Complement factor H-deficient mice develop spontaneous hepatic tumors.

1Department of Medicine, Nephrology and Hypertension, University of Colorado School of Medicine, Aurora, CO, 80045, USA. 2Centre for Inflammatory Disease, Department of Medicine, Division of Immunology and Inflammation, Imperial College of London, W12 ONN, London, United Kingdom. 3Department of Medicine, Radiology, Radiation Oncology, and Anesthesiology, University of Colorado School of Medicine, Aurora, CO, 80045, USA. 4Department of Anesthesiology, University of Colorado School of Medicine, Aurora, CO 80045, USA. PMS-J current correspondence: 118 Brook Road, Port Jefferson, NY, 11777, USA.


Introduction
Key functions of the immune system are the ability to discriminate self from non-self, and to rapidly eliminate invasive pathogens while causing minimal injury to the host. Rapid induction of inflammation is critical for an efficient immune response and its rapid resolution prevents unintended damage. If left unresolved, however, inflammation can damage surrounding tissues, eventually leading to fibrosis, organ failure, and carcinogenesis (1,2). The complement cascade is an important arm of the immune system and is controlled through a balance of activating and regulatory proteins (3). The classical and mannose binding lectin pathways are activated by antibodies and other pattern recognition molecules (4). The alternative pathway (AP), however, is distinct in that it is continually activated in plasma through a process called "tick-over," indiscriminately depositing activated C3b on nearby surfaces. The deposited C3b catalyzes further AP activation, self-amplifying unless adequately controlled on the target surface by complement regulatory proteins (CRPs). CRPs selectively protect host cells but not invasive pathogens, thereby creating a system of rapid and continuous immune surveillance.
CRPs prevent autologous complement-mediated injury by cleaving and inactivating C3b (a component of the C3 and C5 convertases), and/or accelerating the decay of existing C3 and C5 convertases (3). Because the AP is constitutively activated, inadequate regulation can lead to complement-mediated injury. Congenital and acquired defects in complement regulation are associated with severe diseases, including atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G), and protein-losing enteropathy (5)(6)(7). Genetic variants of AP proteins are also associated with accelerated progression of some chronic diseases, including age related macular degeneration (AMD) and IgA nephropathy (8,9). Complement activation has been linked with the development and spread of several cancers (10)(11)(12), raising the possibility that impaired complement regulation could be a risk factor for some types of cancer.
Factor H (CFH) is a soluble CRP that regulates AP activation in plasma and on host surfaces through its ability to bind to self-surface ligands. Regions within the CFH protein bind to anionic molecules, including glycosaminoglycans (GAGs), which are highly expressed on basement membranes and extracellular matrix (ECM), and sialic acid, a common component of cellular membranes (13,14). Engagement of these molecules tethers CFH to host tissues, controlling AP activation at those sites (15). Although sulfated GAGs and sialic acids are ubiquitously expressed, CFH binding and regulatory activity are particularly important for specific tissues (16). For example, CFH is critical for controlling AP activation on ECM and basement membranes, as these surfaces do not intrinsically express other CRPs. Mutations in CFH binding regions are primarily associated with diseases of the kidneys and eyes (8,17), possibly because these organs have specialized basement membranes that are highly exposed to plasma proteins.
While studying complement-mediated kidney disease, we observed spontaneous liver tumor development in fH -/male mice at a significantly higher rate than control mice. We hypothesized that CFH deficiency causes complement-mediated liver inflammation. Chronic inflammation can promote development of HCC (18), and complement activation can also impair the elimination of tumors by the immune system (19). Consequently, inadequate complement regulation within the liver could contribute to carcinogenesis by several mechanisms. CFH is primarily generated in the liver, as are factor B (CFB) and C3 (activating components of the AP) (20,21). Proteins produced by hepatocytes enter the circulation by passage through the ECM-rich space of Disse and fenestrated endothelial cells lining the sinusoids, continually exposing these regions to high concentrations of complement proteins.
The mechanisms of complement activation and regulation within the liver are incompletely understood and a direct connection between CFH dysfunction and liver disease, to our knowledge, has not been previously described.
In the current study, we examined the role of CFH in regulating complement activation in the liver, and the extent to which complement activation is associated with hepatocellular injury.
We examined the link between local complement activation in fH -/mice with changes in the hepatic immune milieu. Finally, we analyzed biopsies and clinical data from patients with HCC to look for evidence of complement dysregulation in human HCC.

Mice lacking complement factor H spontaneously develop hepatocellular carcinoma
While studying aged fH -/mice, we noticed a high incidence of spontaneous liver tumor development, which was especially pronounced in males ( Figures 1A and 1B) compared to WT mice ( Figure 1C). In male mice 15 months and older (≥ 15 months), we observed grossly visible liver tumors in 54% of fH -/-(n=35) compared to 10% in fH -/-fB -/-(which are unable to activate the AP; n=10) and 7% in WT (n=29). The rate of liver tumor incidence was reduced in female mice ≥ 15 months, with 15% incidence in fH -/-(n=26), 3% in WT (n=29), and 0% in fH -/-fB -/-(n=10) mice ( Figure 1D). Histological evaluation of livers from WT and fH -/mice from 3 different age groups (≤ 5 months, 6-14 months, and ≥15 months) revealed notable differences between the two strains. Hepatocyte ballooning (an indicator of hepatocyte stress or injury) was present in fH -/males at all ages ( Figure 1E), with multiple instances per field of view observed in over half of the ≥15-month males, compared to 14% of WT males of the same age. In male mice ≥15-months of age, multi-focal tumor rosettes or nests were seen in all fH -/mice with visible liver tumors ( Figure 1F), and in 50% of those without. Generally normal liver parenchyma was observed in WT males of the same age ( Figure 1G).
Glypican-3 (GPC3) is a heparan sulfate proteoglycan that is overexpressed in HCC and has been used as a tumor biomarker (22,23). Variation in the location of GPC3 expression has been noted in human HCC, with localization occurring on cell membranes in some and in the cell cytoplasm of other tumors (24). As further support that tumors in fH -/mice are HCC, we stained livers collected from animals with and without visible tumors for GPC3 expression. We observed two distinct GPC3 expression patterns in the fH -/mice, which correlated with the macroscopic findings. Livers from fH -/mice with large focal tumors surrounded by otherwise normal appearing parenchyma ( Figure 1A), expressed GPC3 in a continuous, membranous pattern ( Figure 1H). In contrast, GPC3 was predominantly cytoplasmic ( Figure 1I) in fH -/livers with multi-focal tumors extending throughout the parenchyma of each lobe ( Figure 1B, yellow arrows). Only membranous GPC3 deposition was seen in WT livers with tumors ( Figure 1J).

C3 fragments are deposited throughout the livers of fH -/mice
During AP activation, intact fluid-phase C3 is cleaved to form C3b, which covalently attaches to nearby tissues. C3b is subsequently cleaved, generating iC3b and C3d. We immunostained liver and kidney sections from 3-5-month-old WT, fH -/-, CFH heterozygotes (fH +/-), and fH -/-fB -/mice to evaluate hepatic C3 fragment deposition. In WT and fH +/mice, we found only trace amounts of punctate C3b and iC3b/C3d in the liver sinusoids (Figures 2A and Supplementary Figure S1A, respectively). In fH -/mice, however, C3b and iC3b/C3d were deposited extensively throughout the sinusoids ( Figure 2B). C3b-iC3b/C3d deposition was linear and continuous within the sinusoidal spaces, and appeared to deposit in a gradient, with concentrated C3 in the perivenous regions adjacent to the central vein, becoming more diffuse in the periportal regions. C3b deposits colocalized with collagen IV (COL4) [a component of the basement membrane (25)] and adjacent to MECA-32 (an endothelial cell marker), further supporting that complement was activated within the sinusoidal wall (Supplementary Figures   S2A and S2B). Deficiency of both CFH and CFB completely abrogated C3 deposition in livers of fH -/-fB -/mice ( Figure 2C). fH -/mice are most frequently used to study C3G which is characterized by C3 deposition localized in the glomeruli ( Figure 2E), a location known to be particularly vulnerable to AP activation in the absence of CFH. C3b-iC3b/C3d deposition for each strain has been previously described in detail (26), and representative staining is shown ( Figures 2D-2F and Supplementary Figure S1A). These data demonstrate that, like the glomeruli, hepatic C3 deposition in fH -/mice is largely due to AP activation.
CFH is a systemic regulator; therefore, fH -/mice could also be susceptible to AP activation in other organs. To determine other possible locations of AP activation in fH -/mice, we performed whole animal PET imaging to detect localized iodinated (I 124 )-C3d29 (monoclonal antibody specific for the complement fragments iC3b and C3d) (27). 124 I-C3d29 localization was only seen in the livers and kidneys of fH -/mice (non-specific signal in thyroid and bladder is likely due to uptake or clearance of free iodine) ( Figure 2I). Bound 124 I-C3d29 was quantified (28) 96 hours after imaging ( Figure 2H), which confirmed the liver and kidneys are the predominant sites of C3 deposition in fH -/mice.

AP activation leads to hepatocellular injury in fH -/mice
To assess the pathological effects of unregulated AP activation in the liver, we measured levels of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and albumin in 3-month old WT, fH -/-, and fH -/-fB -/mice. ALT and AST were significantly higher in fH -/mice than in WT or fH -/-fB -/mice ( Figure 3A), confirming hepatocellular injury in fH -/mice. ALP and albumin were not different between the strains, indicating that the injury had not caused biliary obstruction or impaired synthetic function of the liver at this timepoint (29).
We stained liver sections from fH -/and WT males at 3, 9, and 18 months of age with Oil Red O, a lysochrome used to detect lipid. Lipid accumulation was mild and limited to the perivenous hepatocytes of 3-month-old fH -/mice but became progressively more widespread with age ( Figure 3B, right panels). Positive staining was seen in the 9-and 18-month-old WT mice ( Figure 3B, left panels), but when quantified, was found to be significantly less than in agematched fH -/mice ( Figure 3C). We next immunostained livers from 14-month old fH -/and WT mice for COL4, a structural component of basement membranes, that when deposited in excess, leads to the development of fibrosis (30). There was greater COL4 deposition in fH -/mice compared to WT ( Figure 3E), occurring in a continuous pattern in the sinusoids of fH -/mice ( Figure 3D, bottom).
Only punctate deposits were seen in age-matched WT livers ( Figure 3D, top). This suggests that the chronic inflammatory environment triggered by uncontrolled AP activation in fH -/livers contributes to the development of steatosis and fibrosis as the mice age.

Hepatic mRNA expression reveals a strong inflammatory signature in fH -/mice
We examined the mRNA expression profile of myeloid innate immune markers in the livers of 3-month-old fH -/mice compared to WT. When comparing the most differentially expressed (DE) mRNA to WT controls, fH -/livers had greater than 4.65-fold higher mRNA expression of S100a9 and S100a8, the pro-inflammatory chemokine Cxcl14, and serum amyloid A 1 (Saa1) (Figures 4A and 4B). Of note, S100A9 is expressed on monocytes and neutrophils (31) and has been implicated in the development of cancer (32,33) and tumor metastasis (34).
Pathway analysis revealed an increase in fH -/liver mRNA expression in 16 of the 17 gene sets relevant to innate myeloid immune function ( Figure 4C). Directed global significance (GS) scores were used to identify the most DE gene sets in fH -/mice as a group. Three of the gene sets with high directed GS scores (indicating high expression compared to WT covariates), were the chemokine, cytokine, and Fc receptor signaling pathways. Composite scores for these three pathways ( Figures 4C and 4D-4F), and the most DE genes corresponding to each of the three pathways with relative expression frequencies ( Figures 4G-4I) are shown. These data provide transcript level evidence of a pro-inflammatory environment in the livers of young fH -/mice.

T-cell infiltration and reduced neutrophils in fH -/livers
To evaluate the immune milieu within the livers of fH -/mice, we stained liver sections for six general leukocyte markers (F4/80, CD8, CD4, CD3, B220, and Foxp3) and collected images with a Vectra quantitative pathology imaging system. Dense clusters of immune cells were observed in all fH -/livers near portal veins ( Figure 5A, left and Figure 5B). Similar immune clusters were not seen in WT livers ( Figure 5A, right). Significantly more F4/80 + and CD8 + T cells were seen in fH -/livers ( Figure 5C), as well as a trend towards greater numbers of CD4 + , CD3 + , and CD4 + Foxp3 + cells.
We assessed hepatic neutrophil populations in young fH -/-, WT, and fH -/-fB -/males by flow cytometry. There was greater than a 50% reduction in neutrophils (CD45 + CD11b + Ly6G + ) in fH -/livers compared to those from WT and fH -/-fB -/-( Figure 5D, left and middle). Because neutrophil infiltration within tumors may influence tumor growth (37), we assessed livers with and without tumors from 22-24-month-old fH -/mice. There was a trend towards a greater number of neutrophils in livers with tumors than in those without ( Figure 5D, right). We stained fH -/liver sections containing tumor foci for neutrophils (Ly6G + cells). Neutrophils were seen within and around tumors ( Figure 5E, arrows), providing evidence of neutrophil infiltration in fH -/liver tumors.

Skewed myeloid cell populations in fH -/livers
Complement fragments are chemoattractants for infiltrating monocytes, which have been shown to play an important role in shaping the tumor immune microenvironment (38). Previous studies have shown that MHC-II + monocytes can inhibit tumor growth (39), whereas MHC-IImonocytes, can be pro-angiogenic, suppress T cell function, and can promote tumor growth (39,40). To examine whether the monocyte population is altered in livers of fH -/mice, we isolated and analyzed hepatic myeloid cells from young fH -/-, WT, and fH -/-fB -/males, as well as from aged fH -/males with and without liver tumors. Compared to WT, fH -/mice had fewer Ly6C high MHC-II -F4/80 low/pro-inflammatory infiltrating monocytes (41) and significantly more Ly6C -MHC-II -F4/80 low/anti-inflammatory patrolling monocytes and Ly6C int MHC-II -F4/80 -( Figure   6A). Only patrolling monocytes (Ly6C -MHC-II -F4/80 low/-) were increased in fH -/livers when compared to fH -/-fB -/mice ( Figure 6B). There were no significant differences in the monocyte populations between aged fH -/mice with liver tumors and those without. It is notable, however, that the Ly6C high MHC-II -F4/80 low/infiltrating monocytes are the most numerous of these cell types in the aged fH -/livers, regardless of tumor status ( Figure 6C), in contrast to what was seen in young fH -/mice.

Recombinant murine factor H binds within the liver sinusoids and inactivates C3b
Given the pattern of C3 fragment deposition in fH -/livers, we examined whether CFH controls AP activation on basement membrane within the sinusoids and hepatic vasculature. We produced and functionally tested a recombinant murine CFH (rmCFH) (42), which was then conjugated with Alexa-647 and injected into male fH -/and WT mice. The mice were euthanized 24 hours later, and livers were examined for bound rmCFH. The rmCFH deposited along the sinusoids of fH -/livers ( Figure 7A left, 7C, and 7D) in a pattern much like that of C3b deposition in unmanipulated fH -/mice ( Figure 2B). We observed a similar pattern of rmCFH deposition in WT sinusoids ( Figure 7A, right), although in significantly lower amounts ( Figure 7B).
To assess the functionality of the rmCFH bound within the sinusoids, we stained liver tissues from the rmCFH-injected fH -/mice for C3b and iC3b/C3d and evaluated the sinusoids for differences in complement deposition ( Figures 7C-7E). In rmCFH-reconstituted fH -/mice, we observed continuous deposition of iC3b/C3d in the sinusoids ( Figures 7C and 7E) and a shift in C3b deposition from linear to punctate ( Figures 7D and 7E). There was colocalization of rmCFH with iC3b/C3d ( Figure 7C), and only sparse colocalization with C3b ( Figure 7D). We quantified C3b and iC3b/C3d deposition in the reconstituted fH -/with that of unmanipulated fH -/-males of the same age. In reconstituted mice, there was a significant decrease in mean percent area C3b deposition (2% compared to 7% in unmanipulated mice) and a two-fold increase in iC3b/C3d (11% compared to 5% in unmanipulated mice) ( Figure 7F). These data demonstrate that the injected rmCFH mediated cleavage of bound C3b in the sinusoids, which generated local iC3b/C3d. Finally, to further characterize the specific location of rmCFH deposition in the sinusoids, we stained liver tissues from rmCFH-injected mice for COL4 and MECA-32. Similar to localization of C3b in the sinusoids, we observed regions of colocalization of rmCFH with both COL4 ( Figure 7G) and MECA-32 ( Figure 7H), indicating that rmCFH binds ECM and endothelial cells within the sinusoids and is critical for controlling alternative pathway activation at these sites.

Evidence of C3 inactivation within fH -/liver tumors
To examine complement regulation within liver tumors, we dual stained livers from tumor bearing mice for C3b and iC3b/C3d. In WT livers, we observed small tumor foci with heavy deposits of both C3b and iC3b/C3d, surrounded by normal appearing parenchyma with very little C3 deposition ( Figure 8A, left panels). In contrast, fH -/tumors and surrounding parenchymal tissue displayed extensive C3b-iC3b/C3d deposition ( Figure 8A, right panels).
There were distinct regions within some of the fH -/tumors with minimal C3b and abundant iC3b/C3d deposition, suggesting that there was cofactor activity and inactivation of C3b within the tumor environment. To examine this further, we dual stained tumor tissues with GPC3 (to indicate tumor boundary) and either C3b or iC3b/C3d ( Figures 8B and 8C). In tumors with membranous GPC3, we observed both C3b and iC3b/C3d within the tumor lesion ( Figure 8B).
However, in tissues with cytoplasmic GPC3, C3b deposits were less apparent suggesting better complement regulation within these tumors ( Figure 8C).
The presence of iC3b/C3d within fH -/liver tumors indicates that another protein serves as a cofactor for cleavage of C3b in the absence of CFH. Studies of human HCC have shown that tumors can upregulate membrane bound complement regulators, such as CD46 (membrane cofactor protein or MCP) (43,44). Similar to CFH, CD46 is a cofactor for the cleavage of C3b, although it is reported to have limited expression in healthy mice (45). We stained fH -/liver tumors for CD46 and C3b and found prominent CD46 expression in some regions where C3b was absent ( Figure 8D, white box). We also dual stained fH -/liver tumors for CD46 and iC3b/C3d. There were regions of high CD46 expression in which iC3b/C3d was not seen, as well as regions in which CD46 and iC3b/C3d colocalized. These results suggest that expression of CD46 within the HCC lesions may inhibit alternative pathway activation and it may also serve as a cofactor for the cleavage of cleavage of C3b.

C3 fragments are deposited in human HCC biopsies
We immunostained liver biopsy samples from six confirmed HCC patients for C3d/iC3b and GPC3 ( Figure 9A), five of which were strongly positive C3d/iC3b. Among these five samples, there was colocalization of GPC3 and C3d/iC3b, indicating that there is complement activation within the tumors.
We next queried the TCGA through cBioPortal for Cancer Genomics (46,47) to determine if CFH mutations or mRNA expression correlate with outcomes for patients with HCC. We found that CFH is altered in 23% (86 of 337) of the patients comprising this data set.
Of those 86 patients, 50 had increased tumor CFH mRNA expression, seven had missense or truncating mutations, and 29 had an amplification in CFH copy number (Supplementary Figure   S4A). Increased expression of CFH is expected to help control complement activation, and we found that high CFH mRNA correlated with improved disease/progression-free survival as well as overall survival when compared to HCC patients with unaltered CFH mRNA ( Figure 9B).
The median disease/progression-free time for patients with high CFH mRNA was 55.06 months compared to 18.43 months for those with unaltered CFH mRNA expression. Conversely, CFH mutations are expected to increase complement activation in the liver and HCC tumors. Patients with CFH mutations had significantly worse overall survival, with a median disease/progression free time of only 9.72 months compared to 20.99 months for those without CFH mutations ( Figure 9C). These findings demonstrate that the complement cascade is activated in the livers of patients with HCC, and that expression levels of CFH mRNA as well as mutations in the CFH gene significantly affect the prognosis for HCC patients.
To determine if these survival and prognostic outcomes relating to CFH mutations and increased CFH mRNA are unique to HCC, we queried CFH for all cancers in the TCGA Firehose Legacy study (Supplementary Table S1). We found that most cancer types could be grouped according to three categories: 1) worse outcome with CFH mutations, improved outcome with increased CFH mRNA, 2) worse outcomes for both CFH mutations and/or increased CFH mRNA, and 3) improved outcomes for both. We found three cancers for which the correlations were similar to HCC (worse outcomes with CFH mutations, improved outcomes with increased CFH mRNA): lymphoid neoplasm diffuse large B-cell lymphoma, lung adenocarcinoma, and esophageal carcinoma.

Discussion
Specific locations such as the eyes and kidneys, are known to be particularly dependent on CFH to control the AP. These sites are rich in ECM, have specialized basement membranes and are susceptible to AP-mediated inflammation in patients with CFH mutations. In the current study, we report that CFH is also critical for controlling AP activation in the liver. There is extensive complement activation throughout the sinusoids of fH -/mice, which is ameliorated through combined deficiency of CFH and CFB, confirming a principal role of the AP. AP activation in male fH -/mice was associated with hepatocellular inflammation and injury at 3 months of age, chronic liver damage and steatosis at 9 months, and the development of liver tumors for more than 50% of those aged to ≥15 months. This suggests that the hepatic inflammation resulting from chronic AP activation promotes a favorable environment for tumorigenesis.
C3b and iC3b/C3d were deposited in a continuous pattern along the sinusoids within the livers of fH -/mice. In mice reconstituted with rmCFH, the injected protein colocalized with COL4 ( Figure 7G) and MECA-32 ( Figure 7H) within the sinusoids and mediated cleavage of the deposited C3b to inactive iC3b/C3d. We developed a whole-body PET method to identify other sites of AP activation in fH -/mice and confirmed that the liver and kidneys are the primary sites of complement activation in the absence of CFH.
There is substantial experimental and clinical research linking genetic defects with CFH in kidney disease. Much less is known, however, regarding the role of CFH in liver disease.
Although aHUS is primarily regarded as a kidney disease, it is noteworthy that abnormal liver function tests may be seen in up to 46% of patients (48). It has also been reported that complement-mediated injury can lead to hepatic failure in aHUS patients undergoing liver transplantation (49). The presumed mechanism for this is inadequately controlled AP activation in the ischemic liver allograft, resulting from dysfunctional endogenous CFH. It is possible that the liver is a target organ in patients with aHUS associated CFH mutations but is overlooked due to the severe renal involvement. It is also possible that the binding mechanism of CFH to liver sinusoids and glomerular capillaries involves slightly different interactions. This is the case for AMD and aHUS, in which distinct genetic variants of CFH impair binding of the protein to either the eyes or the kidneys, thereby predisposing patients to one of these diseases but not the other (50).
CFH mutations have not previously been linked with the risk for HCC, although it has been reported that complement activation fragments are elevated in affected patients (51)(52)(53). In one study, complement fragments were the only independent plasma biomarker predictive of HCC by multivariate analysis (53). In the current study, we found evidence of complement activation in biopsy samples from six patients with HCC. This demonstrates that complement activation can occur within the tumors, similar to observations in mice. Furthermore, examination of the TCGA reveals that expression of CFH mRNA significantly correlates with survival in patients with HCC ( Figure 9B). Although fH -/mice have complete deficiency of CFH, the TCGA data suggests that variations in CFH function and expression levels may also influence the prognosis of HCC. Future studies can examine whether CFH influences the growth of tumors by inhibiting complement within the tumor itself or through interactions in the tumor microenvironment.
It has been estimated that chronic liver inflammation may account for as much as 90% of HCCs, although the mechanisms linking inflammation with carcinogenesis are incompletely understood (54). Multiple studies have also reported that the complement cascade is activated in the livers of patients with chronic hepatitis B (55,56), hepatitis C (51,52,56), and alcoholic hepatitis (57), inflammatory diseases strongly associated with an increased risk of HCC. Based on the results of the current study, it is possible that inflammation resulting from uncontrolled complement activation within the liver is a common downstream pathway that links these chronic liver diseases with carcinogenesis. The heterogeneous pattern of complement activation within the tumors may be due to upregulation of CD46. It is also possible that additional factors promote complement activation once a cancer has formed. Antibodies may recognize tumor neoantigens, for example. Some tumor cells also produce enzymes that are capable of directly activating C3, such as cathepsins B and L (58).
Complement activation within the liver may contribute to the development of HCC by several mechanisms. Complement activation has been shown to trigger NF-kB activation in Kupffer cells and STAT3 activation in hepatocytes (59), which may facilitate recovery of the liver after acute injury, but chronically promote hepatocyte proliferation and the development of HCC (18,60,61). Complement mediated inflammation in the livers of fH -/mice could also promote DNA damage, increasing the rate of cancer formation. Complement activation has also been shown to inhibit anti-tumor immunity in multiple types of cancer (10-12, 62, 63). For example, work has shown that C3a and C5a can induce suppressive myeloid cells or directly inhibit T cell proliferation and function (11,63,64). We found that the myeloid cell population is altered in the livers of fH -/mice, and a greater number of Ly6C int MHC-IImonocytes were present within the livers of these mice ( Figures 6A and 6B). These cells can be immunosuppressive and may reduce anti-tumor immunity in fH -/livers. Thus, complement activation within the liver may both directly accelerate the development of HCC, as well as impair the ability of the immune system to eliminate these tumors once they have formed.
A limitation of our study is that it uses mice that are genetically deficient in CFH from birth. Many different signaling pathways are activated in these mice, and the development of chronic liver damage and HCC takes a relatively long time. An advantage of this model, however, is that it is characterized by spontaneous tumor formation. Most cancer models in immunocompetent mice involve genetic manipulation of tumor suppressor genes, chemical carcinogenesis, or transplant of cancer cells into recipient mice. Furthermore, in tumor transplant models, the cell lines are often derived from advanced stage tumors (65). These tumors therefore start with a high mutational burden and have already undergone immune selection. The fH -/mice, in contrast, provide the opportunity to study spontaneous HCCs as they develop.
In conclusion, we have found that CFH is necessary for controlling spontaneous AP activation in the liver. In the absence of CFH, dysregulated AP activation is associated with hepatocellular injury, steatosis, and development of HCC. We found evidence of complement activation in human HCC biopsies, and that CFH mutations and levels of CFH mRNA correlate with patient survival. There is clinical evidence that the complement system is activated in multiple chronic inflammatory diseases associated with increased risk of HCC. Complement activation may, therefore, be a common pathway linking viral, toxic, or autoimmune injury of the liver with carcinogenesis. Several therapeutic complement inhibitors are currently in development (66). Given the potential role of complement activation in liver carcinogenesis identified in this study, complement inhibition may represent a novel approach for treating HCC.

Materials and Methods
Antibodies, equipment, and software. Manufacture/supplier, catalog number, clone and working concentration for all antibodies is provided in Supplementary Table S2. The mouse IgG2 isotype antibody was generated and validated using previously described methods (27). Specific details regarding equipment and software are provided in Supplementary Tables S3 and S4, respectively.
Study Design. Spontaneous hepatic tumor development was observational; therefore, group sizes were dependent on the number of animals reaching advanced age (≥ 15 months). Ethical and humane endpoints were rigorously followed. All other group sizes were determined based on previous experience (minimal phenotypic variation) and the minimum number of animals required to provide reliable statistical results. Endpoints, when applicable, were determined in advance and were based on age or a time (hours following administration of recombinant protein or imaging reagent). No data have been excluded. A minimum of three independent experiments with successful replication were performed for all studies when possible (except in cases of limited resources). All reported microscopy studies were successfully replicated in at least five independent experiments with varying conditions and equipment. Additionally, microscopy data were collected, background adjusted, converted to binary format, and evaluated in a blinded manner. All data are reported as biological replicates.
Human HCC liver tissues were provided as deidentified, pre-sectioned slides by the University of Colorado Biorepository Core facility and the University of Colorado Cancer Center Tissue Biobanking and Histology Shared Resource. The following BRISQ Tier 1 criteria were provided: Solid tissue was removed from the livers of living patients with confirmed HCC diagnosis by surgical/clinical procedure. Both clinical and pathology diagnosis of HCC were confirmed for each sample. Resected tissues were stabilized and fixed in 10% neutral buffered formalin prior to long-term preservation in paraffin-embedded blocks. Preserved tissues were shipped and stored at ambient temperature; storage duration ranges from 10-20 years. HCC liver samples provided for this study were selected based on >20% tumor composition by the University of Colorado Biorepository Core.
Mice. Male and female C57Bl/6J wild type mice were purchased from the Jackson Laboratory.
The fH -/mice were created and initially provided by MCP (26). Generation of fB -/mice has been previously described (67). Generation of fH -/-fB -/mice was achieved through interbreeding of fH -/and fB -/mice. All mice were bred for at least 7 generations prior to experimentation. Non-specific binding and endogenous Fc receptors were blocked with 1% BSA and 5% heatinactivated goat/rat/rabbit serum diluted in PBS for one hour at RT. Antibodies were diluted in PBS containing 2% heat-inactivated fetal bovine serum (HI-FBS) and 1% BSA, applied to blocked tissue sections, and incubated overnight at 4°C in a humidified chamber. Nuclei were stained with DAPI, washed in cold PBS, and mounted with a 1:1 solution of PBS and glycerol.
Kidney autofluorescence was blocked with 0.05% Sudan Black B in 70% ethanol for 15 minutes at RT, washed twice in deionized water (10 minutes each), and mounted as described above.
Slides were sealed and imaged in a blinded fashion with either an Olympus FV1000 FCS confocal (at either x100, x200, or x600 original magnification) or a Zeiss Axio Observer D1 epifluorescent microscope (x100, x200, or x400 magnification). A minimum of ten fields of view (FOV) were captured per sample. Images were converted from binary data format with either Olympus FV-10ASW (version 04.02.02.09) or Zeiss Zen Blue (version 2.6) software.
Representative isotype images are shown in Supplementary Figure S1B-S1H. Image quantification was performed according to methods described in Oil Red O staining.  Table S5) using the GeNorm algorithm (68) within the Advanced Analysis software. The resulting data set used for analysis consisted of 414 genes. DE and pathway analyses were determined by specifying WT as the baseline covariate. The pathways or gene sets with the greatest differential expression were identified by directed global significance scoring, followed by analysis with composite pathway scoring. The composite pathway scoring algorithm used in nanoString advanced analysis is calculated as the first principal component of the pathway genes' normalized expression and has been described in detail (69).
Multiplex immunofluorescence of hepatic immune populations. Three-micrometer-thick, FFPE liver sections from 3-month old fH -/and WT male mice were processed and stained using the Leica Bond-III Fully Automated IHC Autostainer. Leica ER2-pH 9 antigen retrieval buffer was used for CD3ε, FoxP3, CD4, and CD8 antibodies; Dako-pH 6 was used for the F4/80 antibody; and Leica ER1-pH 6 was used for the B220 (CD45R) antibody. Primary antibodies were visualized using Opal dyes. Stained slides were scanned at low magnification with the Vectra 3.  Immunofluorescent staining of human HCC tissues. Three-micrometer-thick, de-identified FFPE sections from 6 HCC patients were subject to standard deparaffinization and rehydration methods (xylene followed by ethanol gradient). Following 2 washes in deionized water, slides were incubated in antigen retrieval citrate buffer, pH 6 at 96°C for 30 minutes, allowed to cool to RT for 20 minutes, and washed with running deionized water. Tissues were blocked with 5% HI-FBS and 1% BSA in PBS for one hour at RT and washed once with cold PBS. Primary antibodies were diluted in 2% HI-FBS and 1% BSA in PBS, applied to blocked tissues and left to incubate overnight at 4°C in a humidified chamber. Tissues were washed thrice with cold PBS prior to incubation with secondary antibody for one hour at RT, followed by a short incubation with DAPI diluted in PBS and two PBS washes. Slides were dried, mounted with 1:1 PBS and glycerol, sealed, and viewed with an Olympus FV1000 FCS confocal microscope. Images were collected at x100, x200, and x600 original magnification. Isotype images are shown (Supplementary Figure S1B-S1D).