Tumor Cells Express Fcγrl Which Contributes to Tumor Cell Growth and a Metastatic Phenotype

RESEARCH ARTICLE Neoplasia . Vol. 3, No. 2, 2001, pp. 115 ± 124 115 www.nature.com/neo Tumor Cells Express Fc RI Which Contributes to Tumor Cell Growth and a Metastatic Phenotype

M. Bud Nelson, Julie K. Nyhus, Katherine I. Oravecz-Wilson and Emilio Barbera-Guillem

BioCrystal Ltd., Westerville, OH

Abstract High levels of circulating immune complexes containing epitope, and the shed tumor±associated antigen complexes tumor-associated antigens are associated with a poor with this IgG antibody in forming polyvalent immune prognosis for individuals with cancer. The ability of B complexes. The polyvalent immune complexes cross-link cells, previously exposed to tumor-associated anti- Fc RonFc R-expressing immune effector cells, such as gens, to promote both in vitro and in vivo tumor growth monocytes and interferon-gamma±activated polymorpho- formed the rationale to evaluate the mechanism by nuclear cells, in a process that results in release in the tumor which immune complexes may promote tumor growth. stroma of soluble mediators, which promote tumor cell In elucidating this mechanism, Fc RI expression by invasion, angiogenesis, and metastasis. Thus, there is a tumor cells was characterized by flow cytometry, mechanism by which B cells and antitumor antibodies, as polymerase chain reaction, and sequence analysis. mediated by immune effector cells, can induce tumor Immune complexes containing shed tumor antigen and progression [7]. A direct antibody±tumor cell interaction anti±shed tumor antigen Ab cross-linked Fc RI-ex- has been proposed to involve IgM, which binds tumor cell- pressing tumor cells, which resulted in an induction of associated antigen, thereby coating and masking the tumor tumor cell proliferation and of shed tumor antigen cells, protecting them from attack by cytotoxic lymphocytes production. Use of selective tyrosine kinase inhibitors and allowing for progressive growth of the tumor [9]. demonstrated that tumor cell proliferation induced by Independent of these discoveries, it has been proposed that immune complex cross-linking of Fc RI is dependent nonlymphoid tumors, experimental and human, express on the tyrosine kinase signal transduction pathway. A Fc R identified as Fc RIIB1 [10]. Although the mechan- selective inhibitor of phosphatidylinositol-3 kinase also ism(s) by which Fc RIIB1 may confer augmentation of inhibited this induction of tumor cell proliferation. These tumorigenicity remains unknown, it was suggested that findings support a role for immune complexes and Fc RI complexed IgG, bound by Fc RIIB1 on tumor cells, could expression by tumor cells in augmentation of tumor sterically block immune effector mechanisms from eliminat- growth and a metastatic phenotype. Neoplasia (2001) 3, ing the tumor cells. 115±124. We have observed that mucin-secreting tumor cells appeared to be able to interact directly with B cells to Keywords: B lymphocytes, tumor, cellular proliferation, Fc receptors, signal transduction. modulate tumor cell behavior. Here, we characterized this interaction and found that the B cells require prior exposure to tumor-associated antigens, that a tumor cell Introduction receptor for mediating this interaction is Fc RI, and that it Despite the activation of lymphocytes such as NK cells, T is immune complex binding and cross-linking of this cells, and B cells by tumor antigens, the immune system is receptor, which induces both tumor cell proliferation and often inefficient in both eliminating the tumor and preventing production of mucin with polyvalent sialyl-Tn (sTn) antigen its progressive growth. There have been several proposed (sTn-mucin). mechanisms by which tumor cells may evade immune attack by inducing apoptosis of lymphocytes, including by expression of Fas ligand, and by some tumor-associated antigens Materials and Methods such as RCAS1 and Muc-1 [1±4]. Additionally, it has also been proposed that B cells may be involved in lymphocyte Cell Lines suppression, thereby allowing tumor cells to escape T-47D (human ductal breast carcinoma), SW620 (human mechanisms of T cell±mediated cytotoxicity [5,6]. Further, colon carcinoma), B16F1 (C57BL murine melanoma), and we have identified a novel mechanism by which B cells

contribute to the promotion of tumor growth, invasion, and Abbreviations: sTn, sialyl Tn; B - TIL, tumor - infiltrating B lymphocytes; TIL, tumor - metastasis in cooperation with immune effector cells [7,8]. infiltrating T lymphocytes; PI - 3 kinase, phosphatidylinositol - 3 kinase Address all correspondence to: Emilio Barbera - guillem, BioCrystal Ltd., 575 McCorkle Briefly, B cells are activated by shed tumor±associated Boulevard, Westerville, OH 43082 - 8888. E-mail: [email protected] antigens in a humoral immune response resulting in the Received 11 October 2000; Accepted 28 November 2000.

production of IgG antibody against a repeated carbohydrate Copyright # 2001 Nature Publishing Group All rights reserved 1522-8002/01/$17.00 116 Contribution of Fc RI Expression to Tumor Cell Malignancy Nelson et al.

U937 (monocytic) cell lines were obtained from ATCC Fc R Expression (Rockville, MD). The Met 129 (methylcholanthrene-induced B16F1 tumor cells, T-47D tumor cells, and Met 129 tumor mammary carcinoma in C3H mice) cell line was kindly cells were each cultured as an adherent monolayer provided by Dr J. Vaage (Roswell Park Memorial Institute, (378C+5% CO2 ) in serum-free tissue culture medium; Buffalo, NY). SW620 tumor cells were cultured in suspension with agitation (378C) in serum-free tissue culture medium. The Mice respective tumor cells were harvested, washed twice with C3H mice and athymic nude C3H mice (nu/nu) were PBS, centrifuged at 1200 rpm for 10 minutes, and then purchased from Harlan Sprague-Dawley. The use of resuspended in PBS without calcium and magnesium. Cells experimental animals, and animal care, was in accordance were counted, and cell viability was checked using trypan with both the NIH and European Community principles of blue exclusion dye. Cells were aliquoted at a concentration of laboratory animal care. 1 million cells per tube, and resuspended in 50 lofan antibody solution selected from fluoroscein isothiocyanate (FITC)-labeled anti-Fc RI (CD64), phycoerythrin (PE)- In Vivo Tumor Growth labeled anti-Fc RII (CD32), R-phycoerythrin-cyanin 5.1 B cells were isolated as described previously [7] using (PC5)-labeled anti-Fc RIII (CD16), PE-labeled isotype anti-B220 mAb, selected using magnetic beads coated with control IgG1 mAb, or PE-labeled isotype control IgG2 mAb sheep anti-rat IgG (Dynal), and cultured in RPMI. Flow (fluorescent dye±labeled mAbs all obtained from Immuno- cytometry was used to confirm that greater than 90% of tech, Coulter). After staining for 30 minutes on ice, 800 lof these isolated cells were B220+. C3H mice or athymic nude PBS was added, the cells were pelleted by centrifugation at 6 C3H mice were injected intrasplenically with 10 Met 129 3000 rpm for 3 minutes, and the supernatants were removed. tumor cells. Starting 5 days later, the mice were injected The cell pellets were then resuspended in 250 to 350 lof every 2 days for a 14-day period with either B cells (50,000 PBS for analysis by flow cytometry. Each value was cells/100 l) isolated from normal mouse spleen, or isolated corrected for nonspecific binding by subtracting the value from spleens of tumor-bearing mice, or isolated from tumors of the respective isotype control. For analysis of Fc RI of tumor-bearing mice, or received no injection of B cells. expression at the mRNA level, RNA isolation, RT/PCR After the 14-day period, liver metastasis and spleen tumor conditions, primers, PCR conditions for cDNA amplification, was scored by assigning a numerical value where ``0'' and analysis by agarose gel electrophoresis were per- represents no metastasis (as proven by histology); ``1'' formed as described by Ernst et al. [11]. U937 monocytic represents a single metastases in one liver lobe; ``2'' cell RNA was used as a control for expression of all 3 represents several metastases in one liver lobe; ``3'' Fc RI genes. Fc RI-specific primers included 50 -ACAC- represents multiple metastases involving several liver lobes; CACAAAGGCAGTGA-30 and reverse primer 50 -CACCCA- and ``4'' represents greater than 50% of the liver mass is GAGAACAGTGTT-30. DNA sequencing was performed tumor. The degree of spleen tumor was scored by assigning commercially. a numerical value, where: ``0'' represents no tumor (as proven by histology); ``1'' represents a small tumor; ``2'' Immune Complex Induction of Tumor Proliferation represents a large tumor with visible spleen; ``3'' represents a Either B72.3 mAb (IgG1; ATCC) or HB-STn mAb large tumor in which only a portion of the spleen shape is (IgG1; Dako) were used as the anti-sTn mAb. For initial visible; and ``4'' represents a tumor so large that the spleen induction experiments, sTn-mucin±secreting tumor cells shape is no longer visible. Each individual score was were aliquoted into 96-well plates at 1000 cells per well. squared, and then the values for the group being analyzed After culture overnight (378Cin5%CO2 ), the culture were averaged to obtain a single value representative of that medium from each well was removed, and replaced with group. fresh serum-free tissue culture medium alone or with anti-sTn mAb (HB-STn) to a final concentration of either In Vitro Tumor Growth in Presence of B Cells 10, 1, 0.1, or 0.01 g/ml, for a total of six wells per Tumor infiltrating CD8+ lymphocytes were isolated from control or antibody dilution. In blocking Fc RI [12] dispersed tumor tissue from C3H mice using magnetic experiments, 100-fold excess of Fc fragments (prepared separation techniques; and flow cytometry was used to from papain digestion of B72.3 mAb) was added to each demonstrate that greater than 90% of these isolated cells antibody dilution and control. After incubating the 96-well were CD8+. Ten thousand Met 129 cells were cultured in 1.5 plates for an additional 24 hours, proliferation was ml of tissue culture medium supplemented with 10% fetal assessed by removing the culture medium, adding MTT bovine serum per well in 24-well plates alone, or in the (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazolium bro- presence of either 10,000 CD8+ tumor-infiltrating T lym- mide; thiazolyl blue) dye to the cells in each well (final phocytes (TIL), 10,000 tumor-infiltrating B lymphocytes (B- concentration of 0.5 mg/ml), incubating the plate for 4 TIL), or 10,000 B-TIL and 10,000 CD8+ TIL cells. After 72 hours, solubilizing the dye with 0.1 N HCl in absolute hours of coincubation in monolayer culture, Met 129 tumor isopropanol, and measuring the absorbance at 570 and cell growth was quantitated by counting Alcian blue±stained 690 nm. Cell proliferation was expressed as the absor- tumor cells. bance measured at 570 nm minus the absorbance at 690

Neoplasia . Vol. 3, No. 2, 2001 Contribution of Fc RI Expression to Tumor Cell Malignancy Nelson et al. 117 nm. In the case of non±sTn-mucin secreting B16F1, the culture medium, and then incubated with either fresh same procedure was followed, except that the cells were serum-free tissue culture medium (controls) or the incubated for a 72-hour period, and every 24 hours the medium containing anti-sTn mAb (B72.3; 0.1 g/ml) for culture medium was removed from each well and replaced a 24-hour culture period, and then determination of sTn- with either fresh serum-free tissue culture medium mucin production by ELISA. (``without mucin'') or with 90 l of fresh serum-free tissue culture medium containing 10 l of cell-free tissue culture Statistical Analysis supernatant containing shed sTn-mucin from cultured T- Data from each experimental group were subjected to an 47D tumor cells (``with mucin''). In assaying for inhibition analysis of normality and variance. Differences between of induction of tumor proliferation using tyrosine kinase comparative experimental groups were analyzed for statis- inhibitors (genistein, herbimycin A; Sigma, St. Louis, MO) tical significance using Student's t test. or PI-3 kinase inhibitor (wortmannin; Sigma), 1000 T-47D tumor cells were plated as described above, followed by treatment with either: serum-free tissue culture medium Results alone (1 hour); the medium containing genistein (final concentration 100 M; for 1 hour); the medium containing B Cells with Prior Exposure to Tumor-Associated Antigens herbimycin A (final concentration 10 M; for 1 hour); or Promoted In Vivo Tumor Growth the medium containing wortmannin (final concentration of To investigate whether B cells first require exposure to 500 nM; for 10 minutes). After the appropriate treatment tumor-associated antigens to be able to interact with period, the cells were washed with fresh serum-free tumors in promoting tumor growth in vivo, C3H mice were tissue culture medium, and then incubated with either injected intrasplenically with high mucin-secreting, Met fresh serum-free tissue culture medium (controls) or the 129 murine mammary carcinoma cells. The mice were medium containing anti-sTn mAb (HB-STn; 0.1 g/ml) then divided into three groups: one group received B cells for a 24-hour culture period, and proliferation was isolated from spleens of isogeneic mice in which no tumor determined by the addition of MTT dye. was induced; a second group received B cells isolated from the tumor tissue of isogeneicmicethat had been Immune Complex Induction of sTn-Mucin Production implanted with Met 129 cells into mammary pads; and a For initial induction experiments, T-47D tumor cells third group served as a control, which did not receive any were aliquoted into 96-well plates at 1000 cells per well. exogenous B cells. Nineteen days after the tumor

After culture overnight (378Cin5%CO2 ), the culture challenge, the mice were scored for liver metastasis and medium from each well was removed, and replaced with spleen tumor growth. B cells isolated from tumor tissue of fresh serum-free tissue culture medium alone or with antitumor-bearing mice (B-TIL) promoted statistically signifi- sTn mAb (B72.3) to a final concentration of either 10, 1, cant tumor growth and metastasis in vivo compared with 0.1, or 0.01 ng/ml, for a total of six wells per control or no exogenous B cells (as a control), and to B cells antibody dilution. After incubating the 96-well plates for an isolated from spleens of non±tumor-bearing mice spleen additional 2 hours, the culture medium was removed and (N-SpBL) having similar scores compared with the quantitated for sTn-mucin by ELISA [7] using color control (Figure 1A). These results indicate that to be development with 3,30,5,50 -tetramethylbenizidine (TMB; able to interact with tumor cells in promoting tumor growth Gibco BRL), and absorbance readings at 450 nm with a in vivo, B cells must be exposed to tumor-associated correction at 570 nm. sTn-mucin concentration was antigens. To determine whether the promotion of tumor expressed as the corrected absorbance reading. Cell growth was a result of an exclusive interaction between B proliferation was also assessed at the same time by cells and tumor cells, as opposed to an interaction in treating the cells with MTT dye as described above. Thus, which B cells also require a T-cell failure, the experiment the absorbance reading for the sTn-mucin produced in a was repeated in T cell±deficient mice. Athymic nude- well was divided by the absorbance reading of the MTT for beige mice were injected intrasplenically with mucin- the same well, so as to express sTn-mucin production secreting, Met 129 tumor cells, and then divided into relative to the number of tumor cells. For assays for three groups: a group receiving B cells isolated from inhibition of induction of sTn-mucin production using spleens of isogeneic, non±tumor-bearing mice; a group tyrosine kinase inhibitors (genistein, herbimycin A) or receiving B cells isolated from spleens of isogeneic, Met PI-3 kinase inhibitor (wortmannin), 1000 T-47D tumor 129 tumor-bearing mice; and a group receiving B cells cells were plated as described above, followed by isolated from the tumor tissue of isogeneic, Met 129 treatment with either: serum-free tissue culture medium tumor-bearing mice. In the athymic nude-beige mice alone (1 hour); the medium containing genistein (final (without T cells or NK cells), B cells isolated from tumor concentration 100 M; for 1 hour); the medium containing tissue (B-TIL) or spleens (T-SpBL) of tumor-bearing herbimycin A (final concentration 10 M; for 1 hour); or mice promoted statistically significant tumor growth and the medium containing wortmannin (final concentration of metastasis in vivo compared with B cells isolated from 500 nM; for 10 minutes). After the appropriate treatment spleens of non±tumor-bearing mice (N-SpBL) (Figure period, the cells were washed with fresh serum-free tissue 1B ). Thus, B cells in the spleens of tumor-bearing mice

Neoplasia . Vol. 3, No. 2, 2001 118 Contribution of Fc RI Expression to Tumor Cell Malignancy Nelson et al.

Figure 1. ( A ± C ) Promotion of tumor growth by B cells. ( A ) In C3H mice, liver metastasis scores ( black boxes ) and spleen tumor scores ( white boxes ) from injection with either Met 129 tumor cells alone, Met 129 and B cells isolated from spleens of non ± tumor - bearing mice spleen ( N - SpBL ), or Met 129 and B cells isolated from tumor tissue of Met 129 tumor - bearing mice ( B - TIL ). Averages of six mice per group. The means of scores from mice receiving B - TIL is significantly greater than the means of the remaining groups ( P < 0.01 ). ( B ) In nude mice, spleen tumor scores from injection with either Met 129 tumor cells alone, Met 129 and N - SpBL, Met 129 and B cells isolated from spleens of tumor - bearing mice spleen ( T - SpBL ), or Met 129 and B - TIL. Averages of six mice per group. The comparison of means of scores from mice receiving B - TIL and from mice receiving T - SpBL does not show a statistical difference. However, both are significantly greater than the means of the remaining groups ( P < 0.01 ). ( C ) In vitro tumor cell growth ( number of tumor cells ) of Met 129 tumor cells alone, Met 129 mixed with CD8 + T lymphocytes isolated from the tumor tissue of Met 129 tumor - bearing mice ( CD8 + TIL ), Met 129 and B - TIL, or Met 129 with CD8 + TIL and B - TIL. Averages of six wells per experiment. The mean of well values with CD8 + TIL is significantly lower than the control ( P < 0.01 ). The means of well values with B - TIL are significantly greater than the means of the remaining combinations ( P < 0.01 ). The mean of well values with both CD8 + TIL and B - TIL is statistically identical to that of the control.

(i.e., B cells found outside the tumor) can also promote Met 129 cells (Figure 1C; Met129+, CD8+ TIL+, B- tumor growth and, additionally, that promotion is not due TILÀ ) resulted in a statistically significant reduction in to an impairment of antitumor activity of T cells or NK tumor cell growth, and thus appeared to effect growth cells. inhibition or tumor cell death, when compared to the growth of the control of Met 129 cells alone (Figure 1C; B Cells with Prior Exposure to Tumor-Associated Antigens Met 129+, CD8+ TILÀ, B-TILÀ ). A slight reduction in Promoted In Vitro Tumor Growth tumor cell growth, compared with that of the control, was To further test this interpretation, we tested whether observed when Met 129 cells were cocultured in the tumor cells and B cells (B-TIL) could interact directly to presence of B-TIL and CD8+ TIL cells (Figure 1C; Met promote tumor cell growth in vitro. Met 129 tumor cells 129+, CD8+ TIL+, B-TIL+ ). In contrast, a statistically were cultured either alone, in the presence of B-TIL, in the significant increase in tumor cell growth was observed, presence of CD8+ T lymphocytes isolated from the tumor compared with that of the control, when B-TIL were tissue of Met 129 tumor-bearing mice (CD8+ TIL), or in cocultured with Met 129 cells (Figure 1C; Met 129+, the presence of both B-TIL and CD8+ TIL cells; and then CD8+ TILÀ, B-TIL+ ). These results indicate that tumor tumor cell growth was quantitated by counting the number cell growth can be promoted when tumor cells interact of adherent Met 129 cells. CD8+ TIL cells cocultured with directly, without any other cellular participation, with B cells

Neoplasia . Vol. 3, No. 2, 2001 Contribution of Fc RI Expression to Tumor Cell Malignancy Nelson et al. 119

Figure 2. (A±B)Fc R expression by B16F1 tumor cells and sTn - mucin - secreting tumor cells. ( A ) Flow cytometric analysis of human tumor cell lines SW620 and T - 47D, and murine tumor cell lines B16F1 and Met 129, for cell surface expression of Fc RI ( CD64 ), CD64 in comparison to Fc RII ( CD32 ), and Fc RIII ( CD16 ) in comparison to CD32 ( duplicate analyses ). All of these tumor cell lines show certain low percentages ( 10% to 40% ) of cells expressing significant amounts of CD64. None showed evident expression of CD16 or CD32. ( B ) Ethidium bromide ± stained agarose gel showing amplified Fc RI cDNA ( 600 bp ): lane 1, from T - 47D carcinoma cells; lane 2, blank ( negative control ); lane 3, from U937 monocytic cells ( positive control ); lane L, standard 1000 - bp ladder. activated by tumor-associated antigens (``specific recogni- tumor-associated antigens by antibody. Based on our tion''). previous experience, which identified immune complexes as triggering promotion of tumor progression [7], we Fc RI Expression by sTn-Mucin±Secreting Tumor Cells investigated whether the observed promotion of tumor cell Because prior exposure to tumor-associated antigens growth involved Fc R expressed by tumor cells that could be was necessary for the observed promotion of tumor cell cross-linked by complexes of shed tumor±associated growth, suggested is a mechanism of specific recognition of antigens and antibody induced against these shed antigens.

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Figure 3. ( A ± C ) Immune complex induction of tumor cell proliferation as measured by MTT assays ( A570 to A690 nm ). ( A ) Proliferation of sTn - mucin producing T - 47D tumor cells after incubation with various concentrations of anti - sTn IgG1 Ab ( HB - STn ) ( repeated in triplicate ). The mean of well values with 0.01 and 0.1 g / well of Ab are significantly greater than those observed for the remaining concentrations ( P < 0.01 ). ( B ) Proliferation of B16F1 tumor cells after incubation with various concentrations of anti - sTn IgG1 Ab ( HB - STn ) in the absence of sTn - mucin ( black boxes ) or in the presence of sTn - mucin ( white boxes ). Averages of six wells per experiment. The mean of well values without sTn - Ag does not show a significant difference when compared to that of the control. The mean of well values with sTn - Ag + 0.1 g of HB - STn Ab, and sTn - Ag + 1 g HB - STn Ab are significantly greater than the means observed for the remaining concentrations ( P < 0.05 and P < 0.01, respectively ). ( C ) Proliferation of T - 47D tumor cells incubated with various concentrations of anti - sTn IgG1 ( solid black boxes ); with various concentrations of Fc fragment alone ( white boxes ); and with various concentrations of anti - sTn IgG1 and Fc fragments ( hatched boxes ) Averages of six wells per experiment. The means of well values from each of the experiments containing anti - sTn IgG1 are significantly greater compared with that of the combination of anti - sTn IgG1 and Fc fragments or of Fc fragments alone ( control ) ( P < 0.01 ).

To facilitate this investigation, we analyzed Fc R expression the RT/PCR of SW620 tumor cells and T-47D tumor cells in a model involving tumor cells that either express or do not showed a similar banding pattern; hence, T-47D was express a known tumor-associated antigen (sTn); and mAb selected as representative of both. A primary amplified B72.3 having binding specificity for sTn, which could be used cDNA band of about 600 bp was observed (Figure 2B ), and as mediator between sTn-containing shed tumor±asso- sequence analysis of the 600-bp band identified the ciated antigen and Fc R. B16F1 tumor cells (not expressing sequence as that of the Fc RIB gene. sTn), and the sTn-mucin±secreting tumor cells SW620 (human colon carcinoma), T-47D (human ductal breast Immune Complexes Induced Proliferation of Fc Ri-Expres- carcinoma) and Met 129 were first analyzed for FcR sing Tumor Cells expression by flow cytometric analysis for surface expres- Because Fc RIB has greater affinity for binding immune sion of Fc RI (CD64), Fc RII (CD32), and Fc RIII (CD16). complexes than monomeric IgG [13], we investigated This analysis showed that only Fc RI (CD64) was sig- whether immune complexes comprised of shed tumor± nificantly expressed by the four different tumor cell lines, associated antigen and anti-shed tumor±associated anti- whereas expression of Fc RII (CD32) and Fc RIII (CD16) gen Ab mediated the promotion of tumor growth. Because T- was either minimally detected or undetectable (Figure 2A). 47D tumor cells secrete sTn-mucin [7], murine anti-sTn Due to this protein expression pattern, and to determine IgG1 mAb was added to the cells in an in vitro culture, which Fc RI subtypes were expressed, we further char- forming soluble immune complexes with the secreted sTn- acterized the expression of Fc RI at the mRNA level. SW620 mucin. Proliferation was then compared between T-47D tumor cells and T-47D tumor cells were analyzed by reverse tumor cells incubated in various concentrations of anti-sTn transcription±polymerase chain reaction (RT/PCR) using IgG1 mAb, and control T-47D tumor cells incubated with Fc RI-specific primers [11]. By agarose gel electrophoresis, serum-free medium only. A statistically significant induction

Neoplasia . Vol. 3, No. 2, 2001 Contribution of Fc RI Expression to Tumor Cell Malignancy Nelson et al. 121 of tumor cell growth was observed for the tumor cells production by T-47D tumor cells in the presence of anti-sTn incubated with anti-sTn IgG1 mAb compared with the control IgG1 mAb was increased approximately two-fold over tumor cells (Figure 3A). Similar growth induction curves comparative cells to which isotype control mAb was added were also observed for Met 129 and SW620 carcinoma cell (Figure 4). Further, treatment of T-47D tumor cells with an lines. The bell-shaped, tumor growth induction curve is isotype control mAb did not induce a detectable increase in indicative of sTn antigen excess at the lower anti-sTn mAb sTn-mucin production compared with the comparative concentrations, optimal immune complex formation that control cells to which no IgG1 mAb was added (not shown). would therefore be more effective in cross-linking Fc RI, Based on these results, in addition to being a mechanism by and antibody excess at the higher anti-sTn mAb concentra- which tumor cell proliferation is induced, immune complexes tions. Additionally, the results show that the repeated sTn formed between sTn-mucin and anti-sTn Ab can induce antigen of mucin secreted by tumor cells, and IgG antibody production of sTn-mucin by sTn-mucin±secreting, Fc RI- bound thereto, can provide the proper immune complex for expressing tumor cells. cross-linking Fc RI. To confirm that the induction of tumor cell proliferation Induction of Tumor Cells by Fc Ri Cross-linking Is was a response to cross-linking of Fc RI-expressing tumor Dependent on the Tyrosine Kinase Signal Transduction cells by immune complexes, we repeated this in vitro assay Pathway using murine metastaticmelanoma cells,B16F1. We have There is evidence that cross-linking of Fc RI-expressing found that these melanoma cells also express Fc RI, but cells can activate the tyrosine kinase signal transduction do not secrete detectable amounts of sTn-mucin. The pathway [17,18]. To explore the possibility that the induction melanoma cells were treated in in vitro culture with either of proliferation or sTn-mucin production by tumor cells anti-sTn IgG1 mAb in various concentrations and super- having Fc RI cross-linked by immune complexes also natant containing sTn-mucin, with anti-sTn IgG1 mAb, or involved elements of the tyrosine kinase pathway, T-47D with fresh tissue culture medium as a control. A statistically tumor cells were treated with the selective tyrosine kinase significant induction of tumor cell growth was observed for inhibitors genistein or herbimycin A. Cell proliferation or sTn- the melanoma cells incubated with anti-sTn IgG1 mAb and mucin production was compared between T-47D tumor sTn-mucin compared with melanoma cells incubated in cells, T-47D treated with genistein, T-47D treated with either anti-sTn IgG1 mAb or no antibody (Figure 3B ). herbimycin A, T-47D incubated with anti-sTn IgG1 mAb, T- These results further support the observation that it is 47D treated with genistein and then incubated with anti-sTn immune complexes, which cross-link Fc RI-expressing IgG1 mAb, or T-47D treated with herbimycin and then tumor cells, which induce tumor cell proliferation. To further incubated with anti-sTn IgG1 mAb. A statistically significant establish the role of Fc RI in immune complex binding and induction of (about a 60% increase in) proliferation was induction of tumor cell proliferation, inhibition experiments observed for the tumor cells incubated with anti-sTn IgG1 were performed with murine Fcfragments and T-47D mAb compared with the same basal level of proliferation tumor cells. T-47D tumor cells were treated with either exhibited by untreated (T-47D alone) or treated (T- anti-sTn IgG1 mAb alone, or anti-sTn IgG1 mAb with Fc 47D+genistein or T-47D+herbimycin A) tumor cells in the fragments at a concentration 100-fold that of the anti-sTn IgG1 mAb. Coincubation of T-47D tumor cells and anti- sTn IgG1 mAb with Fcfragments effectivelyinhibited tumor cell proliferation induced by the immune complexes (Figure 3C ), further demonstrating the role of Fc RI in the binding of immune complexes to the tumor cells.

Immune Complexes Induced Production of sTn-Mucin by Fc Ri-Expressing Tumor Cells The amount of sTn-mucin produced by adenocarcinomas in vivo is associated with the metastatic potential of the tumor [14,15], may be used as a marker of progression and metastasis [15], and may be used as a prognosticindicator [16]. To investigate if cross-linking of Fc RI expressed by sTn-mucin±secreting tumor cells may also induce sTn- mucin production, T-47D tumor cells were incubated in the presence of either various concentrations of anti-sTn IgG1 mAb, tissue culture medium alone, or an isotype control mAb. The culture supernatants were then assayed for sTn- mucin production by measuring the amount of sTn epitope, Figure 4. Immune complex induction of sTn - mucin production by tumor cells. and sTn-mucin production was then expressed as the ratio Ratio of sTn - mucin to tumor cell proliferation for T - 47D tumor cells incubated in various concentrations of anti - sTn IgG1 Ab ( B72.3 ) or isotype control Ab of the amount of sTn-mucin to the amount of cell proliferation ( 4D9 ). Each point represents the mean of values obtained from six wells per to take induction of cell proliferation into account. sTn-mucin experiment.

Neoplasia . Vol. 3, No. 2, 2001 122 Contribution of Fc RI Expression to Tumor Cell Malignancy Nelson et al.

constitutive level of detectable sTn-mucin production by the tumor cells (T-47D alone) was significantly inhibited by either of the tyrosine kinase inhibitors genistein or herbimycin A (Figure 5B ), thereby preventing us from assessing involvement of the tyrosine kinase pathway in sTn-mucin production induced by cross-linking Fc RI by immune complexes. Activation of tyrosine kinases is followed by events that can include phosphorylation on tyrosine of multiple cellular substrates including phosphatidylinositol-3 kinase (PI-3 kinase) [17], a tyrosine kinase±regulated enzyme. To explore the possibility that the induction of tumor cell proliferation or of sTn-mucin production by cross-linking Fc RI by immune complexes also involved PI-3 kinase, T- 47D tumor cells were treated with wortmannin, a selective inhibitor of PI-3 kinase. As also shown in Figure 5A,a statistically significant induction of tumor cell growth was observed for the tumor cells incubated with anti-sTn IgG1 mAb compared with the same basal level of proliferation exhibited by untreated (T-47D alone) or treated (T-47D +wortmannin) tumor cells in the absence of anti-sTn IgG1 mAb. That pretreatment of the tumor cells with wortmannin completely inhibited the induction of tumor proliferation observed in the presence of anti-sTn IgG1 mAb (Figure 5A) demonstrates that immune complex±induced tumor cell proliferation is also dependent on the PI-3 kinase. However, the constitutive level of detectable sTn-mucin production by the tumor cells (T-47D alone) was significantly inhibited by wortmannin (Figure 5B ); thereby preventing us from assessing involvement of PI-3 kinase on induction of sTn-mucin production by cross-linking Fc RI by immune complexes.

Figure 5. ( A ± B ) Effect of treatment with signal transduction pathway inhibitors. ( A ) Immune complex induction of proliferation of T - 47D tumor cells with anti - sTn IgG1 with no inhibitor, after treatment with genistein, after Discussion treatment with herbimycin, and after treatment with wortmannin. Effect of Because tumor-associated antigens can comprise self or treatment on constitutive level is represented by T - 47D in the absence of anti - sTn IgG1 with no inhibitor, after treatment with genistein, after treatment altered self antigens, a challenge in tumor immunotherapy is with herbimycin, and after treatment with wortmannin. The number of cells / the induction of immune responses, which are capable of well is expressed as 1000Âthe corrected absorbance of the media after the inducing a clinical antitumor effect, rather than a disease- MTT reaction. Averages of six wells per experiment. The mean of well values with sTn - Ag ( first left column ) reflect the induced proliferation of the tumor promoting (e.g., immune evasive or autoimmune) effect. Our cells. The means of well values obtained with the inhibitors all indicate results strongly support the concept of a humoral immune significant inhibition of the induction ( P < 0.01 ). ( B ) Immune complex response that can directly promote tumor progression. The induction of sTn - mucin production of T - 47D tumor cells with anti - sTn IgG1 with no inhibitor, after treatment with genistein, after treatment with results of the in vivo experiments show that B cells exposed in herbimycin, and after treatment with wortmannin. Effect of treatment on vivo to tumor-associated antigens can promote tumor growth constitutive level is represented by T - 47D in the absence of anti - sTn IgG1 in immune-competent animals, sustaining the suggestion of with no inhibitor, after treatment with genistein, after treatment with herbimycin, and after treatment with wortmannin. The number of cells / well an undesired tumor promoting role of a humoral immune is expressed as 1000Â the corrected absorbance of the media after the response induced by tumor [5±7]. Moreover, the results of peroxidase ELISA reaction. Average of six wells per experiment. The means the in vitro experiments, utilizing a model that isolated the of well values with and without sTn - Ag are significantly different ( P < 0.01 ). The mean of well values with inhibitors show significant different degrees of interaction to only tumor cells and B cells, stress the concept inhibition ( P < 0.01 ). that B cells exposed in vivo to tumor-associated antigens can directly interact with certain tumor cells through specific absence of anti-sTn IgG1 mAb (Figure 5A). That pretreat- recognition, and promote tumor cell growth. As shown by flow ment of the tumor cells with either genistein or herbimycin A cytometry tumor cells can express Fc RI. RT-PCR and DNA resulted in a total inhibition of the induction of tumor cell sequencing showed that those tumor cells expressed proliferation observed in the presence of anti-sTn IgG1 mAb Fc RIB-RNA, which encodes a transmembrane receptor (Figure 5A) demonstrates that induction of tumor cell with two extracellular domains, in which the second extra- proliferation by immune complexes is dependent on the cellular domain splices precisely to the transmembrane/ tyrosine kinase signal transduction pathway. However, the cytoplasmic domain, and does not utilize the third extra-

Neoplasia . Vol. 3, No. 2, 2001 Contribution of Fc RI Expression to Tumor Cell Malignancy Nelson et al. 123 cellular domain encoded by the Fc RIA gene [11]. Fc RIB also inhibited Fc RI-dependent induction of tumor cell has higher affinity for immune complexes than free Ab [13]; proliferation is supported by the observation that some this suggests that an specific recognition tumor cell/B cells human tumor cell lines have elevated levels of PI-3 kinase, could be carried out, in some cases, by immune complexes which correlated with the oncogenic growth of these cells produced by binding of tumor cell±secreted antigens with the [23]. correspondent antibodies produced by the lymphocytes. The We also show an induction of sTn-mucin production by experiments with adenocarcinoma cells secreting sTn-mucin tumor cells having Fc RI cross-linked by immune com- incubated in the presence of anti-sTn monoclonal Ab sustain plexes. As related to a metastaticphenotype, productionof the interpretation that tumor-associated antigen secreted by sTn-mucin by human mucinous adenocarcinomas in vivo is tumor cells, and IgG bound thereto, can provide the required associated with the metastatic potential of the tumor [14]. immune complex for cross-linking the tumor cell Fc RI. The Metastases express a decrease in mucin core structures interpretation that cross-linking of Fc RI is the basis of this such as Tn and T antigens, with a reciprocal increase in sTn type of induction of tumor cell proliferation is supported by the antigen structures, and the increased sialylation of mucin- fact that Fc fragments or monomeric IgG can inhibit the associated carbohydrates is believed to increase metastatic induction of tumor cell proliferation, and by the requirement potential because it enhances motility [14]. Thus, an for both exogenous sTn-mucin and anti-sTn IgG1 mAb to induction in sTn-mucin production could lead to an induce proliferation of a non sTn-mucin±secreting tumor cell augmentation in metastaticphenotype by several mechan- line (B16F1 melanoma cells). Further, cross-linking can be isms: when surface expressed, an enhancement of motility; impaired by antibody excess, i.e., higher anti-sTn mono- when shed, the formation of more polyvalent immune clonal antibody concentrations. This suggests the possible complexes that cross-link Fc RonFc R-expressing existence of a feedback mechanism in which the concentra- immune effector cells in activating the cells to promote tion and type of both components (tumor Ag and antitumor matrix degradation, angiogenesis, and metastasis [7]; and Ab) may determine promotion or inhibition of tumor cell when shed, the formation of more polyvalent immune growth. complexes that cross-link Fc RonFc R-expressing tumor The concordance between Fc RI-dependent induction of cells in an amplification loop, which results in more sTn- tumor cell proliferation and its inhibition of induction by mucin production. Considering that inhibition of tyrosine protein tyrosine kinase inhibitors genistein and herbimycin A kinase signal transduction pathway (e.g., by genistein) is supported by the observations that cross-linking of Fc RI decreased constitutive mucin expression by normal epithelial can result in activation of the tyrosine kinase signal cells, and that tyrosine kinase activity is critical for maintain- transduction pathway [17,18], and that activation of tyrosine ing a high level of mucin expression in the epithelial cells kinase signal transduction pathway has been associated [24], it is not surprising that inhibition of the tyrosine kinase with tumor cell proliferation [19,20]. For example, a signal transduction pathway appears to decrease constitu- significant correlation was found between tyrosine kinase tive expression of mucin by tumor cells of epithelial origin. signal transduction pathway activity and the proliferation Taken together, these observations support the possibility capacity of human colon solid tumors and tumor cell lines of a direct antibody-mediated effect of B cells on tumor cells. [21]. Wortmannin is a potent inhibitor of PI-3 kinase, a As illustrated in Figure 6, the mechanism underlying this tyrosine kinase±regulated enzyme [22]. That wortmannin direct effect, which we term ``specific Ab-tumor cell promo-

Figure 6. Model of specific Ab - tumor cell promotion. Tumor cell secrete tumor - associated antigens ( s - TAA, such as sTn mucin; step 1 ). B cells recognize the s - TAA ( step 2 ) and, as activated B cells and plasma cells, respond by producing and secreting anti ± s - TAA IgG( step 3 ). Polyvalent immune complexes ( step 4) comprised of s - TAA bound to anti - sTAA Ab cross - link Fc RI on Fc RI - expressing tumor cells and activate one or more signal transduction pathways ( e.g., protein kinase activation PK; (step 5 ), resulting in the induction of cell proliferation ( step 6 ) and sTn - mucin production ( step 7 ).

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Neoplasia . Vol. 3, No. 2, 2001