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Immunology |
Department of Pathology, New York University School of Medicine, New York, New York 10016 [S. C., T. C., S. T.], and Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [N-K. V. C.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Complement inhibitors have been found on nearly all primary tumors and cancer cell lines that have been examined, and some studies indicate that complement-inhibitory proteins are up-regulated on tumor cells. DAF and the serum complement inhibitor factor H or related proteins have been identified as tumor-associated antigens (1 , 2) , and the overexpression of DAF confers a poor prognosis in colorectal cancer patients (2) . In vitro studies have shown that complement inhibitors expressed on tumor cells can inhibit both complement opsonization and direct cytolysis by the MAC (for recent reviews of immune evasion and complement resistance of tumor cells, see Refs. 3, 4 ). However, there is little information regarding how tumor-expressed complement inhibitors relate to complement deposition in situ, and the in vivo relevance of complement effector mechanisms and the importance of tumor-expressed complement inhibitors in controlling tumor growth remain largely unexplored. One reason for this is that complement inhibitor proteins (particularly CD59) are species selective, and human complement inhibitors are less effective against rat and mouse complement (5 , 6) . Thus, endogenous complement inhibitors expressed on the surface of human tumor cells will not provide the cells with effective protection from complement in rodent models of human cancer. Indeed, the species-selective activity of membrane complement-inhibitory proteins may be a basis for observations that complement-activating mAbs effective at causing regression of human tumors in rodents have, in most cases, proven ineffective in clinical trials.
When investigating the role of complement-inhibitory proteins in immune evasion of tumor cells in vivo, it is therefore relevant to study rodent complement inhibitors in rodent models of cancer. The ubiquitous and high level of expression of membrane complement inhibitors on normal tissues has not allowed for the targeted blocking of complement inhibitors (using current technologies) on tumor cells in syngeneic rodent models of cancer. In the studies described here, we investigated the effect of heterologously expressed rat CD59 on the growth of a human neuroblastoma cell line in nude rats. The neuroblastoma cell line endogenously expressed CD59, but we have previously determined on a quantitative basis that human CD59 is severalfold less effective at inhibiting rat complement compared to human complement (6) . The data show for the first time in vivo that the complement inhibitor CD59 expressed on a tumor cell surface significantly promotes tumor growth. We also show that growth in vivo resulted in the up-regulation of DAF on the tumor cell surface and that the level of DAF expression was further up-regulated by the expression of functional CD59.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies and Complement.
mAbs to human (YTH53.1) and rat (6D1) CD59 and rabbit antirat C9
polyclonal IgG were the gifts of Dr. B. P. Morgan. Human MCP mAb M75
(7)
was a gift of Dr. D. M. Lublin (Washington
University, St. Louis, MO). Antihuman DAF mAb 1A10 was described
previously (8)
, and anti-GD2 3F8 mAb (9)
was
described previously. Goat antihuman C3 IgG cross-reactive with rat C3
was obtained from ICN Pharmaceuticals (Aurora, OH). Anti Ly6A/E mAb D7
was purchased from BD Pharmingen (San Diego, CA).
FITC-conjugated antibodies used for flow cytometry were purchased from
Sigma (St. Louis, MO). Normal human serum was obtained from the blood
of healthy volunteers in the laboratory, and rat serum was obtained
from the blood of normal and immune-deficient rats. Serum was stored in
aliquots at -70°C until use.
Preparation of LAN-1 Transfectants.
Rat CD59 cDNA and Ly6E cDNA were subcloned into the multiple cloning
site of mammalian expression vector pCDNA3 (Invitrogen, Carlsbad, CA).
DNA was transfected into 50–75% confluent LAN-1 cells using
LipofectAMINE according to the manufacturer’s instructions (Life
Technologies, Inc., Grand Island, NY). Stable populations of LAN-1
cells expressing either rat CD59 or Ly6E were isolated by several
rounds of cell sorting using either antirat CD59 mAb 6D1 or anti-Ly6A/E
mAb D7 as described previously (10)
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Complement Lysis Assays.
Complement-mediated cell lysis was determined by both 51Cr
release (11)
and enumeration after trypan blue staining
(12)
, as described previously. Both methods gave similar
results. Lysis assays of LAN-1 cells were performed using detached
cells in both the absence and presence of antitumor
complement-activating antibody. In assays in which cells were
antibody-sensitized to complement, the anti-GD2 monoclonal antibody 3F8
was added at 15 µg/ml, and cells were incubated for 30 min at 4°C
before the addition of rat serum. Experimental details have been
described previously (13)
.
Flow Cytometric Analysis.
Analysis of cell surface protein expression and complement protein
deposition was performed by flow cytometry using appropriate antibodies
(see above), as described previously (10)
. Primary
antibodies and isotype-matched irrelevant control antibodies were used
at a concentration of 10 µg/ml. Analysis was performed on cells
removed from tissue culture using versene (Life Technologies, Inc.) for
cell detachment and on cells isolated from excised tumors. Cell
suspensions were obtained from tumors by gentle teasing of tumor tissue
(in RPMI 1640/10% FCS) with scalpels, followed by low-speed
centrifugation through Ficoll to remove tumor pieces and aggregates
(14)
. Tumor-derived cells were then washed in RPMI
1640/10% FCS by centrifugation before use.
In Vivo Experiments.
Four-week-old male athymic nu/nu (nude) rats were obtained
from the National Cancer Institute (Frederick, MD). The rats were
housed in a clean room, and food and water were sterilized. Rats were
injected s.c. in the right flank with the indicated numbers of LAN-1
cells suspended in 0.2 ml of PBS. Groups of rats received either LAN-1
cells transfected with rat CD59 or control-transfected LAN-1 cells.
Control cells were transfected with Ly6E (a structural but not
functional homologue of CD59) or with empty plasmid. There was no
difference in tumor growth between the different control LAN-1 cells.
Tumor volumes were calculated using the formula
4/3
r3
(volume of sphere). Statistical
analyses were performed using the SAS system (SAS Institute Inc., Cary,
NC).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows that LAN-1 cells are significantly more sensitive to lysis by rat
complement than lysis by human complement after sensitization by
anti-GD2 3F8 mAb. LAN-1 cells express high levels of GD2 antigen, and
the complement-activating properties of 3F8 mAb have been described
previously (13
, 15)
. Of note, LAN-1 cells are also lysed
by rat complement in the absence of 3F8 mAb, albeit less effectively
(Fig. 1b
). These results confirm that endogenous expression
of human complement inhibitors on LAN-1 cells does not provide
effective protection from lysis by rat complement. Similar data were
obtained with serum isolated from either normal or immune-deficient
rats. Lysis of LAN-1 cells in the absence of sensitizing antibody may
be due to the presence of natural endogenous complement-activating
antibodies that bind to LAN-1 cells, and flow cytometric analysis of
cells after incubation in heat-inactivated rat serum revealed that
small amounts of rat immunoglobulin were deposited on the cell surface,
supporting this possibility (data not shown).
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). As a control for in vivo studies (see below), LAN-1 cells
were also transfected with murine Ly6E antigen, a structural but not
functional analogue of CD59, and sorted as described for rat CD59
transfectants. Fig. 3
shows that the expression of rat CD59 on LAN-1 cells significantly
enhanced their resistance to lysis by rat complement, both in the
absence and presence of complement-activating 3F8 mAb.
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. To investigate the effect of CD59 expression and increased complement
resistance on in vivo tumor growth, control-transfected
LAN-1 cells and LAN-1 cells stably expressing rat CD59 were injected
separately into nude rats, and tumor growth was monitored. Groups of
nude rats were inoculated with either 8 x 106 cells, a dose resulting in almost 100% tumor take for
untransfected LAN-1 cells, or 4 x 106
cells, a dose determined to result in tumor growth in approximately
50% of animals (Table 1)
.
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2 analysis). Regression analysis showed that the rate of
tumor growth was also significantly faster in rats inoculated with rat
CD59-transfected cells (P < 0.01). In
addition, analysis of the mean difference in tumor size on each day of
tumor measurement between the two groups of rats showed that tumors
growing in rats inoculated with rat CD59-transfected cells were
significantly larger, with Ps < 0.01 and an
average P value of 0.0021 (Student’s t test;
Fig. 4a
).
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. The
onset of tumor growth, however, occurred significantly earlier in rats
inoculated with rat CD59-transfected cells (Fig. 5
); the mean day of tumor onset was day 13 for rats inoculated with control
LAN-1 cells and day 7.4 for rats inoculated with rat CD59-transfected
LAN-1 cells. One week after inoculation, 7 of 21 rats inoculated with
control LAN-1 cells contained tumors, whereas 15 of 19 rats inoculated
with rat CD59-transfected LAN-1 cells contained tumors. This is a
highly significant difference (P = 0.001,
2 analysis). Similar to the data obtained with an
inoculum of 4 x 106 cells, there was also a
highly significant difference in the mean tumor size between rats
inoculated with either control or rat CD59-transfectants at each day of
tumor measurement, with Ps < 0.01 and an
average P < 0.001 (Student’s t
test; Fig. 4b
).
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). In
this context, our data indicate the presence of low concentrations of
natural endogenous antibodies in nude rats that bind to LAN-1 cells
(see above), and when a high cell inoculum or after a threshold tumor
size is reached, it is possible that endogenous antitumor antibodies
may become depleted. At this point, complement may no longer be
effectively activated at the tumor cell surface, and
complement-sensitive (control-transfected cells) and -resistant
cells (rat CD59-transfected cells) may grow at similar rates.
Complement Deposition and Expression of Complement Inhibitors on
Tumor-derived LAN-1 Cells.
Cells isolated from tumors after 28 days of growth were initially
analyzed for deposition of complement and the continued expression of
transfected rat CD59 by flow cytometry. As shown in Fig. 6
, expression of rat CD59 was maintained on the tumor cells at a level
similar to that seen in in vitro cultured cells used for
inoculation. Interestingly, the level of Ly6E expression on
control-transfected LAN-1 cells was not maintained during in
vivo growth. This finding may be the result of selective pressure
exerted by rat complement on rat CD59 expression.
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), unsensitized LAN-1 cells are lysed by rat
complement in vitro, and, as anticipated, complement
proteins C3 and C9 were both deposited on LAN-1 tumors in
vivo. Less deposited C9 was detected on rat CD59-transfected
tumor-derived cells than on tumor-derived control LAN-1 cells (Fig. 6
),
consistent with the known function of CD59. More surprising was the
finding that rat CD59-transfected tumor cells also had lower levels of
C3 deposited on their surface as compared with control cells; the
difference was small but consistent (Fig. 6
shows the results from a
representative analysis). This was surprising because CD59 does not
inhibit complement activation and is not expected to influence C3
deposition. An explanation for these data was provided, however, when
we analyzed the endogenous expression of complement inhibitors on LAN-1
cells. We compared the relative levels of endogenously expressed DAF,
MCP, and CD59 between in vitro cultured LAN-1 cells and
LAN-1 cells isolated from tumors. Fig. 6
shows that DAF expression was
up-regulated on the surface of tumor-derived control LAN-1 cells by
about twofold compared with in vitro cultured cells. The
relative level of DAF expressed on rat CD59-transfected cells derived
from tumors was even further up-regulated compared with that in cells
grown in vitro (about threefold). Thus, the increased level
of DAF expression is likely to account for the decreased level of C3
deposited on the rat CD59-transfected tumor-derived cells. Of relevance
to this finding, human DAF is known to inhibit rat complement, albeit
less effectively than human complement (see "Discussion"). Multiple
tumors from separate experiments were analyzed by flow cytometry, and
the data shown in Fig. 6
are representative of at least six
determinations for particular antigen groups. Transfection of LAN-1
with rat CD59 did not alter the level of endogenous DAF expression on
cells cultured in vitro, and the level of endogenous CD59
and MCP expression on LAN-1 cells was unchanged after in
vivo growth (Fig. 6
). It is unlikely that the increased levels of
DAF on LAN-1 cells after in vivo growth are due to selection
because populations expressing higher-than-normal amounts of DAF
could not be selected by cell sorting in vitro, and
selection is not consistent with the finding that even higher levels of
DAF are seen on rat CD59-expressing cells grown in vivo. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The expression of membrane-bound complement-inhibitory proteins may benefit tumor cells for several reasons. Complement activation products (particularly C5a and the MAC) are powerful mediators of inflammation and may promote the recruitment of immune effector cells to the site of tumor growth. Cell-bound C3 activation products can promote and enhance antibody-dependent cell cytotoxicity and natural killer effector systems, and formation of the MAC can be directly cytolytic. Therefore, at least conceptually, it is reasonable to consider that up-regulation of complement inhibitors, as we observe here for DAF, may represent a mechanism by which some tumors can escape immune destruction. DAF is an inhibitor of complement activation and will inhibit the generation of C3/C5 activation products as well as the terminal MAC, whereas CD59 inhibits only MAC assembly. Because of the effect of CD59 on DAF expression, the current data do not provide information on the relative roles that these mechanisms may play in controlling tumor growth. However, the data do clearly establish that complement is involved in controlling tumor growth in this model and that CD59 promotes tumor growth whichever complement-associated mechanism(s) is operative.
So how does in vivo growth and, in particular, the expression of functional CD59 modulate DAF expression? Complement activation products and various cytokines have been reported to modulate complement inhibitor expression in vitro, although the effects appear to be variable, particularly for DAF (3 , 4 , 16, 17, 18, 19, 20, 21) . Also, a recent in vitro study reported that assembly of the MAC on endothelial cells directly up-regulated DAF expression and that expression was enhanced by cytokines (16) . Similar mechanisms may be responsible for the up-regulation of DAF on tumor cells in vivo, as reported here. To explain the higher levels of DAF observed on rat CD59-expressing LAN-1 cells derived from tumors, it is conceivable that CD59-expressing cells may be able to survive higher levels of MAC that are initially deposited on the cell surface, thus enhancing the signal for DAF expression. CD59 limits the number of C9 molecules bound per MAC, and complexes containing bound C9, but with abrogated lytic function, may still be able to provide the signal for DAF up-regulation. It is also possible that the signal for induction of DAF expression is delivered via rat CD59 after its engagement by assembling complement complexes. This notion is consistent with the demonstration that CD59 is a signal transducing molecule (22, 23, 24, 25, 26) . Increased endogenous DAF expression on LAN-1 cells correlated with decreased rat C3 deposition, and in this regard, human DAF is able to inhibit rat complement, although it is a less effective inhibitor of rat complement than human complement.4
We show that LAN-1 cells activate rat complement in the absence of exogenously added complement-activating antibody both in vitro and in vivo. This is probably due to the presence of natural endogenous xenogeneic antibodies because rat immunoglobulin is deposited on the LAN-1 cell surface after the incubation of cells in nude rat serum. It may be that tumor cell lines that do not "spontaneously" activate rodent complement will require the administration of exogenous complement-activating antitumor antibodies for an effect of complement inhibitors on tumor growth to be observed in rodent hosts. Indeed, human tumor cell lines transfected with rodent complement inhibitors and grown in rodents may represent good preclinical models relevant for evaluating tumor-specific mAbs. For our studies, we chose to use a rat model because the rat complement system appears to be more robust than the murine complement system and may represent a better model. It is difficult to isolate hemolytically active mouse complement, and there are reports documenting low complement levels in common laboratory mouse strains and nude mouse strains as compared with complement levels found in humans and rats (27 , 28) .
In summary, our results show that a membrane complement inhibitor expressed on the surface of a tumor cell plays a role in determining tumorigenesis and that reversing the effects of tumor-specific complement regulators is likely to enhance immune-mediated clearance of some tumors. The widespread expression of membrane-bound complement inhibitors presents technical difficulties for the selective blocking of complement inhibitors on tumor cells. However, it may be possible to adapt current and developing technologies to permit targeted delivery of antibodies, peptides, or perhaps antisense DNA to block the effects of endogenous complement inhibitors expressed on tumor cells.
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FOOTNOTES |
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1 Supported by NIH Grant AI 34451 and Department
of the Army Grants DAMD17-97-1-7273 and DAMD12-99-1-9325. 
2 To whom requests for reprints should be
addressed, at Department of Microbiology & Immunology, Medical
University of South Carolina, BSB 201, 173 Ashley Avenue, Charleston,
SC 29425, E-mail: tomlinss{at}musc.edu
3 The abbreviations used are: MAC, membrane attack
complex; DAF, decay-accelerating factor; MCP, membrane cofactor
protein; mAb, monoclonal antibody.
4 C. L. Harris, O. B. Spiller, and
B. P. Morgan, personal communication.
Received 2/14/00. Accepted 4/17/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, interleukin-1ß and interferon-
on the expression and function of the complement regulatory protein CD59 on the human colonic adenocarcinoma cell line HT29. Scand. J. Immunol., 41: 350-356, 1995.[Medline]
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