removed by six washes with water using have been investigated for ex vivo purging of marrow grafts a magnetic field. The SBA-PG magnetic beads were quenched with 3 contaminated with leukemia, lymphoma, neuroblastoma, or ml of 0.5% human serum albumin in water and kept in this solution at small cell lung carcinoma cells (5-10). Breast cancer, a most 4Â°Cuntil use. PS magnetic beads (Dynabeads M-450) were kindly frequent malignant disorder in adult females, is one of the supplied by Dynal, Norway. Binding of SBA was carried out by mixing neoplastic diseases in which the availability of cryopreserved 105-mg beads with 5 mg of SBA in a total volume of 6.6 ml of PBS for 16 h at 4Â°Cbyend-over-end rotation. Dynabeads were collected by autologous BM would allow escalation of cytotoxic therapeutic doses, thereby enabling more efficient eradication of metastatic placing the suspension in a cobalt samarium MPC kindly supplied by tumor cells and prolongation of disease-free survival time. Dynal. Coated beads were washed twice immediately and then twice, 2 h each, by end-over-end rotation at 4Â°C.The fifth wash was done Unfortunately, at advanced stages of disease, BM involvement overnight in PBS containing 0.1% BSA. SBA-PS-magnetic beads were occurs in some 40 to 70% of patients (11). resuspended to a concentration of 30 mg/ml with the same buffer We have recently reported a preliminary study in which SBA (PBS/BSA) and kept at 4Â°Cuntil use. Conjugates of SBA-magnetic was utilized to remove cells of a breast cancer cell line from beads were found to be effective for at least 2 mo. artificial mixtures of tumor and human BM cells (12). Our Depletion of Tumor Cells by SBA-Magnetic Beads. SBA-PG magnetic decision to attempt selective removal of breast cancer cells from beads were mixed with the contaminated BM cells (5% and 10% T- human marrow aspirates using SBA was based on the following 47D cells) as follows: 1.3 mg of SBA-PG were incubated with 20 x 10" two observations: (a) binding of SBA to breast cancer cells was cells in a total volume of 0.3 ml for 15 min at room temperature. demonstrated in histolÃ³gica! sections of breast carcinoma tissue Magnetic particles were removed over a MPC, and the unbound cells (13, 14), and only 5% of 130 studied were totally negative; and were collected and washed with 1% BSA in PBS. When 2 cycles of separation were carried out, the SBA-negative cell fraction was sub Received 7/7/87; revised 2/10/88, 5/9/88; accepted 5/12/88. jected to another interaction with new SBA-PG magnetic beads in the The costs of publication of this article were defrayed in part by the payment following ratio: 0.40 mg of SBA-bead/4 x IO6cells. of page charges. This article must therefore be hereby marked advertisement in SBA-PS magnetic beads were mixed with tumor BM cell mixtures accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported by the National Council for Research and Development of the as follows: 12.3 mg of SBA-PS were incubated with 20 x IO6cells in a Ministry of Science and Development, the Israel Cancer Research Fund (Career total volume of 0.8 ml for 15 min at room temperature. Magnetic beads Development Award to S. S.), United States-Israel Binational Science Foundation were removed by MPC, and the cells collected and were washed and (Grant 85-257), and a special grant by the Pep and Jerry Silverstein Family subjected to a second cycle of cell separation. In the second cycle, 0.9 Foundation. 2To whom requests for reprints should be addressed, at Dept. of Bone Marrow mg of SBA-PS beads were mixed with 2 x IO6cells for 15 min at room Transplantation, Hadassah University Hospital, IL-91120 Jerusalem, Israel. temperature, and the SBA-negative cells were collected. 3The abbreviations used are: BM, bone marrow; SBA, soybean agglutinin; Clonogeneic Assay. Various amounts of T-47D cells (10 to 1000) GM-CFU, granuloid macrophage colony-forming unit; PG, polyglutaraldehyde; were plated with 2x10* irradiated (4000 rads) fresh human BM cells PS, polystyrene; BMT, bone marrow transplantation; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; MPC, magnetic suspended in 1 ml of culture medium supplemented with 15% FBS and particle concentrator. 0.5% agar (Difco, MI). Cell suspensions were each plated over a layer 4573

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1988 American Association for Cancer Research. REMOVAL OF BREAST CANCER CELL BY SOYBEAN AGGLUT1NIN of 1% agar in 1 ml of 15% FBS culture medium in 35-mm Petri dishes Removal of 51Cr-radiolabeled T-47D Cells from BM/Tumor (Nunc, Denmark). Plates were maintained at 37Â°Cin a 5% CO2- Cell Mixtures by Two Cycles of Agglutination with SBA. Fresh humidified incubator for 12 to 14 days. human BM cells mixed with 10% 51Cr-radiolabeled T-47D cells Granuloid-Macrophage Colony-formed Units. A standard test for were subjected to agglutination with SBA. In order to improve assaying committed GM-CFU was carried out according to the method the degree of BM purging, the SBA-negative fraction collected of Pike and Robinson (23) with a slight modification, as previously described (12). Briefly, 2 x 105 BM cells suspended in 1 ml of McCoy's in the first step was subjected to a second cycle of SBA agglu Medium 5A (Gibco Laboratories, Grand Island, NY) supplemented tination. The degree of depletion was calculated by comparing with 0.3% agar and 20% FBS were plated over 1 ml of a layer consisting radioactivity left in the second SBA-negative fraction to the of 0.5% agar and 10% conditioned medium supernatant collected from total radioactivity recovered in both SBA-positive fractions of 5637 bladder carcinoma cell line (kindly supplied by Dr. E. Fibach, the 2 cycles and the SBA-negative fraction collected in the Department of Hematology, Hadassah University Hospital. Jerusalem, Israel). Plates were kept at 37Â°Cina 5% CO2-humidified incubator for second cycle. After 2 cycles of agglutination, the residual radio activity left was 0.05% and 0.025% in the 2 experiments pre 10 to 12 days. T-47D cells were irradiated (6000 rads) before mixing with human BM cells in order to avoid colony growth of T-47D origin sented in Table 2. Depletions of tumor cells expressed in orders which might affect the growth of GM-CFU. of magnitude were 3.3 and 3.6, respectively. Log depletions of tumor cells calculated in relation to radioactivity added to the original mixtures were 3.5 and 3.8, respectively. The total RESULTS number of cells recovered in the SBA-negative fraction after 2 cycles of agglutination was between 3.5 and 5%. Binding of Fluorescein Isothiocyanate-conjugated SBA to T-47D Clonogenic Assay. In order to improve the evaluation Breast Cancer Cell Lines. As shown in Fig. 1, fluorescence- of tumor cell depletion, we developed a sensitive biological activated cell sorter analysis of 2 x IO4 T-47D cells showed assay which provides better biological information than the bright fluorescent SBA staining which could be abrogated in radiolabeling assay. Various amounts of T-47D cells (10 to IO3) the presence of the SBA-specific inhibitor o-galactose. A large with or without 2 x 10s BM cells were plated in agar. Tumor fraction of MDA-MB 231 and MCF/7 cells also showed a colonies consisting of more than 10 cells each were formed strong fluorescent stain (data not shown). between Days 12 and 14. Depletion of T-47D Cells from BM/Tumor Cell Mixtures after The number of T-47D colonies formed was not affected by One Cycle of Separation by Soluble SBA or SBA-PG. Artificial the presence of BM cells (data not shown). Irradiation of BM mixtures consisting of normal human BM cells and 5 to 10% cells before mixing with T-47D cells was carried out in order "Cr-labeled T-47D tumor cells were subjected to agglutination to prevent colony growth from BM origin, thus enabling exact by soluble SBA or to interaction with SBA-PG magnetic beads. titration of tumor cells for establishment of a standard clono- After removal of SBA-positive cells (T-47D as well as those genic assay in the experimental model. Double logarithmic present in human BM), radioactivity remaining in the SBA- plots of the number of T-47D colonies formed as a function of negative fraction was measured and compared to the total the number of cells plated gave a straight line with a slope of radioactive recovery. In Table 1 we compared the efficacy of T- 0.91, indicating the clonal origin of the colonies. Clonogenic 47D depletion obtained by soluble SBA with depletion accom efficiency ranged between 30 and 80% when small numbers of plished by SBA-PG magnetic beads. Depletion of the same tumor cells were plated and between 60 and 80% when large order of magnitude was achieved by both means: 98.1% of the radioactivity was removed from the SBA-negative fraction by numbers of cells were plated. Fig. 2 demonstrates a titration soluble SBA and 97.8% by SBA-PG magnetic beads. Total cell curve which summarizes 3 different experiments. A titration recovery in the SBA-negative fraction was between 4 and 10% curve of this type was later performed for each experiment of by direct agglutination and 5 and 10% by SBA-PG magnetic tumor cell depletion, and the number of tumor cells left after beads. These results indicate that SBA-PG magnetic beads are purging was calculated by extrapolation on the titration curve. Depletion of T-47D Cells by Two Cycles of Agglutination with as effective as soluble SBA for tumor cell depletion. None of these separation procedures affected the recovery of GM-CFU SBA as Assessed by Clonogenic Assay. Fresh human BM cells were mixed with 10% T-47D cells and subjected to 2 cycles of hematopoietic cells (data not shown). agglutination with SBA. A sample of 2 x IO5 cells obtained from the second cycle SBA-negative fraction was plated in a T- T-A7D 47D clonogenic assay. From a standard titration curve done on the same day, we extrapolated the number of tumor cells present

Table 2 Depletion of^'Cr-radiolabeled T-47D cells by 2 cycles of agglutination with SBA Mixtures containing fresh human bone marrow cells and 10% "Cr-radiolabeled Fluorescence intensity T-47D cells were subjected to 2 cycles of agglutination. The degree of tumor cell Fig. 1. Cytofluorometry of cells of breast cancer cell line T-47D stained with depletion was determined by comparing radioactivity left in the second SBA- fluorescein isothiocyanate-conjugated SBA in the presence (a) or absence (b) of the specific inhibitor D-galactose. negative fraction, either to total radioactivity recovered in both fractions (SBA positive and SBA negative) or to radioactivity added to the original mixtures.

Table I Depletion of'Cr-radiolabeled T-47D cells from 5 to 10% tumor/bone Experiment 1 Experiment 2 marrow cell mixtures by direct agglutination with SBA or by SBA covalenti)' cpm in SBA-negative fraction 1430 6SS bound to polyglutaraldehyde magnetic beads (SBA-PG) Total cpm recovered in 2 cycles 2.8 x IO4 2.6 x 10' % of radioactivity in SBA-negative frac Radioactivity left in SBA-negative fraction 0.050% 0.025% tion out of total radioactivity recovered out of total radioactivity recovered Log depletion 3.3 3.6 of experiments12 Radioactivity left in SBA-negative fraction 0.030% 0.016% SBA agglutination out of radioactivity added SBA-PGMedian1.92.2Range0.2-9.2 1.3-3.3No. 3 Log depletion 3.5 3.8

4574

1000 assessed by T-47D clonogenic assay. Results of 3 experiments are summarized in Table 4. In Experiment 1, PG magnetic beads were used, and of 2 x IO6 T-47D cells added to the Q LU original mixture, 127 were left after 2 cycles of cell separation. et o In Experiments 2 and 3, PS magnetic beads were used, and u. samples of both steps of cell separation were taken. In Experi n y 100 ment 2, of 1.3 x IO6 T-47D cells added to original mixture, 3 800 tumor cells were left after the first cycle. The second step o o further improved removal of tumor cells; only 15 T-47D cells were left in the SBA-negative fraction. In Experiment 3, of 2 x IO6 T-47D cells in the original mixture, 575 tumor cells were left in the SBA-negative fraction after the first step of separation LL O and 78 tumor cells after the second cycle. o: 10 UJ Efficacy of tumor cell removal after one cycle of separation co with PS magnetic beads was calculated to be 3.2 and 3.5 orders of magnitude in Experiments 2 and 3, respectively. After 2 cycles of separation, depletion of tumor cells achieved with PG magnetic beads was 4.2 orders of magnitude and 4.95 or 4.4 with PS magnetic beads. Total cell yield in the SBA-negative

10 100 1000 fraction after 2 cycles of separation was between 7.5 and 11.5%. NUMBER OF T-47D CELLS PLATED Using SBA-PS magnetic beads for tumor cell depletion did not Fig. 2. Titration curve of T-47D clonogenic assay. Points, mean of 3 different affect the recovery of GM-CFU after 2 cycles of cell separation. experiments (each performed in triplicate); bars, SE. As can be seen in Table 5, the number of GM-CFU postsepa- ration was approximately the same as found in the original Table 3 Depletion of tumor cells by 2 cycles of agglutination with SUA as assessed by the T-47D clonogenic assay untreated fresh human BM cells (58 versus 48 in Experiment 1 Artificial mixtures consisting of 10% T-47D cells and fresh bone marrow cells and 212 versus 233 in Experiment 2). were subjected to 2 cycles of agglutination with soluble SBA. The number of T- 47D cells left in the second SBA-negative fraction was determined in a clonogenic DISCUSSION assay. Experiment 1 Experiment 2 Experiment 3 The present report describes the use of the lectin SBA to No. of T-47D colonies formed 20 Â±0.73Â° 20 Â±1.47 281 Â±15.8 remove tumor cells from artificial mixtures of breast carcinoma in 2 x 10' cells of SBA-neg ative fraction Table 4 Depletion of tumor cells by two cycles of separation with SBA-magnetic beads, as assessed by T-47D clonogenic assay No. of T-47D present* in 2 x 34 29 400 10' cells of SBA-negative Experi Experi Experi ment 1Â° ment 2 ment 3 fraction No. of T-47D colonies present in ND* 64 Â±4.0e 46 Â±0.5 2x 10'cells of first SBA- Total no. of T-47D cells left in 425 188 3260 SBA-negative fraction negative fraction

Total no. of T-47D cells added 8.0x10' 4.0x IO6 3.8x 10' Total no. of T-47D cells in first ND 800 575 SBA-negative fraction to original BM/tumor cell Log depletion'' ND 3.22 3.55 Log depletion 4.3 4.3 3.0 No. of T-47D colonies present in 17 Â±1.8 2 Â±0.8 9 Â±2.5 " Mean Â±SE of triplicates. 2x10' cells of second SBA- * Number of T-47D present was determined from the cloning efficiency titra- negative tion curves. fraction in 2 x 105 cells of the SBA-negative fraction and calculated the Total no. of T-47D cells in second 127 15 78 SBA-negative fraction total number of tumor cells left in this fraction. The numbers of T-47D cells left in 2 x 10s cells were 34, 29, and 400 in 3 Log depletion'' 4.20 4.95 4.40 experiments presented in Table 3. Of the 8 x IO6, 4 x IO6, and Yield of total cells in second SBA- 7.5% 11.5% 8.7% 3.8 x IO6T-47D cells added to the original mixtures in these 3 negative fraction experiments, the numbers of residual T-47D cells after the " Experiment 1 was done with SBA-PG magnetic beads, while Experiments 2 and 3 were done with SBA-PS magnetic beads. separation procedure were calculated to be 425, 188, and 3260, * ND, not determined. respectively. Thus, depletion of 4.3 orders of magnitude was ' Mean Â±SE. achieved in 2 experiments, and only 3 orders of magnitude of d Log depletion was calculated per original number of T-47D. tumor cells were removed in one experiment. Clonogenic effi Table 5 Recovery of GM-CFU in T-47D/bone marrow mixtures after 2 cycles of ciency in all 3 experiments was between 60 and 90%. The total separation with SBA-PS beads cell recovery in the SBA-negative fraction after 2 cycles of T-47D cells were irradiated (4000 rads) before mixing with BM cells. separation was 3 to 4%. GM-CFU/2X 10'cells Removal of T-47D Cells and GM-CFU Recovery from the BM/Tumor Cell Mixture Subjected to Two Cycles of Interaction Experiment 1 Experiment 2 with SBA-Magnetic Beads. Artificial mixtures consisting of Untreated BM cells 48 (xl) 233+16" 10% T-47D tumor cells and fresh human BM cells were sub SBA-negative fraction after 2 58 Â±4 212 Â±26 jected to 2 cycles of interaction with SBA bound to either PG cycles of magnetic beads or PS magnetic beads. Efficacy of tumor cell depletion was 1Mean Â±SE. 4575

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1988 American Association for Cancer Research. REMOVAL OF BREAST CANCER CELL BY SOYBEAN AGGLUT1NIN cell line-derived cells (T-47D) and fresh normal human BM vious studies have indicated that, although 90% of the nucleated cells. This experimental system was developed to simulate the cells are lost following purging with SBA (and rosetting with BM aspirate of metastatic breast cancer infiltrating the marrow sheep RBC) in allogeneic BMT as part of the T-cell depletion in patients with advanced disease, bearing in mind autologous procedure, most GM-CFU are left in the final fraction, and fast BMT as a rational form of therapy. Nevertheless, because of hemopoietic reconstitution of lethally conditioned recipients is the ubiquitous distribution of SBA in a variety of cancer cells, the rule (17). Therefore, most of the cell loss following frac- our method represents a model system for other SBA-positive tionation by marrow with SBA is clinically irrelevant, since tumors as well. Our studies demonstrate that one cycle of human pluripotent stem cells are clearly SBA negative (15-18). separation, carried out either by agglutination with soluble SBA SBA agglutination or SBA bound to Sepharose columns was or by SBA covalently bound to PG magnetic beads, results in a previously tested in 2 experimental models, including neuro depletion of radiolabeled tumor cells of almost 2 logs. Hema- blastoma and T-cell leukemia line cells. Only 1 log of depletion topoietic cells of the GM-CFU lineage were not affected by was achieved for neuroblastoma cells, while a 3-log depletion these procedures (data not shown). Moreover, tumor cell elim was accomplished for a T-cell leukemia line (8). The difference ination of 3.5 orders of magnitude could be achieved by sub in depletion was ascribed to the total number of SBA receptors jecting the mixtures to 2 successive cycles of agglutination with present on the tumor cells tested. Evidence for variable degree soluble SBA (Table 2) or, as we have already shown, by com of SBA binding to breast carcinoma cells was previously tested bining one cycle of agglutination with one cycle of polyacrolein in paraffin-embedded breast carcinoma sections (13, 14) and in magnetic bead separation (12). These results were obtained a number of breast cancer cell lines examined in our laboratory from a radiolabeling assay using 5lCr-labeled tumor cells, which (Ref. 12 and the present study). Based on the positive binding provide an immediate evaluation of the efficacy of tumor cell of SBA to breast cancer cells as well as other tumor cells and depletion. in view of the fact that hemopoietic stem cells are SBA negative, In order to evaluate the effect of our purging techniques on SBA can serve as a tool for selective elimination of breast residual SBA-negative clonogenic tumor cells, we established cancer cells as well as other SBA-positive cancer cells without the conditions for a tumor colony growth assay in agar. Clon compromising hematopoietic marrow reconstitution following ogenic efficiency was mostly above 65%, with the exception of autologous BMT. In view of the heterogeneity of breast cancer a few cases of low efficiency (<50%) for low doses of T-47D cells, low affinity of SBA binding to certain cancer cell subsets cells (10 cells) on the titration curve. Contamination as low as in comparison with normal marrow cells might interfere with 0.005% of tumor cells seeded in 2 x 10s BM cells could be effective depletion of a low concentration of tumor cells in detected by our clonogenic assay. This provided us with a more tumor/BM mixtures. Nevertheless, the use of 2 subsequent precise, biologically meaningful evaluation of residual tumor cycles of SBA separation is expected to give the cancer cell with cells than could be obtained from the radiolabeling assay. The low-affinity binding a better chance of interaction with SBA in degree of tumor cell depletion after 2 cycles of SBA agglutina the second separation cycle after removal of more avidly binding tion was 3.5 logs, as determined by immediate radiolabeling, cells. whereas the clonogenic assay indicated a depletion of 4.3 logs Previous reports on the use of magnetic beads for marrow in 2 experiments and 3 logs in one experiment. The clonogenic purging in experimental models as well as in clinical BMT (6, assay is of practical relevance, since it can provide indication 24) suggested the use of beads bound to monoclonal antibodies of the growth potential of residual tumor cells following marrow directed against the target cells or against a primary antibody purging in clinical autologous transplantation. bound to the target cells. In contrast to the need for tumor- Various types of SBA-bound magnetic beads were tested in specific antibodies required for each disease (which can be an attempt to identify the optimal purging technique. In a extremely variable in itself), SBA, a lectin which is commer previous study (12) we compared direct agglutination by soluble cially available, can be bound to magnetic beads and might SBA and SBA covalently bound to polyacrolein magnetic beads serve as a general reagent to remove many types of tumor cells of various sizes (0.7 to 5 Mm). No difference was found in the which bear the receptor for SBA, since SBA receptors are degree of tumor cell depletion achieved by these 2 techniques. ubiquitous on a variety of tumor cells. Nevertheless, due to the In the present investigation we used PG magnetic beads (0.2 heterogeneity of tumor cell populations (in some cases investi i/m) covalently bound to SBA, as well as the commercial PS gated, in tissue sections only a small proportion of breast cancer magnetic beads (4.5 um) bound to SBA. As with the case with cells react with SBA; 14), a combination of various approaches direct agglutination by soluble SBA, 2 cycles of interaction with will probably provide the most promising strategy for the most SBA-magnetic beads improved elimination of tumor cells when effective removal of tumor cells from heavily contaminated BM compared with one cycle of separation. A comparable degree aspirates in conjunction with autologous BMT. of depletion was obtained by SBA-PS beads and SBA-PG beads. However, PS beads displayed somewhat more efficient tumor cell depletion (4.95 and 4.4 logs as compared to 4.2 logs) and ACKNOWLEDGMENTS better total recovery of nucleated cells. Both types of beads The authors wish to acknowledge grant support from those sources spare the GM-CFU hematopoietic cells. listed in Footnote 1. The use of SBA magnetic beads has several advantages over direct agglutination by soluble SBA. (a) Separation of the SBA- negative fraction is technically simple and faster. This aspect is REFERENCES of particular importance when large volumes are involved, as is the case in clinical situations in preparation for autologous 1. Gorin, N. G., David, R., Stachowiak, J., Salmon, C, Pettit, J. C, Parlier, Y., Najman, A., and Duhamei, G. High dose chemotherapy and autologous BMT. (/>)The yield of total nucleated cells was better than with bone marrow transplantation in acute leukemias, malignant lymphomas, and soluble SBA. It is likely that nonspecific cell loss in large-scale solid tumors. A study of 23 patients. Eur. J. Cancer, 17: 557-568, 1981. 2. Ritz. J.. Sallan, S., Basel. R. C., Upton. J. M., Clavella, L. A., Feeney, M.. separation necessary for clinical BMT would be proportionately Hercend, T., Nathan, D. G., and Schlossman, S. F. Autologous bone marrow even less than that observed in small-scale experiments. Pre transplantation in CALLA-positive acute lymphoblastic leukemia after in 4576

vitro treatment with J-5 monoclonal antibody and complement. Lancet, 2: cells in preparation for autologous bone marrow transplantation. Bone Mar 60-63, 1982. row Transplant., /: 357-363, 1987. 3. Philip, T., Biron, P., Maraninchi, D.. Goldstone, A. H., HervÃ©,P.,Souillet. 13. Franklin, W. A. Tissue binding of lectins in disorders of the breast. Cancer, G., Gastaut. J. L., Plouvier, E., Flesh, Y., Philip, I., Harousseau. J. L., Le 51: 295-300, 1983. 14. Walker, R. A. The binding of peroxidase-labelled lectins to human breast Mevel, A., Rebattu, P., Linch, D. C, Freycon, F., Milan, J. J., and Souhami, R. L. Massive chemotherapy with autologous bone marrow transplantation epithelium. IV. The reactivity of breast carcinomas to peanut, soybean, and in SO cases of non-Hodgkin's lymphoma with poor prognosis. In: K. A. Dolichos biflorus agglutinins. J. Pathol., 145: 269-277, 1985. 15. Reisner, Y., O'Reilly, R. J., Kapoor. N.. and Good, R. A. Allogeneic bone Dicke. G. Spitzer, and A. R. Zander (eds.), Autologous Bone Marrow Transplantation, pp. 89-107. Houston, TX: The University of Texas-M. D. marrow transplantation using stem cells fractionated by lectins. VI. In vitro analysis of human and monkey bone marrow cells fractionated by sheep red Anderson Hospital and Tumor Institute of Houston, 1985. blood cells and soybean agglutinin. Lancet, 2: 1320-1323, 1980. 4. Ratanathrathorn, V., Karanes, C., Samson, M. K., CorbeÂ«,T. H., and Baker, 16. Cowan, M. J., Wara, D. W., Weintrub, P. S., Pabst, M., and Ammann, A. L. H. High dose chemotherapy with autologous bone marrow transplantation J. Haploidentical bone marrow transplantation for severe combined immu in solid tumors: a Wayne State University experience. In: K. A. Dicke, G. nodeficiency disease using soybean agglutinin-negative, T-depleted marrow Spitzer, and A. R. Zander (eds.). Autologous Bone Marrow Transplantation, cells. J. Clin. Immunobiol., 5: 370-376, 1985. pp. 249-254. Houston, TX: The University of Texas-M. D. Anderson Hos 17. Reisner. Y., Kapoor, N., Kirkpatrick, D.. Pollack. M. S., Dupont, B., Good, R. A., and O'Reilly, R. J. Transplantation for acute leukemia with HLA-A pital and Tumor Institute of Houston, 1985. 5. Uckun, F. M.. Stong, R. C., Youle, R. J., and Vallera, D. A. Combined ex and B non-identical parental marrow cells fractionated with soybean agglu vivo treatment with immunotoxins and mafosfamid: a novel immunochem- tinin and sheep red blood cells. Lancet, 2: 327-331, 1981. otherapeutic approach for elimination of enoplastic T-cells from autologous 18. Slavin, S., Waldmann, H., Or, R., Cividalli, G., Naparstek, E., Steiner-Salz, marrow grafts. J. Ininnino!., 134: 3504-3515, 1985. D., Michaeli, J., Galun, E., Weiss, L., Samuel, S., Morecki. S., Bar, S., Brautbar, C., Weshler, Z., Hale, G., Rachmilewitz, E. A., and Reisner, Y. 6. Kemshead, J. T., Treleavern, J. G., Gibson, F. M., and Ugelstad, J. Removal Prevention of graft vv host disease in allogeneic bone marrow transplantation of malignant cells from bone marrow using magnetic microspheres and for leukemia by T-cell depletion in vitro prior to transplantation. Transplant. monoclonal antibodies. Prog. Exp. Tumor Res., 29: 249-255, 1985. Proc., / 7: 465-467, 1985. 7. Berenson. R. J.. Bensinger, W. I.. Kalamasz. D., and Martin. P. Elimination 19. Kedar, I.. Chen, L., Karby. S.. Weiss. F. R.. Delarea, J., Radu, M., Chaitcik, of Daudi lymphoblasts from human bone marrow using avidin-biotin ini S., and Brenner, H. J. Establishment and characterization of a cell line of munoadsorption. Blood, 67: 509-515, 1986. human breast carcinoma origin. Eur. J. Cancer, 15:659-670, 1979. 8. Reisner, Y., and Gan. J. Differential binding of soybean agglutinin to human 20. Cailleau, R., Young, R., Olive, M., and Reeves, W. J., Jr. Breast tumor cell neuroblastoma cell lines: potential application to autologous bone marrow lines from pleural effusions. J. Nati. Cancer Inst., 53: 661-674, 1974. transplantation. Cancer Res., 45: 4026-4031, 1985. 21. Soule, H. D., Vazquez, J., Long, S., Albert, S., and Brennan, M. A human 9. Okabe, T., Kaizu, T., Ozawa, K.. Urabe, A., and Takaku, F. Elimination of cell from a pleural effusion derived from a breast carcinoma. J. Nati. Cancer Inst., 5/: 1409-1416, 1973. small cell lung cancer cells in vitro from human bone marrow by a monoclonal antibody. Cancer Res., 45: 1930-1933, 1985. 22. Margel, S., Zisblatt, S., and Rembaum, A. Polyglularaldehyde: a new reagent for coupling proteins to microspheres and for labeling cell-surface receptors. 10. Sieber, F., Rao, S., Rowley, S. D., and Sieber-Blum, M. Dye-mediated II. Simplified labeling method by means of nonmagnetic and magnetic photolysis of human neuroblastoma cells: implications for autologous bone polyglutaraldehyde microspheres. J. luminimi. Methods. 28:341-353,1979. marrow transplantation. Blood. 68: 32-36, 1986. 23. Pike, B. L., and Robinson, W. A. Human bone marrow colony growth in 11. DeVita, V. T., Jr., Hellman. S., and Rosenberg, S. A. (eds.). Cancer: Princi agar-gel. J. Cell Physiol.. 76: 77-84, 1970. ples and Practice of Oncology. Ed. 2, pp. 1119-1177. Philadelphia: Lippin- 24. Reynolds, C. P., Black, A. T., Saur, J. W., Seeger. R. C., Ugelstad, J., and cott, 1985. Woody, J. N. An immunomagnetic flow system for selective depletion of cell 12. Morecki, S., Pavlotzky, F.. Margel, S., and Slavin, S. Purging breast cancer population from marrow. Transplant. Proc.. 77:434-436. 1985.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1988 American Association for Cancer Research. Removal of Breast Cancer Cells by Soybean Agglutinin in an Experimental Model for Purging Human Marrow

Shoshana Morecki, Shlomo Margel and Shimon Slavin

Cancer Res 1988;48:4573-4577.

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