# THE ROLE OF COMPLEMENT IN TUMORS

EDITED BY : Barbara Rolfe, Ruben Pio, Trent M. Woodruff, Maciej M. Markiewski and Helga D. Manthey PUBLISHED IN : Frontiers in Immunology

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ISSN 1664-8714 ISBN 978-2-88963-576-4 DOI 10.3389/978-2-88963-576-4

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# THE ROLE OF COMPLEMENT IN TUMORS

Topic Editors: Barbara Rolfe, University of Queensland, Australia Ruben Pio, University of Navarra, Spain Trent M. Woodruff, University of Queensland, Australia Maciej M. Markiewski, Texas Tech University Health Sciences Center, United States Helga D. Manthey, University of Queensland, Australia

Citation: Rolfe, B., Pio, R., Woodruff, T. M., Markiewski, M. M., Manthey, H. D., eds. (2020). The Role of Complement in Tumors. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-576-4

# Table of Contents

Emily K. Kleczko, Jeff W. Kwak, Erin L. Schenk and Raphael A. Nemenoff

Anna Lenkiewicz, Kamila Bujko, Katarzyna Brzezniakiewicz-Janus, Bing Xu and Mariusz Z. Ratajczak

*99 The Complement Receptors C3aR and C5aR are a New Class of Immune Checkpoint Receptor in Cancer Immunotherapy*

Yu Wang, Hui Zhang and You-Wen He

# Editorial: The Role of Complement in Tumors

Barbara E. Rolfe1 \*, Ruben Pio2 , Trent M. Woodruff 3 , Maciej M. Markiewski 4 and Helga D. Manthey 1

*1 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia, 2 Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain, 3 School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia, 4 Department of Immunotherapeutics and Biotechnology, School of Pharmacy, Texas Tech University Health Sciences Center, Abilene, TX, United States*

Keywords: complement, cancer, metastasis, C5b-9, C1q, C3a, C5a, complement regulatory proteins

#### **Editorial on the Research Topic**

#### **The Role of Complement in Tumors**

Activation of the complement system is one of the earliest responses to invading pathogens and tissue damage (1). Complement activation leads to production of a range of effectors including the opsonin C3b, the anaphylatoxins C3a and C5a, and the C5b-9 complex (membrane attack complex; MAC) (2, 3). In addition to potent innate immune activities, complement effector systems also contribute to efficient adaptive immune responses (4). While critical to proper immune function, inappropriate or excessive complement activation contributes to many pathological inflammatory conditions (5), including cancer. As described in this issue, the complement system is increasingly recognized as a double-edged sword: on the one hand contributing to the anti-tumor response, but on the other protecting the tumor against immune attack and promoting metastasis.

#### Edited and reviewed by:

*Catherine Sautes-Fridman, INSERM U1138 Centre de Recherche des Cordeliers, France*

> \*Correspondence: *Barbara E. Rolfe<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology*

> Received: *14 January 2020* Accepted: *20 January 2020* Published: *11 February 2020*

#### Citation:

*Rolfe BE, Pio R, Woodruff TM, Markiewski MM and Manthey HD (2020) Editorial: The Role of Complement in Tumors. Front. Immunol. 11:139. doi: 10.3389/fimmu.2020.00139*

# COMPLEMENT-DEPENDENT CYTOTOXICITY AND C5b-9 (MEMBRANE ATTACK COMPLEX)

As described by Macor et al., the complement system has long been recognized to contribute to anti-tumor defense mechanisms via complement dependent cytotoxicity (CDC) (6) and antibodydependent cell mediated cytotoxicity (ADCC) (7). The introduction of recombinant antibodies for cancer treatment has led to renewed interest in complement as an anti-tumor defense system. The protective role of complement in cancer is discussed, with focus on the beneficial effect of complement-fixing antibodies which initiate cancer cell killing via CDC.

The cytotoxic activities of C5b-9, and the mechanisms by which it damages cancer cells, are further discussed by Fishelson and Kirschfink, along with the multiple mechanisms that tumor cells employ to resist C5b-9-induced death. They discuss the potential for therapeutic approaches to counter tumor escape mechanisms and potentiate antibody-based immunotherapies, but caution that intervention strategies to augment complement activation could also worsen outcomes.

Although C5b-9 has traditionally been attributed an anti-tumoral role through CDC, Vlaicu et al. review the evidence that C5b-9 at a sub-lytic dose stimulates tumor growth. Hence strategies to counteract the tumor-promoting traits of C5b-9 and potentiate anti-tumoral actions (including enhanced efficacy of antibody-based immunotherapy) may be the next major direction for immuno-oncology.

**4**

## COMPLEMENT REGULATORY PROTEINS

As described by Geller and Yan, membrane and soluble complement regulatory proteins (CRPs) prevent excessive complement activation. Therefore, over-expression of CRPs by tumor cells protects them against complement-mediated attack, interferes with anti-tumor therapies, and enhances metastatic potential. The application of CRPs as prognostic biomarkers and therapeutic targets is discussed, along with the potential for combinatorial approaches with other anti-tumor therapies.

## COMPLEMENT C1q

The first subcomponent of the classical complement pathway, C1q is a pattern recognition molecule locally synthesized by macrophages and dendritic cells (8). Bioinformatics analysis by Mangogna et al. suggests C1q as a new prognostic biomarker for several cancers.

#### ANAPHYLATOXINS C3a AND C5a

Since the seminal paper of Markiewski et al. (9) identifying a role for C5a in promoting tumor progression, similar effects have been demonstrated in a range of murine cancer models. Wang et al. propose C3aR and C5aR1 as a new class of immune checkpoints. They discuss findings suggesting that C3aR/C5aR signaling regulates T cell mediated antitumor immunity via transcriptional suppression of interleukin (IL)- 10. Given resistance of the majority of patients to the current forms of immunotherapy, and adverse reactions associated with these approaches, the authors suggest the manipulation of the C3aR/C5aR/IL-10 pathway as an alternative strategy for cancer.

Lenkiewicz et al. discovered that C3 and C5 cleavage fragments enhance trafficking, motility and, therefore, dissemination of malignant cells in hematologic malignancies through a p38 MAPK and inducible heme oxygenase 1 (HO-1) manner. They propose that activation of the complement cascade in patients with these malignancies (e.g., triggered by infection) can contribute to faster dissemination of disease. Thus, targeting this pathway may ameliorate dissemination of leukemic cells and improve clinical outcomes in these patients.

Kleczko et al. discuss the potential for complement targeting therapies for the treatment of intractable cancers, in particular lung cancer. They review the mechanisms by which the anaphylatoxins C3a and C5a influence tumor growth and promote epithelial-mesenchymal transition and tumor metastasis. Since complement proteins can regulate both proand anti-tumorigenic pathways, the authors stress the need to better understand the effects of complement activation within tumor tissue, and how this may be influenced by different oncogenic drivers.

Cancer metastasis is estimated to be responsible for greater than 90% of cancer deaths (10). As discussed by Ajona et al., distorted complement homeostasis not only remodels the tumor microenvironment by inhibiting anti-tumor immune responses, but is also crucial to metastasis, endowing tumor cells with properties required for metastatic dissemination and establishment. Complement activation products (primarily C3a and C5a) induce a range of mediators which promote epithelialmesenchymal transition, tumor growth, invasion, dissemination via lymphatic and circulatory systems, and also protect cells within the metastatic niche. The authors highlight the potential of complement-targeting drugs to augment the clinical efficacy of current immunotherapies and effectively eradicate both primary tumors and distant metastases.

# CONCLUSIONS

Despite recent clinical advances in cancer immunotherapy, the estimated percentage of patients responding to checkpoint inhibitors in the United States in 2018 was only 12.46% (11). Hence there remains an urgent need for novel therapeutic strategies to boost response rates. As a critical link between the innate and adaptive immune systems, the complement system is a promising therapeutic target. However, knowledge is the key to realizing the clinical potential of complementtargeting therapeutics. Only a thorough understanding of the role of complement pathways in the tumor microenvironment will enable development of strategies to selectively (and safely) target the pro-tumor effects of complement, while simultaneously augmenting the anti-tumor effects. To quote Fishelson and Kirschfink, "currently we perceive only the tip of the ice-berg of . . . interactions between cancer cells and complement components." Indeed, given the diversity of responses, therapeutic protocols will likely need to be optimized for each cancer type and stage, and possibly for individual cancer patients, depending on their "immune history."

# AUTHOR CONTRIBUTIONS

All authors contributed to the conception and design of this work. BR drafted the manuscript, the other editors RP, TW, MM, and HM provided critical revisions and approved the submitted manuscript.

# FUNDING

BR, RP, TW, and HM acknowledge support from grants from the Australian National Health and Medical Research Council (NHMRC; APP1103951 and APP1164202) and Cancer Council Queensland (CCQ). RP was supported by the AECC Scientific Foundation, and Fondo de Investigación Sanitaria-Fondo Europeo de Desarrollo Regional Una manera de hacer Europa (PI17/00411) and MM by the National Institute of Health (R01CA190209).

#### ACKNOWLEDGMENTS

We thank all the authors for their contributions to this Research Topic. We also thank the reviewers for their evaluation of the manuscripts.

# REFERENCES

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Rolfe, Pio, Woodruff, Markiewski and Manthey. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Complement as a Biological Tool to Control Tumor Growth

Paolo Macor 1 , Sara Capolla1 and Francesco Tedesco2 \*

*1 Department of Life Sciences, University of Trieste, Trieste, Italy, 2 Immunorheumatology Research Laboratory, Istituto Auxologico Italiano, IRCCS, Milan, Italy*

Deposits of complement components have been documented in several human tumors suggesting a potential involvement of the complement system in tumor immune surveillance. *In vitro* and *in vivo* studies have revealed a double role played by this system in tumor progression. Complement activation in the cancer microenvironment has been shown to promote cancer growth through the release of the chemotactic peptide C5a recruiting myeloid suppressor cells. There is also evidence that tumor progression can be controlled by complement activated on the surface of cancer cells through one of the three pathways of complement activation. The aim of this review is to discuss the protective role of complement in cancer with special focus on the beneficial effect of complement-fixing antibodies that are efficient activators of the classical pathway and contribute to inhibit tumor expansion as a result of MAC-mediated cancer cell killing and complement-mediated inflammatory process. Cancer cells are heterogeneous in their susceptibility to complement-induced killing that generally depends on stable and relatively high expression of the antigen and the ability of therapeutic antibodies to activate complement. A new generation of monoclonal antibodies are being developed with structural modification leading to hexamer formation and enhanced complement activation. An important progress in cancer immunotherapy has been made with the generation of bispecific antibodies targeting tumor antigens and able to neutralize complement regulators overexpressed on cancer cells. A great effort is being devoted to implementing combined therapy of traditional approaches based on surgery, chemotherapy and radiotherapy and complement-fixing therapeutic antibodies. An effective control of tumor growth by complement is likely to be obtained on residual cancer cells following conventional therapy to reduce the tumor mass, prevent recurrences and avoid disabilities.

Keywords: complement system activation, tumor control, antibody-based immunotherapy, combination therapies, antibody

# INTRODUCTION

Cancer development is a complex biological process that starts with the malignant transformation of normal cells caused by genetic alterations and somatic mutations leading to unrestricted cell proliferation (1). The local microenvironment plays an important role in this process providing favorable conditions for the seeding of cancer cells in a protective niche that allows the growth and expansion of the tumor mass (2). Changes in the structural and organizational properties of

#### Edited by:

*Maciej M. Markiewski, Texas Tech University Health Sciences Center, United States*

#### Reviewed by:

*Ronald Paul Taylor, University of Virginia, United States Fabian Benencia, Ohio University, United States*

> \*Correspondence: *Francesco Tedesco<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology*

> Received: *29 June 2018* Accepted: *05 September 2018* Published: *25 September 2018*

#### Citation:

*Macor P, Capolla S and Tedesco F (2018) Complement as a Biological Tool to Control Tumor Growth. Front. Immunol. 9:2203. doi: 10.3389/fimmu.2018.02203*

**7**

extracellular matrix favor adhesion and migration of cancer cells from the initial tumor site (3). Active angiogenesis equally contributes to these environmental changes with the formation of new leaky vessels that supply growing cancer cells with nutrients and promote their metastatic spread to distant organs (4, 5).

Tumor development is constantly controlled by the immune system that recognizes cancer cells as potential threats to body homeostasis and mounts a response leading to local recruitment of effector cells of both innate and adaptive immunity (6). Although cell-mediated immunity has long been recognized to play a critical role in tumor eradication through the action of natural killer cells and cytotoxic T lymphocytes (7, 8), studies reported in recent years have shown that the complement (C) system is also an important player in cancer immune surveillance and these studies have revealed the complex interaction of C with cancer cells. C components are synthesized by resident and recruited cells including fibroblasts, endothelial cells, tissue specific cells, and macrophages (9, 10) and are released in the tumor microenvironment. Biologically active products generated as a result of C activation may directly kill cancer cells or favor their eradication by promoting an inflammatory process. However, it is important to emphasize that C does not always provide an effective protection against tumor growth since its damaging effect on cancer cells can be prevented by C regulatory proteins (CRPs) over-expressed on the cell surface or by other mechanisms of cell resistance to C attack. These evasion strategies are more likely to be operating under conditions of fast tumor growth.

Recent studies have elucidated a novel aspect of C interaction with cancer cells showing that it is able to promote rather than to inhibit tumor development. Markiewski et al. (11) made the original observation that C5a, released in the microenvironment as a result of C activation, recruits and activates myeloid derived suppressor cells that suppress antitumor T-cell responses against HPV-induced cancer. Similar findings have been reported in other syngeneic models of mouse tumors invariably associated with C activation (12, 13). Importantly, C5aR1-deficiency and pharmacological blockade of C5aR1 by selective C5aR1 antagonists have been shown to impair tumor growth, pointing to the C5a/C5aR1 signaling axis as an effector mechanism of C-mediated tumor-promoting functions (14).

More recently, C1q secreted in the tumor microenvironment was reported to favor tumor progression by enhancing adhesion, proliferation, and migration of cancer cells and promoting angiogenesis independently on C activation (15).

Given these restraints in C-dependent tumor control, the system has apparently limited chances to provide an effective defense barrier against cancer development unless the C protective functions are made more efficient by optimizing the conditions of its activation and effector activities. In this review, we shall discuss the strategies that may turn the C system into a more efficient therapeutic tool by enhancing its activation on the surface of cancer cells and overwhelming the mechanisms adopted by tumor cells to evade C attack.

# COMPLEMENT ACTIVATION AT TUMOR SITE

Immunohistochemical analysis of tumor tissue has provided useful information on the contribution of the C system to the immune response to cancer revealing the presence of C components in several solid tumors of different tissues and organs. Various cell types in tumor tissue including cancer cells represent the main source of these components which may also derive, at least in part, from the circulation as a result of the increased permeability of the tumor vessels. C deposits have been observed in a number of tumors (16) and in one study that examined the deposition of various C components in glioblastoma, C1q was found to be the most highly expressed component (17). We have recently shown that C1q is present in various tumors in the absence of other C components and exerts functions unrelated to C activation (15). However, this does not exclude C activation at tumor sites as suggested by tissue deposition of known markers of C activation including C4d, C3d, and SC5b-9 (18, 19). Local changes in tumor tissue due to necrosis and apoptosis and, more importantly, inflammation are responsible for C activation to a degree related to the extent of these changes, in particular of the inflammatory process. This suggests that C deposits are likely to be negligible in the initial stages of tumor growth when the inflammatory reaction is hardly detectable and is probably more evident at a later stage of tumor expansion associated with an overt inflammatory reaction. Tumor cells may partly contribute to C activation using cellbound proteases exposed on their surface to cleave C5 and to generate C5a, which in turn enhances cancer cell invasion (20).

It is not easy to evaluate the impact of C activation at tissue sites on tumor development because the immunohistochemical data have mostly been obtained from well-established cancers. Importantly, C activation products are mainly localized in the tumor microenvironment and found to be weakly or moderately bound to some but not all cancer cells, suggesting that they have limited effect in reducing cell survival. However, it is possible that C exerts a protective effect in the early phase of cancer growth, contributing to induce tumor regression, although this is difficult to ascertain in patients. One way to address this issue is to utilize mice that develop spontaneous tumors and analyze the effect of C activation on its progression at tumor sites. Using BALB/c females expressing the activated rat Her2/neu oncogene, Bandini et al. (21) have shown that the mammary carcinoma developing in C3−/− mice manifests faster growth rate and earlier lung metastasis than the tumor in wild type animals, suggesting that C activated by antibodies (Abs) directed against Her2/neu oncogene and/or other tumor-associated antigens may control tumor growth. Different results were obtained using a syngeneic mouse model of ovarian cancer which showed similar growth in wild type and C3−/− mice due to secretion of C3 by tumor cells that exerts a stimulating effect on cell proliferation (22). Overall, the available data support a dual role of C in tumor immune surveillance and its ability to either prevent or promote tumor progression depends on the characteristics of cancer cells and the anti-tumor efficiency of the C system.

*INN, International Non-proprietary Name; NHL, Non Hodgkin Lymphoma; CLL, Chronic Lymphocytic Leukemia; MM, Multiple Myeloma.*

#### CANCER CELLS AS POTENTIAL TARGET OF COMPLEMENT ATTACK

Expression of tumor-associated molecules on cells undergoing malignant transformation can lead to C activation on the cell surface by all three activation pathways. The lectin pathway has been implicated in C activation on glioma cells which express, like many other malignant cells, high mannose glycopeptides that bind MBL and trigger consumption of C4 and C3, but this reaction fails to induce cell lysis (23). Virus transformed cells express novel antigens that are able to activate the alternative pathway, as is the case of EBV-infected B lymphoblastic cell lines (24–26) and T and monocytic cell lines infected by HIV (27). The classical pathway of C can be activated on cancer cells by natural Abs, preferentially of IgM isotype, that recognize carbohydrate moieties on cell surfaces (28, 29). Cytotoxic Abs reacting with carbohydrate epitopes of gangliosides GD2 and GD3 on neuroblastoma and melanoma cell lines have been detected in a small number of sera from normal individuals (30). Unfortunately, besides the low frequency, the natural Abs are not efficient in promoting C-mediated cell killing due to their low titer and affinity.

Attempts have been made to vaccinate cancer patients with the aim to induce production of therapeutic Abs. The antitumor response has not always been satisfactory, although a novel vaccination procedure has recently been developed in rabbits to stimulate the generation of IgG Abs that cause strong C-mediated lysis of myeloma cells carrying the CD38 antigen (31).

Despite this improvement, the development of recombinant Abs against tumor antigens remains the preferential approach to stimulate selective C activation on cancer cells, although the identification of specific tumor-associated antigens able to discriminate cancer cells from healthy tissue still represents a major limitation in Ab-mediated cancer therapy. A major progress has been made in the immunotherapy of hematologic malignancies, in particular those derived from B cells, with the generation of monoclonal Abs directed against target antigens, such as CD19 and CD20, present only on B-cells at late stages of development, and not on hematopoietic stem cells that are therefore unaffected by the treatment. Conversely, the development of therapeutic Abs against solid tumors has been limited by the difficulty to identify specific target antigens on cancer cells, whether overexpressed self-antigens, or neoantigens due to tumor-specific mutations or oncogenic viruses.

Only 15 monoclonal Abs have been approved by FDA for the treatment of all different solid tumors (32), and only 3 of them are C-fixing molecules, as described in **Table 1**.

#### FACTORS AFFECTING THE EFFICIENCY OF THE ANTIBODIES TO ACTIVATE COMPLEMENT: THE ANTIBODIES STRUCTURE

Among the molecular characteristics of recombinant Abs responsible for C activation, the Ig class is critically important since human IgM, IgG1, and IgG3 are known to be the most effective C activators whereas IgG4 fails to bind C1q (33). The structure of the Fc region of these Abs has been extensively investigated to improve their therapeutic efficiency and changes of some amino acids in this region were found to enhance the Ab activity (34). In particular, computational design followed by high-throughput screening techniques has allowed the identification, production, and characterization of Fc variants with increased ability to bind C1q and to promote C-dependent cytotoxicity (CDC) (35).

Glycosylation is an important secondary modification of immunoglobulins that has a significant impact on their capacity to activate different arms of the immune system. The addition of conserved glycans, in particular α (1,6) linked core fucose, to the Fc region, was shown to be critical for the interaction of the Ab with the C system (36). This observation has raised strong research interest in several biotech companies, resulting in the commercialization of the anti-CD20 Ab obinutuzumab (Genentech, San Francisco, CA, USA). Terminal mannosylation is another important post-translational modification that prolongs the half-life of the Abs in the circulation and favors binding of mannose-binding lectin (MBL) (36). Importantly, the terminal glycosylation of IgG has been shown to influence CDC without affecting Ab-dependent cell cytotoxicity (ADCC). In addition, an increased content of terminal galactose potentiates CDC activity by enhancing the binding of C1q to the modified Ab (37).

The discovery that hexamerization of Abs after binding to target antigens leads to a successful activation of the classical pathway represents a major advance in the development of new strategies to enhance C activation by IgG (38, 39). The critical role of this process in C activation is supported by the finding that some mutations of Fc amino acid sequence of anti-CD20 IgG result in impaired hexamer formation and reduced cell lysis. Conversely, other mutations have the opposite effect and similar results were obtained introducing the same mutations in the IgG4 isotype. A certain degree of flexibility of antigen-bound Abs allows a conformational change required for hexamerization. The ability of anti-CD20 Abs to exhibit a more efficient CDC after hexamer formation is shared also by anti-CD52 and anti-HLA Abs (38, 39).

# FACTORS AFFECTING THE EFFICIENCY OF THE ANTIBODIES TO ACTIVATE COMPLEMENT: THE ANTIGEN

Irrespective of the C-activating capacity of anti-tumor Abs, the characteristics of the target antigen remain of pivotal importance for a successful tumor cell lysis. The beneficial effects of Abs in cancer immunotherapy depend on the expression pattern and the tissue specificity of tumor antigen that should be present exclusively or predominantly on cancer cells to allow selective or almost exclusive targeting of tumor cells. It is equally important that the tumor antigens are expressed also on metastatic cells which represent the main target of Ab-based immunotherapy since other therapeutic approaches including surgery, radiotherapy, and chemotherapy can be used to obtain an effective control of primary tumor. Moreover, the tumor antigens should be stably expressed on the cell surface to serve as useful targets for immunotherapy, whereas intracellular antigens, though specific for tumor cells, can only be used for diagnostic purposes. It is important to point out that tumor cells are often heterogeneous in the expression level of tumor antigens that may influence their susceptibility to CDC. We have observed that cells expressing low levels of CD20 isolated by cell sorting from a population of either chronic lymphocytic leukemia (CLL) or cancer B-cell lines and kept in culture for over a week give rise to cells expressing higher level of CD20 that more easily undergo C-mediated lysis (40, 41). This observation suggests that repeated injections of Abs administered at appropriate time intervals can be used to allow the emergence of cell clones expressing higher levels of CD20 and more susceptible to CDC. Finally, the release of antigen in the tumor microenvironment and in the circulation may lead to blockade of therapeutic Ab and contributes to reduce its expression level on the cell surface, making cancer cells less susceptible to Ab-mediated C-dependent killing (9).

The number of antigenic sites does not always account for the capacity of a monoclonal Ab to cause CDC. In this regard, the impact of the different distribution of two tumor antigens, the alpha isoform of folate receptor (42) and CD20 (43), on Ab binding and C activation has been compared. The folate receptor is associated with epithelial ovarian carcinoma cells and is expressed on several cell lines at a concentration of about 1 × 106 molecules/cells (44). CD20 is present on cancer Bcells at a substantially lower expression level of around 40,000– 70,000 molecules/cell (45). Despite the marked difference in the number of cell-associated antigenic sites, the chimeric anti-CD20 Rituximab is able to activate C (46) and to kill B cells whereas a chimeric anti-folate receptor Ab fails to do so (42).

Additional factors may play a relevant role in promoting a more efficient C activation by recombinant Abs, including the proximity of the target epitopes to the cell surface (47), the density of target antigen (48), and the Ab-induced movement of the antigens across the cell membrane (49).

A recent study by Cleary and colleagues provided convincing evidences that the efficacy of C-mediated killing of cancer cells induced by Ab is largely influenced by the distance of the target epitope from the cell membrane and the greater the distance from the cell surface, the lower the efficiency of cell lysis (47). They used target cells transfected with fusion proteins containing either CD20 or CD52 epitopes attached to various CD137 scaffolds and showed that the cells displaying the target epitopes closer to the membrane were more susceptible to CDC than those expressing the epitopes furthest away from the cell surface. These data clearly suggest that the position of the epitope in the target antigen is an important factor to consider in the selection of therapeutic Abs. The surface expression level of the antigen has been shown to be equally important for an efficient C activation on both hematological and solid tumors. Golay et al. (45) analyzed freshly isolated cells from patients with B-CLL and prolymphocytic leukemia for CDC induced by Rituximab and found that the C sensitivity of these cells correlated with the surface expression of CD20. Derer et al. (48) reached similar conclusions using a fibroblast cell line expressing different levels of Epithelial Growth Factor Receptor (EGFR) and reported data indicating that the cell susceptibility to CDC progressively increased at higher expression level of EGFR. An increased antigen density resulting from Ab-induced movement of the tumor-associated antigen across the cell membrane can also contribute to enhance Ab-dependent C activation. CD20 is an example of a membrane antigen, that is induced by the type 1 Abs rituximab and ofatumumab to translocate to the lipid rafts (49). As a consequence, the immune complexes reach a critical concentration required for hexamer formation and C1q binding (50).

## FACTORS AFFECTING THE EFFICIENCY OF THE ANTIBODIES TO ACTIVATE COMPLEMENT: THE COMPLEMENT SYSTEM

C is an important player in Ab-induced tumor cell death and has therefore a major impact on the efficacy of therapeutic Abs. A clinical observation in patients with CLL treated with Abs is that the depletion of cancer and normal cells in the blood of patients is impaired in the presence of reduced levels of C components (51). Clearance of CLL cells induced by Abs to CD20 has been shown to be associated with C consumption, particularly of the early components, which persist for several days to weeks (52). This would cause a reduced therapeutic effect of subsequent infusions of the same Abs to control the malignant cells that circulate in blood in increasing number due to migration from bone marrow or lymph node. Using an in vitro model to evaluate the CDC of Burkitt's lymphoma cell lines induced by ofatumumab and rituximab, Beurskens et al. (51) have investigated the effect of different concentrations of anti-CD20 Abs on cell killing in two consecutive steps. They found that the dose of anti-CD20 Abs tested in the first step was critical for the degree of cell killing in the second step. In particular, using the maximal dose of anti-CD20 Abs in the first step, the cell lysis did not exceed 30% in the second step, while the percentage of cell killing increased to over 80% using a lower Ab concentration in the first step. These data suggest that the best therapeutic option would be to use the minimal concentration of Ab to trigger C-mediated killing of a relatively high number of cells leaving a C level sufficient to clear newly emerging malignant cells treated with an additional administration of Ab.

The critical role of C in CDC induced by recombinant Abs is supported by other uncontrolled studies suggesting that the killing of cancer B cells could be enhanced based on supplementation with purified C components or fresh frozen plasma (53, 54).

The response to immunotherapy of tumors that develop extravascularly is likely to be different from that of circulating cells. Unfortunately, it is difficult to evaluate the concentration of the Ab at cancer site, nor is it easy to measure the activity of the C system in the tumor microenvironment. However, the amount of Abs that reaches tumor sites (55) should be sufficient to activate C if the Abs tend to form hexamers that require limited amount of C components to activate the system (39).

Evidence supporting local C deposition was obtained by our group using a mouse xenograft model of B-cell lymphoma established in SCID mice with the intraperitoneal injection of a lymphoma cell line (56). This model is characterized by the development of peritoneal tumor masses and formation of foci of lymphoid cells in the spleen, liver, and bone marrow. Injection of rituximab into tumor-bearing mice resulted in the deposition of the Ab, C3, and C9 on tumor cells and in prolonged survival of these animals.

#### COMPLEMENT-MEDIATED CANCER CELL DAMAGE AND REGULATION

The importance of late C components in tumor development has recently been investigated by Verma et al. (57) in a xenogenic mouse model of B-cell lymphoma. They showed that tumorbearing C5 deficient animals treated with rituximab died within the 52 days period of observation whereas all C5 sufficient mice survived. Although the tumor tissue was not examined for complement deposition, the membrane attack complex (MAC) is likely to have contributed to the C protective effect in this model.

MAC assembly on the cell membrane is the final step of C activation. Tumor cell killing caused by Ab-mediated C activation takes a few minutes to complete under standard in vitro conditions (52) and is largely mediated by increased Ca2+ influx and rapid activation of a large variety of enzymes as a result of MAC insertion (58, 59). C5a and other C activation products can also contribute to tumor control by recruiting to the tumor microenvironment inflammatory cells that cause cell death via C-dependent cell cytotoxicity and phagocytosis (60).

A large body of evidence has been collected showing that cancer cells can resist CDC by several different mechanisms acting either on the cell surface or intracellularly.

Removal of MAC from the cell surface is one of these mechanisms observed in different tumor cell types after the activation of the C system by mAbs (61–64). This removal is usually mediated through membrane vesiculation, directed both to the inner and the outer sides of cell surface (65).

Overexpression of the membrane-associated C regulatory proteins (mCRPs) CD46, CD55, and CD59 is another mechanism by which cancer cells can evade undesired C attack due to spontaneous or Ab-induced C activation. The mCRPs act at different steps of the C sequence by favoring the decay of the C3 convertases (CD55), promoting the degradation of C3b and C4b (CD46), and preventing the assembly of MAC (CD59) (66, 67). Because of their high expression level on several tumors, mCRPs are considered promising targets for cancer immunotherapy.  Elevated levels of CD55 have been documented in a wide range of cancers including lung, colorectal, gastric, breast, and cervical cancers as well as in leukemia (66). CD59 is also overexpressed on different types of carcinoma and sarcoma and on melanoma cells (70).

An important point to emphasize is that hyper-expression of mCRPs on the surface of tumor cells does not necessarily mean that they are equally involved in cell protection from C attack. Almost two decades ago, we analyzed various Blymphoma cell lines for their susceptibility to CDC and found that all expressed increased levels of CD55, CD46, and CD59 and were variably resistant to C lysis (46). However, using neutralizing Abs to mCRPs, we were able to show that the resistance to C-dependent cell lysis was abrogated by blocking the inhibitory activity of CD55 and CD59 whereas inhibition of CD46 was totally ineffective (46). In contrast, CD46 appears to play a more prominent role in protecting ovarian cell lines from C attack as suggested from the substantial increase in C-mediated cell lysis observed inhibiting CD46 activity with anti-CD46 neutralizing Abs (42). These findings have important clinical implication for the selection of mCRP to inhibit in the immunotherapy of different tumors.

#### THERAPEUTIC STRATEGIES

#### Antibodies and Complement Activation

Over the past 20 years, therapeutic Abs have rapidly become the leading product in the biopharmaceutical market. Currently, there are more than 30 FDA-approved therapeutic Abs for cancer treatment and some of them are C-fixing Abs that mediate CDC (**Table 1**).

Rituximab was the first C-fixing Ab to receive FDA approval and has been used successfully to treat a large number of patients with CD20-expressing B-cell malignancies. Because CD20 is expressed on several B cell-derived cancer cells and also on normal cells from the late pro-B cell through memory cells, while absent on plasma cells and precursors hematopoietic stem cells, it is understandable why treatment with anti-CD20 Abs induces depletion of cancer cells but does not interfere with the repopulation of the B-cell compartment (71). Analysis of the binding mode of anti-CD20 Abs and their epitope specificity has led to the identification of two types of Abs that differ in their ability to form distinct complexes with CD20. Type I Abs stabilize CD20 in lipid rafts leading to stronger C1q binding and increased C activation whereas type II Abs exhibit reduced C1q binding that results in lower levels of cell death mediated by CDC (72).

Rituximab, ofatumumab and ibritumomab tiuxetan are examples of type I Abs known to be efficient activator of the C cascade (71). On the contrary, type II Abs like tositumumab performed poorly in CDC (49) and the same was observed for the optimized type II Ab obinutuzumab which fails to induce CDC (72, 73). However, Bologna et al. have reported that C plays a role in cell killing induced by high dose of the type II glycoengineered anti-CD20 mAb obinutuzumab on B-CLL expressing high levels of CD20, as suggested by the ability of the anti-C5 Ab eculizumab to totally prevent cell lysis (74).

In addition to anti-CD20 Abs, other approved C-fixing Abs directed against various tumor antigens have been recognized to activate C both in vitro and in vivo including anti-CD52 alemtuzumab (75), anti-CD38 daratumumab (76, 77), anti-EGFR cetuximab (78, 79), anti-GD2 dinutuximab (80) and anti-HER2 pertuzumab (81).

#### Strategies to Improve Complement Activation by Therapeutic Antibodies Combination of Different Antibodies

The efficiency of C activation on the cell surface is largely influenced by the epitope density of the antigen recognized by recombinant Abs which can affect the formation of an adequate number of immune complexes capable of binding C1q. Two different strategies have been reported to increase the formation of C1q-fixing dimers. One approach is to use a combination of two Abs recognizing different epitopes on the same antigen sufficiently close to allow juxtaposition of the IgG Abs which is critical for C1q binding. Spiridon et al. (82) were the first to analyze C-mediated lysis of Her-2+ human breast cancer cell lines induced by several mAbs and showed an enhanced killing using a mixture rather than individual mAbs. Our group has investigated the C-fixing ability of two Abs, cMOV18, and cMOV19, that bind to distinct epitopes of the alpha isoform of folate receptor, highly expressed on epithelial ovarian cancer cells. Interestingly, the mixture of these two Abs was able to activate C and to cause death of ovarian cancer cells while individual Abs were totally ineffective (42). A similar pattern of C activation was obtained using combination of the anti-EGFR Abs cetuximab and matuzumab, which recognize different nonoverlapping epitopes of EGFR (79). Cetuximab was reported to induce some degree of CDC in lung cancer cell lines only at high concentrations (40µg/mL) (79), whereas lower amount (10µg/mL) of either cetuximab or matuzumab failed to trigger C activation. Interestingly, the mixture of the two Abs was able to induce C1q and C4c fixation leading to strong activation of CDC (50 and 80% of lysis of squamous cell carcinoma and glioblastoma cells, respectively) (78).

Although this approach has not yet been introduced in clinical practice, it represents a promising future development in immunotherapy with C-fixing Abs.

#### Neutralization of membrane Complement Regulatory Proteins

Different approaches based on anti-mCRP Abs or silencing mCRP expression in combination with therapeutic Abs have been evaluated in vitro and in vivo by various groups to prevent the C-inhibitory effect of mCRP. We initially reported an increased susceptibility of follicular and Burkitt's lymphoma cell lines to CDC induced by Rituximab in the presence of Abs to CD55 and CD59 (46). These findings were later confirmed using an in vivo model of human CD20+ Blymphoma established in severe combined immunodeficient mice treated with rituximab in combination with anti-CD55 and anti-CD59 Abs that resulted in a significant animal survival (56). Similar enhancing effect of anti-CD55 and anti-CD59 Abs was reported on C-mediated killing of two human lung carcinoma cell lines induced by Herceptin (trastuzumab) (83). Neutralizing Abs to CD46 and CD59 were instead required to enhance CDC of ovarian carcinoma cells induced by the mixture of cMOV18 and cMOV19 (42). Down-regulation of all three mCRPs obtained with cationic liposomes (AtuPLEXes) loaded with siRNAs proved effective in inducing substantial increase of CDC of HER2 positive breast, lung and ovarian adenocarcinoma cell lines stimulated by trastuzumab and pertuzumab (84).

Although lysis of C-resistant tumor cells can be restored by the addition of Abs neutralizing mCRPs, their use is limited by the ubiquitous expression of mCRPs on both normal and tumor cells. One way to avoid undesired side effects that may derived from the binding of Abs to normal cells resulting in decreased expression of mCRPs is to selectively deliver the Abs to tumor cells. To this end, our group has generated two bispecific Abs containing binding specificity to CD20 and either CD55 or CD59. These Abs were able to recognize CD20 expressed on Burkitt's lymphoma cell lines and to neutralize membrane-bound CD55 and CD59 enhancing cell susceptibility to C-mediated lysis. An in vivo model of Burkitt's lymphoma developed in SCID mice was used to investigate the tissue distribution of bispecific Abs that were found to target selectively the tumor mass due to the high affinity of the anti-CD20 portion as opposed to the lower affinity of the anti-CD55 or anti-CD59 arms and to prevent tumor development (60). The therapeutic effect of the Abs was largely dependent on C activation, as revealed by the increased deposition of C3 and C9 in tumor masses, and also by local recruitment of macrophages and NK cells. Importantly, the combination of these two bispecific Abs resulted in the survival of 100% of treated mice whereas treatment with a single bispecific recombinant Ab (MB20/55 or MB20/59) induced the survival of only 20% of animals (60).

An interesting approach aimed at inhibiting mortalin, an heat shock protein over-expressed in many cancer types, has been proposed by Fishelson and his group (85) to interfere with MAC formation and its release from cell surface. The level of mortalin is inversely related to MAC deposition and its over-expression in erythroleukemic cells protects from C activation through the classical pathway, while protein down-regulation using specific siRNA increases the level of cell-bound C9.

# COMPLEMENT ACTIVATION POTENTIATES STANDARD THERAPIES

#### Complement and Radiotherapy

Together with surgery and chemotherapy, radiotherapy is a clinical mainstay of treatment for many malignancies especially for aggressive tumors with poor prognosis.

Recent studies support an association between radiation therapy for both human and murine cancers and C activation. An elegant study by Surace et al. (86) showed that local irradiation of melanoma and colon carcinoma developing in mice with a single dose of 20 or 5 Gy resulted in rapid and transient C activation triggered by tumor cells undergoing necrosis, apoptosis, and mitotic catastrophe with the possible contribution of natural IgM Abs bound to necrotic cells. In addition, they documented tumor deposition of C3 activation products and local increases in C3a and C5a, which induce maturation and activation of tumorassociated dendritic cells expressing the receptors for these anaphylatoxins and in turn promoting the anti-tumor activity of CD8+ T lymphocytes. The important role of C activation in the control of tumor growth was supported by the finding that radiotherapy failed to exert a protective effect against the tumor in mice deficient in either C3, or in C3a or C5a receptors, suggesting the critical contribution of locally released C3a and C5a.

Somewhat different results were obtained by Elvington et al. (87) who used a lymphoma model in mice receiving low dose radiotherapy fractionated over a period of approximately 5 months. They found that C inhibition induced by the administration of CR2-Crry resulted in longer survival and reduced tumor mass in tumor-bearing mice. A possible explanation for these contrasting results is that a radiation treatment administered over a prolonged period of time induces a C-independent inflammatory response that contributes to promote tumor growth. Overall, these data indicate that dose and fractionation in the radiation therapy need to be further investigated to find optimal conditions that combine the beneficial anti-tumor effects of radiotherapy and C activation.

## Complement and Chemotherapy

Limited information is available on the interplay between the C system and chemotherapeutic agents and the effect of this interaction on tumor control.

Levels of C3 and C4 were measured in patients with breast cancer treated with epirubicin/docetaxel-based neoadjuvant chemotherapy and found to be substantially reduced (88). This finding cannot be explained by C consumption because the low concentrations of C3 and C4 were not associated with a parallel increase in the level of the C activation product C4d. The relevance of this observation is unclear since the levels were equally reduced in responders and nonresponders to chemotherapy. A similar conclusion was reached in another study that examined the changes in C activity in patients with various types of cancer and revealed a significant reduction in C activity which was not accompanied by a corresponding increase in the level of C3d (89). More direct evidence supporting the beneficial effect of a combination therapy with a chemotherapeutic agent and a recombinant Ab was obtained from a multicenter clinical trial conducted in patients with advanced non-small-cell lung cancer (90). The patients receiving the monoclonal Ab cetuximab directed against epidermal growth factor receptor (EGFR) in combination with cisplatin/vinorelbine survived longer than those treated with the chemotherapeutic agents alone. In a subsequent study, cetuximab was found to bind to a lung cancer cell line expressing EGFR and to activate C resulting in the assembly of

Macor et al. Complement and Tumor Control

the membrane attack complex and cell death. C involvement in the killing of tumor cells was further documented by the finding that the inhibitory effect of cetuximab on tumor growth in an in vivo xenogeneic model of A549 lung cancer cells in nude mice was abolished in tumor-bearing mice treated with cobra venom factor to deplete C (79). These are promising results that need to be confirmed using a similar approach in the study of other tumors because the effect of chemotherapy on C activation and the consequent impact of these treatments on cancer cell killing may be different in various tumors.

#### CONCLUSION

The introduction of recombinant Abs into the clinic to control tumor growth has fostered the interest in C as an antitumor defense system acting in close collaboration with other components of both innate and acquired immunity. This has prompted the development of various strategies to optimize their therapeutic efficiency including structural modifications of the Abs to promote C activation and also control C inhibitors expressed on the tumor cell surface to enhance Ab-induced Cmediated cell killing. Major efforts are being made to selectively deliver mCRPs neutralizing agents to tumor cells and the recently generated bispecific Abs that target cancer cells and inhibit mCRP appear to move in this direction.

#### REFERENCES

An important point to consider when adopting this therapeutic approach is that C activated in the tumor microenvironment, particularly in the case of slow growing tumors associated with an inflammatory process developing in the surrounding tissue, may promote cancer expansion due to recruitment of suppressor cells by locally released C5a (**Figure 1**). We believe that this undesired effect may be prevented or markedly reduced by focusing Ab dependent C activation on residual tumor cells after surgical removal or substantial reduction of tumor mass after radio and/or chemotherapy. It is important, though, that the protocols for radiation therapy and chemotherapeutic treatment are selected to be highly effective in the control of tumor growth with limited proinflammatory side effects and negligible C activation in the tumor microenvironment.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

#### ACKNOWLEDGMENTS

This work was supported by grants from Italian Ministry of Health, IRCCS Istituto Auxologico Italiano, Ricerca Corrente and by Italian Association for Cancer Research.

in C3-deficient Her2/neu transgenic mice. Oncoimmunology (2013) 2:1–14. doi: 10.4161/onci.26137

mAb-mediated complement-dependent cytotoxicity of B-cell lines and of chronic lymphocytic leukemia B-cells. Mol Immunol. (2016) 70:13–23. doi: 10.1016/j.molimm.2015.12.007

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Macor, Capolla and Tedesco. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Complement in Metastasis: A Comp in the Camp

Daniel Ajona1,2,3,4, Sergio Ortiz-Espinosa1,4, Ruben Pio1,2,3,4 \* and Fernando Lecanda1,2,3,5

1 Program in Solid Tumors, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain, 2 Navarra Institute for Health Research (IdISNA), Pamplona, Spain, 3 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain, 4 Department of Biochemistry and Genetics, School of Sciences, University of Navarra, Pamplona, Spain, 5 Department of Pathology, Anatomy and Physiology, School of Medicine, University of Navarra, Pamplona, Spain

The complement system represents a pillar of the innate immune response. This system, critical for host defense against pathogens, encompasses more than 50 soluble, and membrane-bound proteins. Emerging evidence underscores its clinical relevance in tumor progression and its role in metastasis, one of the hallmarks of cancer. The multistep process of metastasis entails the acquisition of advantageous functions required for the formation of secondary tumors. Thus, targeting components of the complement system could impact not only on tumor initiation but also on several crucial steps along tumor dissemination. This novel vulnerability could be concomitantly exploited with current strategies overcoming tumor-mediated immunosuppression to provide a substantial clinical benefit in the treatment of metastatic disease. In this review, we offer a tour d'horizon on recent advances in this area and their prospective potential for cancer treatment.

#### Edited by:

Nurit Hollander, Tel Aviv University, Israel

#### Reviewed by:

Uday Kishore, Brunel University London, United Kingdom Chiara Agostinis, IRCCS Materno Infantile Burlo Garofolo, Italy Michael Kirschfink, Universität Heidelberg, Germany

> \*Correspondence: Ruben Pio<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology

> Received: 10 January 2019 Accepted: 12 March 2019 Published: 03 April 2019

#### Citation:

Ajona D, Ortiz-Espinosa S, Pio R and Lecanda F (2019) Complement in Metastasis: A Comp in the Camp. Front. Immunol. 10:669. doi: 10.3389/fimmu.2019.00669 Keywords: cancer, metastasis, complement, tumor microenvironment, anaphylatoxin, bone colonization

#### INTRODUCTION

The complement system represents a master component effector of innate immunity. Complement activation and regulation encompasses more than 50 soluble and membrane-bound proteins.

The function of complement, which entails the recognition and removal of pathogens and harmful entities, is accomplished by a multistep and sequential serine proteases-mediated cascade. The release of proteolytic fragments mediates key homeostatic and effector functions including: opsonization, inflammation, adaptive immune regulation, coagulation, tissue repair, neural development, bone homeostasis, angiogenesis, and host–microbiota symbiosis (1). Owing to the potentially deleterious effects of the complement system, its activity is tightly regulated at different levels by a number of soluble and membrane-bound proteins (2). Inappropriate complement activation underlies a variety of physiopathological conditions including inflammatory diseases and cancer (3).

Because many of the complement functions modulate tumor progression, their preeminent roles in promoting tumor cell dissemination are not surprising. This review focuses on recent findings on the major role of the complement system in tumor progression and highlights its key contribution to the different steps of the metastatic cascade.

#### COMPLEMENT ACTIVATION

Complement is mainly activated via three different recognition pathways: the classical, the lectin, and the alternative pathways. These three modes of complement activation converge into the generation of C3 convertases, which cleave C3 into C3a and C3b. C3a is an anaphylatoxin displaying an inflammatory regulation role. C3b can act in the opsonization process and as a component of the C5 convertase (4).

The classical pathway is triggered by the binding of C1q to antigen-antibody complexes, dying cells, extracellular matrix proteins, pentraxins, amyloid deposits, prions, or DNA (5).

The lectin pathway starts through binding of proteins homologous to C1q (mannose-binding lectin and H-, L-, or Mficolins) to carbohydrate structures on pathogens (6). Both the classical and the lectin pathways then sequentially cleave C4 and C2 for the generation of the classical/lectin C3 convertase (C4bC2b) (4).

Finally, the alternative pathway is initiated by the spontaneous hydrolysis of C3, also known as the "tickover" of C3, which after the formation of C3(H2O) can bind to factor B. Cleavage of factor B by factor D forms the initial alternative pathway C3 convertase, C3(H2O)Bb (7).

Although these three routes of activation differ in their mechanisms of target recognition and initiation, they converge at C3 cleavage, yielding the active fragments C3a and C3b. C3b binding to C3 convertases assembles the C5 convertase that cleaves C5 into the anaphylatoxin C5a, and C5b. The latter fragment is indispensable to assemble the membrane attack complex which mediates targeted lysis (8).

Additional pathways of complement activation include C3 and C5 extrinsic protease cleavage (9–11), the C2-bypass pathway (12), and the properdin-mediated direct convertase formation on microbial surfaces (13).

Among the complement-derived downstream effectors, C3a and C5a play diverse roles in both homeostasis and disease. These molecules bind to their cognate seven-transmembrane domain receptors C5a receptor 1 (C5aR1; CD88) and C3a receptor (C3aR), respectively. C5a can also bind to C5aR2 (14). The role of C5aR2 remains poorly understood. Recently, it has been reported that the binding of C5a to C5aR2 in carcinoma-associated fibroblasts promotes tumor formation and chemoresistance by providing a survival niche for cancer stem cells (15).

Recent discoveries have also revealed that complement activation is not only restricted to the extracellular space, as originally thought, but also occurs in the cytoplasm. The intracellular components of complement (the so-called complosome) modulate metabolic processes during T cell effector differentiation (16, 17) but so far, their intracellular functions remain largely unexplored.

#### COMPLEMENT IN CANCER PATIENTS

Neoplastic transformation involves complex genomic and epigenomic alterations perturbing normal cell homeostasis. Local or distant dissemination of tumor cells, one of the hallmarks of cancer, represents a multistep process that entails the gain of novel cellular functions which include invasion, increased cell locomotion, intravasation, survival in the circulation, overcoming immune attack, and colonization in foreign cellular niches to form secondary tumors (18).

Overcoming immune attack is a key step in tumor progression. Altered immune recognition is achieved by a variety of mechanisms (19), including the modulation of the complement system. Complement activation has been described in cancer patients with hematological malignancies such as lymphomas (20), and in a plethora of solid tumors (21–23). Furthermore, intact complement proteins were found increased in blood of patients with lung cancer (24, 25), neuroblastoma (26), and digestive tract tumors (27). However, complementmediated cytotoxicity is circumvented by different mechanisms, most of which include the upregulation of complement regulatory proteins (28–30). These regulators normally protect tumor cells from complement-mediated destruction, and can be grouped into two categories: membrane-bound complement regulatory proteins (mCRPs) and soluble regulators. High expression of the mCRPs membrane cofactor protein (CD46), decay-accelerating factor (CD55), and CD59 (protectin) on tumor cells is associated with increased metastatic potential, and poor prognosis in a range of tumors (31–34). Similarly, the soluble regulators factor H and FHL-1 have been found elevated in biological fluids from ovarian (35), bladder (36) and lung cancer patients (37), and are also associated with poor prognosis (38). Other soluble regulators as clusterin (39), C1 inhibitor (40), factor I and C4b-binding protein (C4BP) (41) are secreted by tumor cells into the tumor milieu and could also be detected in the circulation.

Activation of the complement system by tumor cells was long believed to only benefit the patient. Preclinical data suggest that complement can evoke potent complementdependent cytotoxicity against tumor cells, and a range of therapeutic strategies have been designed to potentiate complement activation and overcome the protection mediated by complement inhibitors. This approach has been specially tailored to enhance the therapeutic efficacy of monoclonal antibodies (42). However, recent findings have challenged this view, providing evidence of the cancer-promoting potential of complement activation and the utility of complement inhibition as an anticancer therapy (43). Complement components coopted by tumor cells can lead to the acquisition of self-advantageous functions tilting the balance toward tumor progression. For instance, lung cancer cells are recognized by the complement system more efficiently than their normal counterparts. This effect is mediated by the direct binding of C1q and leads to the subsequent activation of the classical complement pathway (44). This activation is compensated by the expression of factor H/FHL-1 and CD59 (45, 46). This equilibrium in complement activity would explain the elevated levels of complement fragments found in biological fluids from these patients. Thus, C4d, a split product of the classical complement pathway, is increased in biological fluids of lung cancer patients. Detection of C4d is associated with poor prognosis, and has been proposed as a potential biomarker of clinical value in the management of lung cancer patients (44, 47, 48). Similar results were obtained in oropharyngeal tumors by detecting C4d in saliva (49). Moreover, other complement factors have been associated with cancer. Anaphylatoxin C5a is increased in plasma from lung cancer patients (50, 51), and is associated with metastatic potential in lung and gastric cancer patients (52, 53). Similarly, C1QB is one of the top-scoring genes associated with lung metastases in osteosarcoma patients (54).

Taken together, these studies indicate an association between complement activation and malignant progression.

# COMPLEMENT IN THE TUMOR MICROENVIRONMENT

Of all complement proteolytic fragments derived from complement activation, anaphylatoxins are by far, the best described in cancer. Anaphylatoxins C5a and C3a trigger spurious tumor intracellular signaling pathways by binding to their cognate receptors expressed in tumor and immune cells. These signaling events deeply perturb the tumor milieu by inducing the recruitment and/or tumor-promoting abilities of myeloid-derived suppressor cells (MDSC), macrophages, neutrophils, and mast cells, preventing efficient T cell-mediated responses (55).

Elevation of C5a or C5aR1 levels has been observed in solid tumors including lung (50, 53), gastric (56), ovarian (57), breast (58), urothelial (59), and clear cell renal cancers (60).

C5a induces the recruitment of MDSCs into the tumor microenvironment, and markedly dampens anti-tumor T-cell responses. C5aR1 mediates these effects on two subpopulations of MDSCs. On one side, C5a is a potent chemoattractant for granulocytic MDSCs (a neutrophil-like subpopulation) and on the other, C5a stimulates the monocytic MDSC subpopulation with the concomitant production of reactive oxygen and nitrogen species (61).

C5aR1 expressed on MDSCs is also able to bind ribosomal protein S19 (RPS19), which is released from apoptotic tumor cells into the tumor microenvironment, leading to a shift toward Th2 cell responses with increased levels of immunosuppressive TGF-β (62). Accordingly, pharmacological blockade of C5aR1 in a syngeneic model of lung cancer impaired tumor growth, decreased the percentage of splenic MDSCs, and downregulated immunosuppression-related genes including ARG1, IL6, IL10, CTLA4, LAG3, and PDL1 within the tumor milieu (50).

Besides MDSCs, C5a affects the biology of other leukocytes present in the tumor microenvironment. C5a elicits a strong pro-inflammatory infiltration with secretion of MCP-1, responsible for the recruitment of immunosuppressive macrophages, and increase of arginase-1 and IL-10 (63). Similarly, fibrinolytic enzyme-mediated generation of C5a regulates the protumorogenic properties of C5aR1+ mast cells and macrophages, leading to hampered antitumor CD8 T-cell responses in a model of squamous carcinogenesis. Interestingly, the combined treatment based on cytotoxic chemotherapy and the blockade of C5aR1 synergistically increased the recruitment and the cytotoxic properties of CXCR3+ effector memory CD8 T cells by IFNγ-dependent mechanisms (64). Ablation of PTX3, an important negative regulator of inflammation and complement activation, resulted in amplification of complement activation, MCP-1 production, and tumor-promoting macrophage recruitment. Consistently, pharmacological blockade of C5aR1 reversed these pro-tumorogenic effects (65).

Although far less studied than C5a, the anaphylatoxin C3a also preconditions a tumor-promoting microenvironment. Signaling mediated by C3a binding to C3aR contributes to melanoma tumorigenesis by inhibiting neutrophil and CD4 T-cell responses (66). Autocrine complement C3 inhibits IL-10-mediated cytotoxic properties of tumor-infiltrating CD8 T lymphocytes through complement receptors C3aR and C5aR1, and enhances melanoma and breast cancer growth (67).

Moreover, complement activation may underlie the ability of tumors to evolve and adapt to different cues of the microenvironment increasing tumor progression. Thus, under hypoxic conditions, lung cancer cells downregulate complement inhibitors, factor H and factor I, to increase their susceptibility to complement activation (68). This phenomenon may fuel the generation of C5a which in turns may contribute to hypoxic stress in the tumor milieu to promote tumor progression through the inhibition of cell-mediated immunity. Indeed, in a syngeneic lymphoma model the impact of C5a in tumor microenvironment is dose-dependent (69).

Complement effectors can also affect tumor progression independently of complement activation. Factor B and factor I promote squamous cell tumor growth upon the activation of ERK1/2 (70, 71). C1q promote angiogenesis and lung metastasis in a syngeneic model of murine melanoma (72). In malignant pleural mesothelioma, C1q binds to hyaluronic acid in the tumor microenvironment and enhances tumor proliferation (73). C1q secreted by mesenchymal stromal cells mediates the activation of β-catenin in chronic lymphocytic leukemia and enhances malignant progression (74). On the other hand, properdin, a positive regulator of complement activity, induces endoplasmic reticulum-stress response and exerts a tumor suppressive role in breast cancer (75).

In summary, tumors are able to perturb complementrelated immune effectors favoring tumor progression. Distorted complement homeostasis remodels the tumor microenvironment by inhibiting the anti-tumor immune responses and contributes to the metastatic dissemination of cancer cells (**Figure 1**).

### EARLY METASTASIS STEPS: COMPLEMENT EFFECTS ON TUMOR CELLS

Epithelial–mesenchymal transition (EMT), loss of cellcell adhesion, and increase of motility, invasiveness, and intravasation of tumor cells are at the core of the early metastatic events (18). Tumor-associated complement-activation modifies the tumor cell behavior endowing early metastatic traits. Perturbed complement activation leads to the generation of growth factors, proangiogenic factors, and other mediators that promote tumor growth and dissemination. These acquired pro-metastatic functions are mediated by the C3a and C5a stimulation of C3aR and C5aR1 in tumor cells, respectively, which triggers spurious intracellular signaling pathways. For instance, C5aR1 in lung tumor cells activates the p44/42 MAPK and NF-κB signaling pathways leading to the secretion of IL-8, VEGF, and MCP-1 to the tumor milieu (53). Complement

FIGURE 1 | The role of complement in metastasis. Tumor-associated complement activation generates anaphylatoxins C5a and C3a in the tumor microenvironment. Binding of these molecules to their cognate receptors promote a range of tumor-promoting functions. C5a, through its receptor C5aR1, facilitates the recruitment and the activity of suppressive leukocyte subsets such as MDSCs, neutrophils, and Tregs in the tumor microenvironment. C3a also contributes to a suppressive tumor microenvironment by recruiting neutrophils. C5aR1 signaling affects endothelial function and tumor-associated angiogenesis, and the binding of C5a to C5aR2 in carcinoma-associated fibroblasts promotes tumor formation by providing a survival niche for cancer stem cells. In tumor cells, C5a/C5aR1 axis modulates tumor-induced MMP expression, increases tumor cell migration and invasiveness, enhances the release of pro-angiogenic factors, and induces EMT. Binding of C1q to tumor cells enhances tumor cell proliferation and favors angiogenesis in a complement activation-independent manner. Complement anaphylatoxins also facilitate tumor dissemination by stimulating a hyper-coagulation state and NETs, and adapt specific organ environments to the metastatic spread. This includes the disruption of the CSF barrier, the induction of CXCL16-mediated osteoclastogenesis, and the generation of an immunosuppressive microenvironment.

components facilitate tumor dissemination by inducing an EMT in tumor cells which leads to the acquisition of a motile and less adherent phenotype. C5a/C5aR1 axis mediates the upregulation of transcription factor Snail and a concomitant decrease in E-cadherin and claudin-1 gene expression levels with increased invasiveness in hepatocellular carcinoma (76). In ovarian cells, TWIST1 enhances C3 expression and mediates EMT (77). According to these findings C5aR1-tumor expression was associated with tumor invasiveness, vascular and lymphatic invasion, liver metastasis, and poor outcome in patients with gastric tumors (78). Furthermore, C5aR1 inhibition hampers lung cancer cell migration, and up-regulates the expression of E-cadherin, suppressing EMT and invasiveness. Consistently, a negative correlation between the expression of C5aR1 and E-cadherin was found in lung primary tumors (79).

Initial steps for the acquisition of a metastatic phenotype also involves the secretion of stromelysins and other matrix metalloproteinases (MMPs) able to degrade different extracellular matrix components, especially the basal membrane, allowing for tumor cell intravasation and dissemination to local or distant sites (33). C5a markedly enhances cancermediated MMP activities and migratory and invasive tumor cell activities (80). C5a stimulation also decreases tumor adhesion to extracellular matrix proteins including collagens I and IV (53). Aberrantly expressed C5aR1 increases cell locomotion, cytoskeletal rearrangements with the formation of lamellipodia and membrane ruffling in liver bile duct malignant cells (80). C5aR1 signaling promotes motility and invasiveness through the activation of RhoA, and leads to enhanced invasion and vascular invasion in gastric cancer cells (56). ERK and PI3K, downstream C5aR1 activation, mediate an increase in cell invasiveness in renal cancer cells (81).

C3a-mediated stimulation elicits an increase in p42/44, p38 MAPK, and PKB/AKT activation and downregulates inducible hemeoxygenase-1 (HO-1) in leukemic cells (82). Autocrine stimulation of C5aR1 and C3aR upon C5a and C3a binding leads to PI3K/AKT signaling and regulates the proliferation and invasiveness of ovarian tumor cells (57).

In summary, complement-mediated effects are crucial in the early stages of metastasis, involving changes in tumor cell adherence to surrounding stroma and neighboring cells, increasing local invasiveness and promoting lymphatic and hematogenous dissemination.

## COMPLEMENT EFFECTS ON DISSEMINATION

The host microenvironment at local or distant sites provides signals permissive for tumor promotion. Critical pathways triggered in the surrounding stroma and/or endothelial or lymphatic cells are required for proper cell-cell and cell-matrix engagement and for the secretion of a panoply of protumorogenic factors (83, 84). In addition, vascular or lymphatic vessels provide a major route by which tumor cells exit the primary tumor site, enter the circulation and establish metastasis (85). Furthermore, tumor vascular density is a prognostic indicator of metastatic dissemination. In cancer, complement may be involved in the modulation of the angiogenic program in the tumor microenvironment, although the specific role of complement in angiogenesis is highly dependent on the tumor type. For instance, C5aR1 blockade does not affect tumor angiogenesis in murine models of lung or cervical cancer (50, 61). In contrast, genetic inhibition of C3 and C5aR1 impairs endothelial cell function in an ovarian cancer model (86). C5a also supports an angiogenic program displayed by infiltrating macrophages in squamous cell carcinoma (64). C1q deposition on melanoma cells increases tumor vascular density and facilitates tumor progression (72). The evidence that complement has a role in endothelial homeostasis might have implications also at secondary metastatic sites, a possibility which remains largely unexplored.

Once in the circulation, tumor cells have to overcome the mechanical constraints imposed by sheer-stress, anoikis induced by cell anchorage-independency, and the immune attack. A role of platelets, together with fibrin and thrombin, has been invoked for the establishment of distant metastasis by protecting circulating tumor cells from mechanical stress and facilitating engraftment at target sites (87).

Complement components contribute to a hyper-coagulation state allowing tumor cell survival in the circulation. C3a induces platelet activation and aggregation favoring a prothrombogenic state (88). Similarly, C5a stimulates neutrophils to release Tissue Factor, inducing a prothrombotic phenotype (89). C3aR in neutrophils stimulates neutrophils extracellular traps (NETs) (90), extracellular structures composed of chromatin and degrading enzymes (myeloperoxidase, cathepsin G, and elastase) that contribute to form a three-dimensional scaffold that supports fibrin deposition and thrombus stabilization and entraps platelets, erythrocytes and tumor cells, driving a protumorogenic state (91).

This pro-tumorogenic milieu also favors the subsequent dissemination of tumor cells to neighboring or distant sites. Homing of tumor cells to target sites could also be actively mediated by factors released by target organs that act as potent tumor cell chemoattractants (92). But tumors also precondition target organs creating a hospitable niche by the mobilization of bone marrow-derived myeloid cells, tumor secreted factors such as VEGF, TGF, TNF (93–95), and tumor releasedexosomes which also modulate the tumor microenvironment (96, 97). These nanometer-sized vesicles, which contain a complex cargo of membrane receptors, nucleic acids, cytoskeletal components, and intracellular proteins, act as unique vehicles for transport to local or distant organs. Tumor derived exosomes, which are more abundantly released in inflammation, represent another mechanism of immunosuppression. As observed for tumor cells, exosomes display CD55 and CD59, conferring resistance against complement-mediated lysis (98), and potentially regulating the exosome-mediated cross-talk associated with the metastatic program.

These events largely studied in murine models collectively contribute to prepare the "fertile soil" invoked by the Paget's hypothesis (99), and crystallize the concept of "premetastatic niche" (100). The premetastatic niche consists in the accumulation of aberrant immune cells and extracellular matrix proteins in target organs (101). Emerging data demonstrate that C5a contributes to the lung premetastatic niche by regulating the expression of TGF-β and IL-10 by immature myeloid cells and the subsequent accumulation of regulatory T cells, the proliferation of resident alveolar macrophages in the premetastatic lungs, and a decrease in the number and the maturation status of lung dendritic cells. As a consequence, effector CD4 T-cell responses skew toward Th1 responses (102, 103).

# LATE STEPS OF METASTASIS

A similar paradigm to which occurs in the primary tumor could also influence metastatic behavior in the target organ. Tumor cells need to overcome the constraints imposed by the "foreign soil" and require compatibility with the hosting milieu. Each organ provides unique opportunities which could be exploited in the benefit of tumor cells by propelling the growth of micro to macrometastases (104). An increasing body of evidence indicates that complement is involved in this process, resulting in tumor outgrowths at secondary sites.

Genetic abrogation of C5aR1 in the host dampens M2 polarized tumor associated macrophages, leading to a decrease of liver and lung metastases in a syngeneic colon cancer model (52). Pharmacological inhibition of C5aR1 increases the infiltration of CD8 cytotoxic T cells in metastatic nodules, and impairs lung and liver metastatic processes with no effect detected in primary tumors. Thus, genetic or pharmacological inhibition of C5aR1 results in impaired metastasis (103).

Moreover, activation of C5aR1 in tumor cells leads to an increased prometastatic activity. For instance, in a lung cancer model of bone metastasis, C5a/C5aR1 axis induced the production of pro-osteoclastogenic factors favoring skeletal metastases. Among these factors, CXCL16 released upon C5aR1 signaling led to osteoclastogenic activation and osteolytic lesions. These effects were blocked by C5a inhibition or genetic silencing of C5aR1 in tumor cells, suggesting its implication in skeletal metastases (53). Indeed, complement is involved in bone homeostasis and turnover (105). Bone-forming osteoblasts and bone-resorbing osteoclasts are tightly regulated to ensure a balanced bone mass. Receptor activator of nuclear factor k-B ligand (RANKL), which is secreted by osteoblasts, binds to its receptor on the membrane of committed monocytes to differentiate into osteoclasts (106). Complement modulates osteoclasts differentiation in vitro and in vivo through C5aR1, but no effects were exerted in osteoblast differentiation (107). However, C3aR and C5aR1 signaling by C3a and C5a in osteoblasts modulates the release of pro-inflammatory proosteoclastogenic cytokines IL-6 and IL-8, and C5a increases RANKL in osteoblasts, overall favoring a pro-osteoclastogenic milieu (108). Because of these bone-specific mechanisms, the complement system might be specially relevant in skeletal metastases (53). Indeed, lung primary tumors that metastasize to bone show higher C5aR1 levels than those that metastasize to other locations, suggesting its major role in the tumorinduced skeletal lesions. Nevertheless, this axis also mediates lung

metastases, since lung tumor colonization was decreased when lung cancer cells were devoid of C5aR1 (53).

 C3 was upregulated in four leptomeningeal metastatic models and proved necessary for tumor growth within the leptomeningeal space. C3a, generated after C3 cleavage and bound to the C3aR expressed on the choroid plexus, was able to disrupt the bloodcerebrospinal fluid barrier. This effect was critical since blockade of this step provided a survival benefit in these models. However, C3 did not mediate cancer cell entry into the cerebrospinal fluid but other determinants were required for full tumor cell colonization (109).

Inhibition of complement-related proteins, and specially anaphylatoxins (14), has been proposed as a therapeutic option for maximizing the clinical efficacy of current immunotherapies. Recent studies have provided support of this idea after combined inhibition of anaphylatoxins and PD-1 signaling for the treatment of metastatic cancer. Administration of PD-1/PD-L1 blocking antibodies resulted in intratumoral complement activation and the subsequent accumulation of C5a within the tumor milieu (110). Importantly, the combination of C5a and PD-1 blockade reversed CD8 T-cell exhaustion, and markedly reduced lung cancer metastasis in two syngeneic animal models (111).

#### CONCLUSIONS

The complement system represents an important player in tumorigenesis and metastasis. Its relevance stems from its ability

#### REFERENCES

to foster a protumorogenic milieu by modulating tumor-immune responses. It also endows tumor cells with cell functions required for metastatic dissemination. Preclinical studies support the idea that the therapeutic blockade of complement has potential in combinatorial immunotherapy to effectively eradicate primary tumors and distant metastases. A better understanding of the mechanisms of interaction of the complement system with tumor cells and their microenvironment is required for designing combined novel immunotherapeutic regimens able to effectively target established tumors.

#### AUTHOR CONTRIBUTIONS

DA, RP, and FL designed the concept. DA and FL wrote the manuscript. SO-E prepared the figure. All authors read and approved the final version of the manuscript.

#### FUNDING

Authors' work is supported by FIMA (Foundation for Applied Medical Research), and CIBERONC (CB16/12/00443). DA and RP are funded by Fundación Científica de la Asociación Española Contra el Cáncer, Fundación Ramón Areces, Juan Serrano, and Fondo de Investigación Sanitaria-Fondo Europeo de Desarrollo Regional Una manera de hacer Europa (FEDER, PI17/00411). FL is funded by La Caixa Foundation, Caja Navarra Foundation and the Spanish Ministry of Economy and Competitiveness (SAF2015-71606R). SO-E was supported by a predoctoral fellowship from the Asociación de Amigos de la Universidad de Navarra and now is supported by an FPU fellowship.

C3 during murine liver regeneration. Mol Immunol. (2008) 45:3125–32. doi: 10.1016/j.molimm.2008.03.008

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Ajona, Ortiz-Espinosa, Pio and Lecanda. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Complement C5b-9 and Cancer: Mechanisms of Cell Damage, Cancer Counteractions, and Approaches for Intervention

#### Zvi Fishelson1 \* and Michael Kirschfink 2

*1 Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 2 Institute of Immunology, University of Heidelberg, Heidelberg, Germany*

#### Edited by:

*Maciej M. Markiewski, Texas Tech University Health Sciences Center, United States*

#### Reviewed by:

*Michelle A. Dunstone, Monash University, Australia Jamie Honeychurch, University of Manchester, United Kingdom*

> \*Correspondence: *Zvi Fishelson<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology*

> Received: *31 January 2019* Accepted: *20 March 2019* Published: *10 April 2019*

#### Citation:

*Fishelson Z and Kirschfink M (2019) Complement C5b-9 and Cancer: Mechanisms of Cell Damage, Cancer Counteractions, and Approaches for Intervention. Front. Immunol. 10:752. doi: 10.3389/fimmu.2019.00752* The interactions of cancer cells with components of the complement system are highly complex, leading to an outcome that is either favorable or detrimental to cancer cells. Currently, we perceive only the "tip of the iceberg" of these interactions. In this review, we focus on the complement terminal C5b-9 complex, known also as the complement membrane attack complex (MAC) and discuss the complexity of its interaction with cancer cells, starting with a discussion of its proposed mode of action in mediating cell death, and continuing with a portrayal of the strategies of evasion exhibited by cancer cells, and closing with a proposal of treatment approaches targeted at evasion strategies. Upon intense complement activation and membrane insertion of sufficient C5b-9 complexes, the afflicted cells undergo regulated necrotic cell death with characteristic damage to intracellular organelles, including mitochondria, and perforation of the plasma membrane. Several pro-lytic factors have been proposed, including elevated intracellular calcium ion concentrations and activated JNK, Bid, RIPK1, RIPK3, and MLKL; however, further research is required to fully characterize the effective cell death signals activated by the C5b-9 complexes. Cancer cells over-express a multitude of protective measures which either block complement activation, thus reducing the number of membrane-inserted C5b-9 complexes, or facilitate the elimination of C5b-9 from the cell surface.  Blockage of complement activation is mediated by the complement membrane regulatory proteins CD46, CD55, and CD59 and by soluble complement regulators, by proteases that cleave complement proteins and by protein kinases, like CK2, which phosphorylate complement proteins. C5b-9 elimination and inhibition of cell death signals are mediated by caveolin and dynamin, by Hsp70 and Hsp90, by the mitochondrial stress protein mortalin, and by the protein kinases PKC and ERK. It is conceivable that various cancers and cancers at different stages of development will utilize distinct patterns of these and other MAC resistance strategies. In order to enhance the impact of antibody-based therapy on cancer, novel precise reagents that block the most effective protective strategies will have to be designed and applied as adjuvants to the therapeutic antibodies.

Keywords: complement, C5b-9, complement-dependent cytotoxicity, regulated necrosis, cancer immune resistance

# PREFACE

The complement system may affect cancer in several forms, ranging from promotion of cancer growth and metastasis, on the one hand, to antibody-based cancer eradication, on the other. Upon encounter of the cancer cells with the complement system, activation may proceed via the classical, alternative, and/or lectin pathways (1) (**Figure 1**). This initiation step leads to formation of a C3 convertase (C4b2a or C3bBb) that deposits C3b molecules on the cells, followed by formation of a C5 convertase (C4b2a3b or C3bBb3b) that cleaves C5 and initiates formation of the C5b-9 complexes, termed the membrane attack complexes (MAC). Here, we will focus on the anti-cancer cytotoxic activity of complement, with an emphasis on the mode of action of the MAC. Reviews on the cancer-promoting activities of complement (2–4) and on complement activation by clinical anti-cancer antibodies (5–7) have been published recently; therefore, these topics will not be covered in this review. Another topic recently reviewed is the insights into the fine structural details of the complement MAC (8–11). MAC expresses a plethora of nonlytic and sublytic activities that have been reviewed elsewhere (12–15) and are thus excluded from this review. Here we will describe the current status of research on the cytotoxic effects of MAC, emphasizing the findings, dogmas, and open questions in our quest to better understand the fine mechanistic details of MAC-induced cancer cell death. Next, we will present the currently recognized counter-mechanisms utilized by cancer cells to resist complement-dependent cytotoxicity (CDC). Finally, we will discuss several potential therapeutic approaches for the intervention and potentiation of antibody-based anti-cancer immunotherapy that have been proposed and tested.

#### MECHANISMS UNDERLYING COMPLEMENT-MEDIATED CANCER CELL DAMAGE

#### Perspective: The Early Studies on Osmotic Cell Death

Studies on cancer cell killing by complement have been conducted long before the identification of the complement terminal pathway responsible for mediating cell damage and death. As early as 1950s, Kalfayan et al. (16), Ellem (17), and Green et al. (18) investigated the action of antibody and complement on rabbit Brown-Pearce carcinoma cells, rat Ehrlich and mouse Krebs ascites tumor cells, respectively. They observed cell swelling and increased plasma membrane leakiness. They proposed that complement impairs cell membrane integrity, increases cell permeability to anions, cations, and water, and causes osmotic cell swelling up to the point that the membrane collapses, culminating in osmotic cell lysis (19). The leakage from the cells was proposed to occur through functional, stretching, and possibly reversible "holes" in the swelling cells, which could be blocked, to some extent, by increasing the osmotic pressure of the extracellular medium (20). The concept of complement-induced osmotic lysis of target cells is still popular today but, as discussed later, it must be viewed with a grain of salt. Kim et al. (21) subjected Ehrlich ascites tumor cells to CDC and demonstrated that osmotic protection effectively prevented cell swelling but did not rescue the cells from death. They hypothesized that the cells died following activation of metabolic events that were detrimental to cell survival or through activation of a "suicidal" mechanism of programmed cell death. In conclusion, osmotic burst of inflated complement-damaged cells may occur, but these bursts are most likely a consequence of metabolic collapse of the cell rather than the cause of cell death.

### The Complement Cell Death Mediator: A Concerted Action of Toxic Moieties

Membrane pores caused by complement were first visualized by electron microscopy on red blood cell membranes as large ring structures (22). Similar lesions were viewed on E. coli cell walls (23). Over the years, ample information on the fine ultrastructure of the MAC that can activate cell death has been gathered (24) and has been recently further examined (8–11, 25–27). For a complete updated view of the MAC structure, the reader is referred to those publications. The observed ring structure apparently corresponds to the structure of polymerized C9 molecules attached to their polymerization accelerator, the C5b-8 complex (28). However, even today we have only a partial view of the fine details of the cytotoxic mechanisms activated by MAC, eventually leading to the point of no return and cell death. Besides the paucity of investigations on the subject, several reasons account for that. First, the early dogmas were based on investigations with complement-targeted artificial membranes and red blood cells, which are clearly different, largely passive targets, compared with nucleated cells (29–34). Second, very large variation exists in refractoriness to the MAC, even among closely related cancer cell lines and even within a supposedly homogenous population of cultured cancer cell lines. Third, in target cells MAC activates concomitantly several signaling pathways and biochemical events, some cytotoxic and others prosurvival, and it is the particular balance among them that dictates cell fate, survival, or death. Finally, activation of the terminal complement pathway may result in generating, in the target cell membrane, a cocktail of membrane-inserted protein complexes: C5b-8, C5b-91, C5b-92, C5b-93, and so on, up to C5b-9 with 12- 18 polymerized C9 molecules (28, 35). Each of these complexes

FIGURE 1 | Activation and regulation of the complement pathways. Activation: Complement activation proceeds through four converging pathways shown in this simplified scheme, i.e., the classical (CP), lectin (LP), alternative (AP), and terminal (TP) pathways. Activation of the CP and LP can be potentiated by components of the AP (Amplification loop). Binding of C1 (a complex of C1q, 2C1r, 2C1s) via C1q to antigen-bound antibodies initiates the CP, whereas binding of MBL or ficolin (in complex with MBL-associated serine proteases, MASP) to carbohydrates (e.g., microbial) initiates activation of the LP. The AP is initiated by C3 spontaneously hydrolyzed at a low rate into C3(H2O) or following another C3-tickover event. The three pathways generate a C3 converting enzyme, a C3 convertase (that cleaves C3 into C3a and C3b), by activation of C4 and C2 (CP and LP: C4b2a), or of factors B and D (AP: C3bBb). AP activation is facilitated by properdin (P). The resulting C3b not only opsonizes target cells but also joins the C3 convertases and turns them into C5 convertases, which convert C5 into C5a and C5b. Subsequent TP activation by assembly of C5b with C6, C7, C8 and multiple C9 molecules, generates the membrane attack complex, (C5b-9, MAC). By binding to specific receptors, C3a and C5a exert multiple cell stimulatory activities, ranging from allergy and anaphylaxis to promotion of acquired immunity by stimulation of lymphocytes and antigen presenting cells. Regulation: Complement activation is tightly regulated by multiple soluble and membrane proteins. Soluble inhibitors include: C1 inhibitor (C1-INH), C4 binding protein (C4BP), factor H (FH), factor I (FI), Clusterin and Vitronectin. The membrane regulatory proteins are: Decay Accelerating Factor (DAF, CD55), Membrane Cofactor Protein (MCP, CD46), Complement Receptor 1 (CR1, CD35), and CD59. As shown in the figure, C1-INH interferes with activation of C1r, C1s, and MASP. C4BP, FH, CD55, and CD35 restrict formation and stability of the CP and AP C3/C5 convertases or promote FI-mediated inactivation of C4b (CD35/CR1, CD46/MCP, C4BP) or C3b (CD35/CR1, CD46/MCP, FH). Clusterin and vitronectin prevent the association of the forming C5b-9 complexes with the membrane, whereas CD59 limits cell damage by preventing MAC complex formation.

may induce in the target cell slightly different signals that have not yet been discretely characterized. Detailed analysis of the effect of the terminal complement complex size on the lysis of rat Ehrlich ascites tumor cells by human complement indicated that complexes containing more C9 per C5b-8 are cytolytically more potent. Nevertheless, the kinetics of cell death appeared similar in cells bearing C5b-9 complexes that have either 1 or 4 C9 molecules per C5b-8 (36). Moreover, some human cancer cells, such as U938, HL60, and B-CLL cells, could be lysed by C5b-8 alone, in the absence of C9, when a sufficient number of complexes were deposited on them (37, 38). Hemolysis of sheep red blood cells could be efficiently activated by C5b-9 complexes generated with thrombin-cleaved C9, which cannot undergo classic ring-like polymerization, but forms apparently, string-like oligomeric structures that may lead to leakage of membranes (39, 40). Hence, it is improbable that MAC, with its various intermediary complexes, activates a unified mechanism of cell death in all cell types. An additional level of complexity has been introduced by reports of apoptotic cell death induced by MAC (41), but this has not been observed so far with cancer cells undergoing CDC.

## Calcium Ions Influx: Dose-Dependent Dichotomy

At non-toxic or sublytic doses, MAC has been shown to trigger numerous signals in many types of cells, normal and malignant. This topic has been extensively discussed recently and will not be covered here (12–15). Initially, measurements with pigeon erythrocyte sealed "ghosts" revealed an increase in intracellular calcium ions, which begins within seconds after binding of MAC and supposedly precedes the cell death process activated by lytic doses of MAC (42). This transient rise of intracellular free Ca2+ in target cells was thought to be required for cell death. However, later it became apparent that the rise in the level of intracellular calcium ions is essential for cell survival and recovery (43). Reduction of the extracellular Ca2+ concentration by chelation delays the onset of cell death, as measured by LDH release, but the cells eventually die like control cells (44, 45). Similarly, increasing the concentration of Ca2+ around the cells accelerates the rate of cell death without affecting the final percentage of dead cells (36). An intriguing question is: can CDC be blocked by intracellular chelation of the calcium ions? Intracellular Ca2+ chelation with BAPTA-AM was shown to efficiently block mitochondrial distress in human lung epithelial cells responding to a non-lytic dose of MAC, cells that do not undergo cell death (46). Furthermore, calcium ionophores that pump Ca 2+ into the cell induce in K562, human erythroleukemia cells, a state of resistance to CDC (47). Can BAPTA-AM block CDC when cells are exposed to lytic MAC doses? BAPTA-AM reduced the release of LDH from rat hepatocytes subjected to lytic antibody and complement by ∼40% without affecting the rate of cell death (48). Clearly, MAC activates a surge of [Ca2+]i in target cells but its exact impact on the process of cell death still awaits clarification. Furthermore, based on earlier findings, the involvement of calcium-independent processes in the critical events determining cell death cannot be ruled out.

## Beyond Calcium Ions: The Cell Death Propagators in a Regulated Necrotic Process

The molecular checkpoints that tilt the balance within MACbearing cells between a protective state and cell collapse have not yet been identified. It is well-accepted that exposure of nucleated cells to multiple ("lytic") MAC hits (34) is needed to overcome the cells' innate resistance (described below) and to kill the cells by necrotic-type cell death. Intensive research on apoptosis, and more recently on necroptosis induced by numerous effector molecules, has clearly revealed that compound regulated molecular processes accompany and/or lead to cell death (49–52). Those findings have prompted adopting a similar research approach in the analysis of the mechanism underlying CDC. Recently, MAC was shown to activate RIPK1, RIPK3, and MLKL, known transducers of necroptotic cell death activated by several exogenous ligands of TNF receptor, Fas, TLR, and other membrane receptors (53). Necroptotic cell death, also termed regulated necrosis, is characterized by increased membrane permeabilization and mitochondrial damage (49, 50, 54, 55), much like CDC. Inhibitors of RIPK1, RIPK3, and MLKL reduce the extent of CDC, whereas overexpression of these proteins enhances cell sensitivity to CDC (53). Two additional intracellular proteins that may play a role in the multi-factorial cell death process activated by lytic MAC are the c-Jun kinase JNK (56) and the BH3-only protein Bid (57). Apparently, in some cells, the RIP kinases, MLKL, JNK, and Bid, act as components in one or more lined cascades of intracellular molecular interactions activated by sublytic and lytic MAC concentrations (53, 57). At lytic MAC concentrations, this cascade may promote a regulated necrotic cell death (**Figure 2**). Blocking any of these five proteins markedly lowers the extent of CDC but does not block it completely. Therefore, it appears that this cascade acts in concert with other death-promoting processes, calcium-dependent or independent, which still await characterization. Of note, activated MLKL was shown in necroptotic cells to oligomerize at the plasma membrane, increase membrane permeabilization, and induce a Ca2+ influx (55, 58–60). Co-localization of MAC with MLKL at the plasma membrane (53) suggests that they may collaborate in mediating cell death. In general, cancer cells that express sufficient levels of the RIPKs, MLKL, and Bid might be sensitive to this necroptotic-like pathway once activated by MAC. In contrast, cancers that suppress the expression or function of any or all of these proteins are expected to be protected from this cytotoxic pathway even if triggered by MAC.

# Lytic MAC: Mitotoxicity and Metabolic Depletion

Mitochondria play a pivotal active role in activating the intrinsic pathway of apoptotic cell death, mostly after mitochondrial outer membrane permeabilization (61). Mitochondrial swelling and damage were observed in cells undergoing necrotic cell death induced by complement (62, 63). The cellular ATP level drops rapidly in cells attacked by MAC in copy numbers that are above the lytic threshold, apparently after mitochondrial dysfunction, accompanied by leakage of cytosolic ATP from the cells through pores in the plasma membrane (64–66). In theory, mitochondrial damage and cellular metabolic depletion beyond a point of "no return" may induce cell collapse and death. However, to date, there is no strong evidence supporting an active role for mitochondria in CDC. Perhaps, they are mere innocent bystanders damaged by the necrosis executioners? Reactive oxygen species are generated throughout the necrotic

FIGURE 2 | Schematic presentation of the cytotoxic pathways, induced in cancer cells by the complement C5b-9, the counteractive cellular resistance mechanisms, and postulated approaches to overcome this cancer evasion. Following the binding of antibodies to cancer cells, the complement system is activated and deposits C4b and C3b molecules that serve as initiators of C3/C5 convertase activation. The C5 convertases initiate the activation of the terminal complement pathway and the formation of the C5b-9 complexes (24). Upon insertion of the C5b-9 complexes into the plasma membrane of cancer cells, they induce calcium ion influx and activate pro- and anti-lytic signals. This scheme depicts the proteins proposed to be involved in the ensuing cancer cell death (encircled) and the proteins protecting the cancer cells from the lytic processes. Extracellular (gray boxes) and intracellular (purple boxes) protective proteins are indicated. Several reagents (white boxes) that will block the protective proteins are indicated and proposed for adjuvant therapy to therapeutic antibodies. Ab, antibody; Bcl-2, B-cell lymphoma/leukemia-2; BH3, Bcl-2 homolog domain-3; Bid, BH3 interacting domain death agonist; CK2, casein kinase 2; ERK, extracellular signal-regulated kinase; HSP90, heat shock protein 90; HSP70, heat shock protein 70; JNK, c-jun N-terminal kinase; MAC, complement membrane attack complex; MLKL, mixed lineage kinase domain-like protein; MMP, matrix metalloproteinase; PKC, protein kinase C; RIPK1, receptor-interacting protein kinase 1; RIPK3, receptor-interacting protein kinase 3; serine-, serine protease; siRNA, small interfering RNA.

process (15). Still, whether they take part in the MAC-induced cell death process is also an open question. The involvement of mitochondria and mitochondrial ROS in necroptosis triggered by various necroptosis inducers was extensively investigated in several types of target cells (67). Ample earlier findings have supported a pivotal role for mitochondria in necroptosis, but more recently, several investigations casted doubt on that notion (67). Thus, for example, mitochondrially deficient cells were shown to be responsive to TNF/zVAD treatment and to undergo necroptosis (68). It will be of interest to examine the relative sensitivity of these cells to CDC. At present, we can conclude that whether mitochondria are dispensable or essential for MACinduced necrotic cell death remains to be further investigated. Similar to necroptosis (67), it is likely that different cell types may mediate CDC by an array of distinct mitochondria-dependent and -independent strategies.

# NORMAL CELLS AND EVEN MORE SO, CANCER CELLS CAN RESIST MAC-INDUCED CELL DEATH

The fate of a target cell attacked by MAC is dictated by two mutually exclusive processes: (a) the rate and extent of the formation of the C5b-9 complexes and their insertion into the target cell membrane, and (b) the capacity of the target cell to block C5b-9 complex formation and to resist cell damage inflicted by C5b-9. Cancer cells can resist CDC by using a plethora of extracellular and intracellular mechanisms (**Figure 2**). The number of membrane-inserted C5b-9 complexes may be restricted by inhibiting the complement activation cascade earlier at the C3/C5 activation stage, by blocking complex assembly and/or membrane insertion, and by facilitating complex removal from the cell surface. All these protective strategies have been identified in cancer cells and are described below. It is generally accepted that cancer cells are more resistant to CDC than are normal cells due to their elevated expression of protective mechanisms. Apparently, during the tumorigenic process, the complement system reacts against the transformed cells by eliminating or modifying the complement-sensitive malignant cells, thus enriching the cancer cell population for complementresistant cells. This process resembles the selection of antibioticresistant bacteria (69). This hypothesis remains to be supported by in vivo evidence; however, in vitro studies show that sensitive cancer cells may be transformed into cells expressing increased complement resistance, transiently, after a brief treatment with a sublytic dose of MAC (70) and more stably, following several cycles of exposures to cytolytic MAC (71). A MAC-resistant phenotype may be acquired upon the elevation or reduction in the expression level of microRNAs such as miR-200, miR-217 (72), and others that are currently under investigation.

#### Basal Physiological Cell Resistance to CDC

As shown already in 1974 (73, 74), nucleated cells can resist MAC-induced damage. Several inhibitors of protein synthesis were shown to increase the cell's susceptibility to CDC. Since the elevated sensitivity to CDC was acquired hours after the complete shutdown of protein synthesis (75), it is likely that the treated cells became sensitive only after catabolism of longlived protective proteins. These earlier findings were followed by the development of the concept of multi-hit characteristics of nucleated cell death by MAC, implicating the cooperation of multiple MACs in cell death (34). Another interesting earlier finding was that damaged tumor and mast cells could be rescued by exogenous application of cAMP (76, 77). Consequently, activation of cAMP by sublytic MAC supports cell recovery from MAC damage (78). These findings were confirmed in leukemia cells treated with dibutyryl cAMP or with activators of intracellular cAMP (3-isobutyl 1-methyl xanthine and forskolin), which were shown to reduce cell death (79). In contrast, H-89, an inhibitor of the cAMP-dependent kinase PKA, enhanced carcinoma cell sensitivity to CDC. Apparently, phosphorylation events mediated by several protein kinases dictate the basal capacity of cells to resist MAC damage (79, 80). Protein phosphorylation events involving PKC, MEK, and ERK support the survival of cancer cells undergoing a complement attack (81– 83). Protein phosphorylation may upregulate the expression of the complement membrane regulators on cancer cells (84–86) and facilitate MAC elimination from K562 cells (87, 88). The transcription factor NF-κB also plays a role in cell protection from CDC (89). One of its postulated functions is upregulation of a protein phosphatase that inactivates JNK, thus reducing cell death signaling. However, further investigation is required to fully identify the pro-survival phosphoproteins and phosphatases and their precise mode of action.

Proteins of the heat shock protein family (HSPs), wellknown general house keepers, damage/ repair proteins and targets in cancer therapy (90–92), most probably also contribute to the basal resistance of cancer cells to CDC. Thus, far, a role for Hsc70/Hsp70 (93) and Hsp90 (94, 95) in cell protection from CDC has been shown. Pharmaceutical inhibitors of Hsp70 and Hsp90 sensitize cancer cells to CDC. Hsc70 relocates within minutes from the cytoplasm to the cell surface after exposing K562 cells to sublytic complement (93). Upon inhibition of Hsp90, Ramos cells become more sensitive to the action of Rituximab and complement (95). Additional thorough experimentation is required to fully comprehend how Hsc70/Hsp70, Hsp90, and other HSPs regulate CDC. The fact that Hsp90 can directly interact with C9 and that Hsp90 inhibitor enhances MAC deposition (95) suggest that Hsp90 down-regulates MAC deposition by blocking its assembly and/or facilitating its rate of removal from the cell surface. Hsp90 can potentially reduce CDC by suppressing mitochondria-initiated calcium-mediated stress responses (96).

### Anticomplementary Response on the Cancer Cell Surface

Like all normal cells, cancer cells are protected from autologous complement attack by several specific cell-surface complement inhibitors: CD55 (decay accelerating factor, DAF), CD46 (membrane cofactor protein, MCP), CD59, and CD35 (complement receptor type 1, CR1) (1, 97–99) (**Figure 1**). In addition, certain proteolytic enzymes, protein kinases, and sialic acid residues (described below) confer on the cells elevated resistance to CDC (100).

#### Membrane Complement Regulatory Proteins

Immunohistochemical analysis revealed the expression of CD59, CD55, and CD46 on uveal melanoma (101), thyroid carcinoma (102), lung and kidney cancer (103, 104), colon adenocarcinoma (105), and prostate cancer (106). This was supported by analysis of human tumor cell lines derived from human malignant gastrointestinal tumors (107), melanoma (108), breast cancer (109, 110), renal tumor (110), Burkitt lymphoma (111), neuroblastoma (112), and ovarian (113), and prostate carcinoma (79). In primary uterine cervix tissue, the expression of CD46, but not of CD55, was found to increase during transition from normal to premalignant to malignant cells (114). CR1/CD35 was identified in malignant endometrial tissue (115) and on leukemic blasts (116). Increased membrane regulator expression relative to the corresponding normal tissue has been reported in many tumors (103, 105, 114, 115, 117–122). Colorectal and gastric carcinomas and osteosarcoma have increased the expression of CD55 (123), whereas gastric carcinoma exhibits high levels of both CD55 and CD59 (124). Overexpression of CD59 was also identified by expression profiling for pancreatic cancer (125). Upregulation of membrane regulator expression on tumor cells is often correlated with increased complement resistance (86, 111). In ovarian cancer, resistance to complement correlated with high levels of CD55 expression (113). In melanoma cell lines with variable CD59 expression, resistance to death by anti-ganglioside antibody and homologous complement positively correlated with the expression level of CD59 (126).

Several clinical studies support a postulated function of membrane complement regulatory proteins (and thus, the extent of complement resistance) in cancer progression. Poorer prognosis in colorectal carcinoma correlates with the expression level of CD59 (127). Local tumor progression and tissue dedifferentiation of prostate cancer also correlate with CD59 expression (128). Analysis of 120 breast cancer patients revealed a worse prognosis associated with CD59 overexpression (129). In contrast, another report concluded that loss of CD59 correlated with poor survival in 520 breast cancer patients (130). In colorectal cancer patients, a 7-year survival was significantly reduced when the tumors expressed high levels of CD55 (131). CD55 overexpression was also reported as an independent risk factor for recurrence of breast cancer in patients receiving postoperative adjuvant therapy containing trastuzumab (132).

The expression level of the membrane complement regulators may also be shaped by cytokines, growth factors, or hormones, which are released into the tumor microenvironment (83, 133, 134). For example, TNFα and IL-1ß enhanced the expression of CD55 and CD59 in colon adenocarcinoma cells (135). TNFα, IL-1α, and INFγ enhanced CD55 expression in lung cancer cells (136). In hepatoma cells, TNFα, combined with IL-1ß and IL-6, enhanced CD55 and CD59 expression but decreased CD46 expression (134). Transcription abnormalities (137) and the microRNA level of expression (72) may also affect the expression level of the membrane complement regulators. Evidently, the factors and molecular mechanisms that determine the expression level of each of the membrane regulator proteins in vivo in each cancer type (and in normal cells) remain to be further investigated.

Exposure to chemotherapeutic drugs may also modify the level of the regulators′ expression. 5-azacytidine was shown to elevate the levels of CD55 and CD59 in Burkitt lymphoma cell lines (111) but only of CD59 in melanoma cells. In contrast, levamisole reduces CD59 levels in colon adenocarcinoma cell lines (138) and after pretreatment of breast carcinoma cells with tamoxifen, trastuzumab-induced CDC was enhanced due to CD55 down-regulation (132). Conversion of cancer cells from being drug-sensitive to drug-resistant is also associated with modification of their complement sensitivity. Doxorubicinresistant human colon carcinoma cells are more sensitive to CDC than are doxorubicin-sensitive cells (139). KB-V1, a multidrugresistant variant of KB-3-1, the human oral carcinoma cell line, exhibits a higher susceptibility to CDC than do its parental multidrug-sensitive cells (140). The increased complement sensitivity was associated with a reduced expression of CD55. Inversely, drug resistance was associated with CDC resistance in the HL60 myeloid leukemia cell lines (141). In ovarian carcinoma cells, drug resistance was associated with complement resistance and with membrane complement regulator overexpression (142). Hence, the impact of any drug on the expression of membrane regulators and on CDC resistance needs to be determined for each drug and cancer type.

Released or secreted membrane complement regulators in the cancer microenvironment may also support cell resistance to CDC. Soluble forms of membrane regulators have been identified in several body fluids, even under normal conditions. They are either produced by alternative splicing or released from the cell surface through enzymatic cleavage. Thus, sera of cancer patients contain active, soluble forms of CD46 (143). Elevated CD55 concentrations in stool specimens have been proposed to have diagnostic value for patients with colorectal cancer (144). A constitutive release of soluble CD59, which retains its activity as well as its GPI-anchor from human melanoma cells, was reported (145). In primary tumor sections, CD55 and/or CD59 were found in the stroma of breast, colorectal, lung, renal, and cervical carcinomas (103, 123, 146). In vitro, endothelial cells, HeLa cells (147) as well as osteosarcoma and colorectal cells (123, 148) release CD55 in a soluble form or deposit it into their extracellular matrix. K562 erythroleukemia cells (83) and breast, ovarian, and prostate carcinoma cell lines (79) secrete soluble CD59. Elevated plasma levels of soluble CR1 were found in leukemia patients (149).

The observed correlations between elevated expression or secretion of one or more of the membrane complement regulatory proteins on cancer cells and (a) enhanced resistance to CDC or (b) poor cancer prognosis, suggests that the membrane complement regulatory proteins have an effect on prognosis through their impact on complement resistance. Thus, by suppressing C3 deposition on the cancer cells, CD46 and CD55 can lower, on one hand, the extent of MAC generation and CDC, and on the other hand, reduce immune protection through complementdependent cellular cytotoxicity. CD59 can down-regulate MAC generation and CDC. However, a direct correlation between cancer patients' prognosis and the complement resistance level of their cancer, still remains to be established. We cannot rule out non-complement-mediated effects of the membrane complement regulatory proteins of cancer cells on the patients' immune response. Thus, the membrane complement regulatory proteins on cancer cells, through intracellular signaling, or cooperation with other cell surface receptors may potentially modulate cell resistance to immune effector cells such as natural killer cells and cytotoxic T lymphocytes (150, 151).

#### Membrane Surface Proteases, Protein Kinases, and Sialic Acid

Cancer cells become increasingly protected from CDC by expression on their cell surface of proteases that proteolytically degrade the deposited complement proteins (152). Thus, degradation of bound C3b by a C3-cleaving serine (153) or cysteine protease (154), respectively, was demonstrated on human and murine melanoma cells. C3-cleaving serine protease activity was also identified on the surface of U937 cells (155). Membrane serine proteases on K562 erythroleukemia cells also appear to contribute to their complement resistance (156). Matrix metalloproteinases (MMP) membrane type-1 (MT1) can cleave bound C3b off breast cancer cells and protect in vitro breast carcinoma and melanoma cells from CDC (157). Transfection of B16F1 melanoma cells with MT1-MMP enhanced their capacity to form lung metastases in normal but not in C3-deficient C57BL/6 mice (157). The effect of those proteases on proteins of the terminal complement pathway has not been tested. However, it is conceivable that these and other membrane proteases have a similar degradative impact on C5-C9. This still awaits determination.

Ecto-protein kinases (ecto-PK) are extracellular protein kinases that can phosphorylate both cell-surface and external proteins. Serine/threonine and tyrosine ecto-PKs were found on the surfaces of K562, U937, and HL-60 cells (158), and an ecto-casein kinase 2 (CK2)-like activity was associated with breast and ovarian carcinoma cells (159). C9 phosphorylation by ecto-CK2 was shown to be protective in K562 cells against CDC, possibly by inhibiting MAC formation or by leading to the production of an inactive or unstable MAC (160). Further investigation of this strategy of CDC evasion is warranted.

Brief treatment with sialidase, which removes sialic acid from the cell surface, has been shown to confer on several cell types increased sensitivity to CDC. Thus, removal of sialic acid from red blood cells (161, 162), murine sarcoma cells (163), and human bladder carcinoma cells (164) sensitized them to lysis by complement. Human prostate, breast, and ovarian carcinoma cells also utilize surface sialylation for protection from complement (79). High sialic acid expression correlates with lower complement activation, probably because of inactivation of C3b by factors H and I, which is more efficient on surfaces rich in sialic acid (162). The sialic acid inhibitory activity on CDC of mouse erythrolukemia MEL cells is apparently abrogated by O-acetylation at its 9-hydroxyl group (165). α2- 6 hypersialylation apparently lowers the response of CLL cells to Rituximab therapy through its action on complement (166). Thus, by limiting the extent of C3 deposition, sialic acid may also control the assembly of C5b-9 complexes on the cancer cells.

#### Soluble Complement Regulators in the Cancer Microenvironment

Soluble complement inhibitors such as C1 Inhibitor, factor H, and factor I are predominantly synthesized by hepatocytes and macrophages but can also be released from other tissues, although in considerably smaller amounts. In the cancer microenvironment, these secreted inhibitors may contribute to protection of cancer cells from complement attack by blocking complement activation at the C1 and C3 activation steps (99). In support of this, a growing number of reports indicate that cancer cells of various origins secrete one or more complement inhibitor. Synthesis of C1 Inhibitor has been described in astroglioma and neuroblastoma (112), breast cancer cell lines, and in a primary ovarian carcinoma cell line (156). Factor H is expressed both in lung adenocarcinoma and cutaneous squamous cell carcinoma (167, 168) and high levels of factor H and factor H-like protein-1 were shown to be secreted by ovarian tumor cells (83, 169). Additionally, factor H was found to be elevated in bronchoalveolar lavage fluids and the sputum of patients with lung cancer (170). Chronic lymphocytic leukemia (CLL) cells that bind factor H to their surface resisted Rituximabmediated CDC (171). Factor H was coexpressed with factor I in glioma and rhabdomyosarcoma cells in its plasma form and in a truncated form (172). Tumor-associated factor I is postulated to promote the progression of cutaneous squamous cell carcinoma (173) and positively correlates with poor survival and recurrence of breast cancer (174).

# Active Removal of the Membrane-Inserted MAC