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The omics era made possible the quest for efficient markers for cancer progression and revealed that macrophage populations are much more complex than just the M1/M2 dichotomy. Complement C1q pops up as a marker of a tolerogenic and immunosuppressive macrophage populations in both healthy and tumor tissues, but the specific role of C1q+ tumor-associated macrophages (TAM) is poorly understood. C1q is co-expressed in healthy and tumor macrophages with human leukocyte antigen DR (HLA-DR), Apolipoprotein E (APOE), and mannose receptor C-type 1 (MRC1) (CD206), suggesting a resident origin of this population. TAM expressing C1q correlate with T cell exhaustion and poor prognosis in numerous cancers. Herein, we discuss the plural roles of C1q in these macrophages and how it could drive cancer progression.

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The complement system plays a complex role in cancer. In clear cell renal cell carcinoma (ccRCC), local production of complement proteins drives tumor progression, but the mechanisms by which they do this are poorly understood. We found that complement activation, as reflected by high plasma C4d or as C4d deposits at the tumor site, was associated with poor prognosis in two cohorts of patients with ccRCC. High expression of the C4-activating enzyme C1s by tumor cells was associated with poor prognosis in three cohorts. Multivariate Cox analysis revealed that the prognostic value of C1s was independent from complement deposits, suggesting the possibility of complement cascade–unrelated, protumoral functions for C1s. Silencing of C1s in cancer cell lines resulted in decreased proliferation and viability of the cells and in increased activation of T cells in in vitro cocultures. Tumors expressing high levels of C1s showed high infiltration of macrophages and T cells. Modification of the tumor cell phenotype and T-cell activation were independent of extracellular C1s levels, suggesting that C1s was acting in an intracellular, noncanonical manner. In conclusion, our data point to C1s playing a dual role in promoting ccRCC progression by triggering complement activation and by modulating the tumor cell phenotype and tumor microenvironment in a complement cascade–independent, noncanonical manner. Overexpression of C1s by tumor cells could be a new escape mechanism to promote tumor progression.See related Spotlight by Magrini and Garlanda, p. 855.See article by Daugan et al., p. 909 (40).

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SARS-CoV-2 infects cells by attachment to its receptor – the angiotensin converting enzyme 2 (ACE2). Regardless of the wealth of structural data, little is known about the physicochemical mechanism of interactions of the viral spike (S) protein with ACE2 and how this mechanism has evolved during the pandemic. Here, we applied experimental and computational approaches to characterize the molecular interaction of S proteins from SARS-CoV-2 variants of concern (VOC). Data on kinetics, activation- and equilibrium thermodynamics of binding of the receptor binding domain (RBD) from VOC with ACE2 as well as data from computational protein electrostatics revealed a profound remodeling of the physicochemical characteristics of the interaction during the evolution. Thus, as compared to RBDs from Wuhan strain and other VOC, Omicron RBD presented as a unique protein in terms of conformational dynamics and types of non-covalent forces driving the complex formation with ACE2. Viral evolution resulted in a restriction of the RBD structural dynamics, and a shift to a major role of polar forces for ACE2 binding. Further, we investigated how the reshaping of the physicochemical characteristics of interaction affect the binding specificity of S proteins. Data from various binding assays revealed that SARS-CoV-2 Wuhan and Omicron RBDs manifest capacity for promiscuous recognition of unrelated human proteins, but they harbor distinct reactivity patterns. These findings might contribute for mechanistic understanding of the viral tropism, and capacity to evade immune responses during evolution.

International audience; C3 is the central effector molecule of the complement system, mediating its multiple functions through different binding sites and their corresponding receptors. We will introduce the C3 forms (native C3, C3 [H2 O], and intracellular C3), the C3 fragments C3a, C3b, iC3b, and C3dg/C3d, and the C3 expression sites. To highlight the important role that C3 plays in human biological processes, we will give an overview of the diseases linked to C3 deficiency and to uncontrolled C3 activation. Next, we will present a structural description of C3 activation and of the C3 fragments generated by complement regulation. We will proceed by describing the C3a interaction with the anaphylatoxin receptor, followed by the interactions of opsonins (C3b, iC3b, and C3dg/C3d) with complement receptors, divided into two groups: receptors bearing complement regulatory functions and the effector receptors without complement regulatory activity. We outline the molecular architecture of the receptors, their binding sites on the C3 activation fragments, the cells expressing them, the diversity of their functions, and recent advances. With this review, we aim to give an up-to-date analysis of the processes triggered by C3 activation fragments on different cell types in health and disease contexts.

The complement system plays a complex role in cancer. In clear cell renal cell carcinoma (ccRCC), local production of complement proteins drives tumor progression, but the mechanisms by which they do this are poorly understood. We found that complement activation, as reflected by high plasma C4d or as C4d deposits at the tumor site, was associated with poor prognosis in two cohorts of patients with ccRCC. High expression of the C4-activating enzyme C1s by tumor cells was associated with poor prognosis in three cohorts. Multivariate Cox analysis revealed that the prognostic value of C1s was independent from complement deposits, suggesting the possibility of complement cascade–unrelated, protumoral functions for C1s. Silencing of C1s in cancer cell lines resulted in decreased proliferation and viability of the cells and in increased activation of T cells in in vitro cocultures. Tumors expressing high levels of C1s showed high infiltration of macrophages and T cells. Modification of the tumor cell phenotype and T-cell activation were independent of extracellular C1s levels, suggesting that C1s was acting in an intracellular, noncanonical manner. In conclusion, our data point to C1s playing a dual role in promoting ccRCC progression by triggering complement activation and by modulating the tumor cell phenotype and tumor microenvironment in a complement cascade–independent, noncanonical manner. Overexpression of C1s by tumor cells could be a new escape mechanism to promote tumor progression. See related Spotlight by Magrini and Garlanda, p. 855. See article by Daugan et al., p. 909 (40).

Tumors contain a complement rich microenvironment in which all cell types (e.g., tumor cells and stromal cells) are able to produce different proteins. We developed immunohistochemistry (IHC) assays allowing to identify on paraffin embedded tumor sections, not only the complement producing cells but also the complement activation fragments which result from activation of complement cascade within the tumor. The local production of complement can be detected by cytoplasmic staining, whereas the activation fragments are localized at the surface of the cells. There is a high heterogeneity of the staining within tumors but also between patients. Semi-quantification of the staining in large cohorts of patients allows to investigate the prognostic impact of the local complement production and activation. Here we explain the staining process for C1q, C4, and C3 in human paraffin-embedded tumor sections by immunofluorescence and immunohistochemistry.

Heme (Fe protoporphyrin IX) serves as a prosthetic group of numerous proteins implicated in oxidative metabolism. This molecule is abundantly present in the red blood cells where it serves as a cofactor of hemoglobin. As consequence of various pathological conditions, the membrane of red blood cells can be damaged and therefore large quantities of hemoglobin and subsequently heme released in the extracellular space. Since heme is a highly reactive compound, when released extracelluarly it can influence the functional activity of different plasma components. Thus, previous investigations have demonstrated that heme can interact with components of complement system and immunoglobulins, profoundly affecting their functions. Here we propose two basic protocols that can be used for characterization of interaction of free heme with complement proteins and immunoglobulins. The first technique is based on UV-Vis absorbance spectroscopy. It allows general characterization of the heme binding to the protein and estimation of the number of heme binding sites. The second protocol consists in the use of biosensor assay based on surface plasmon resonance. This protocol would be useful for evaluation of heme binding kinetics and equilibrium affinity. Besides for complement components and immunoglobulins, the presented protocols can be utilized for characterization of the interaction of heme with other proteins.

Heme (Fe protoporphyrin IX) serves as a prosthetic group of numerous proteins implicated in oxidative metabolism. This molecule is abundantly present in the red blood cells where it serves as a cofactor of hemoglobin. As consequence of various pathological conditions, the membrane of red blood cells can be damaged and therefore large quantities of hemoglobin and subsequently heme released in the extracellular space. Since heme is a highly reactive compound, when released extracelluarly it can influence the functional activity of different plasma components. Thus, previous investigations have demonstrated that heme can interact with components of complement system and immunoglobulins, profoundly affecting their functions. Here we propose two basic protocols that can be used for characterization of interaction of free heme with complement proteins and immunoglobulins. The first technique is based on UV-Vis absorbance spectroscopy. It allows general characterization of the heme binding to the protein and estimation of the number of heme binding sites. The second protocol consists in the use of biosensor assay based on surface plasmon resonance. This protocol would be useful for evaluation of heme binding kinetics and equilibrium affinity. Besides for complement components and immunoglobulins, the presented protocols can be utilized for characterization of the interaction of heme with other proteins.

Tumors contain a complement rich microenvironment in which all cell types (e.g., tumor cells and stromal cells) are able to produce different proteins. We developed immunohistochemistry (IHC) assays allowing to identify on paraffin embedded tumor sections, not only the complement producing cells but also the complement activation fragments which result from activation of complement cascade within the tumor. The local production of complement can be detected by cytoplasmic staining, whereas the activation fragments are localized at the surface of the cells. There is a high heterogeneity of the staining within tumors but also between patients. Semi-quantification of the staining in large cohorts of patients allows to investigate the prognostic impact of the local complement production and activation. Here we explain the staining process for C1q, C4, and C3 in human paraffin-embedded tumor sections by immunofluorescence and immunohistochemistry.

The complement system is a powerful and druggable innate immune component of the tumor microenvironment. Nevertheless, it is challenging to elucidate the exact mechanisms by which complement affects tumor growth. In this study, we examined the processes by which the master complement regulator factor H (FH) affects clear cell renal cell carcinoma (ccRCC) and lung cancer, two cancers in which complement overactivation predicts poor prognosis. FH was present in two distinct cellular compartments: the membranous (mb-FH) and intracellular (int-FH) compartments. Int-FH resided in lysosomes and colocalized with C3. In ccRCC and lung adenocarcinoma, FH exerted protumoral action through an intracellular, noncanonical mechanism. FH silencing in ccRCC cell lines resulted in decreased proliferation, due to cell-cycle arrest and increased mortality, and this was associated with increased p53 phosphorylation and NFκB translocation to the nucleus. Moreover, the migration of the FH-silenced cells was reduced, likely due to altered morphology. These effects were cell type–specific because no modifications occurred upon CFH silencing in other FH-expressing cells tested: tubular cells (from which ccRCC originates), endothelial cells (human umbilical vein endothelial cells), and squamous cell lung cancer cells. Consistent with this, in ccRCC and lung adenocarcinoma, but not in lung squamous cell carcinoma, int-FH conferred poor prognosis in patient cohorts. Mb-FH performed its canonical function of complement regulation but had no impact on tumor cell phenotype or patient survival. The discovery of intracellular functions for FH redefines the role of the protein in tumor progression and its use as a prognostic biomarker or potential therapeutic target. See article by Daugan et al., p. 891 (36).

International audience; Properdin (FP) is a positive regulator of the immune system stimulating the activity of the proteolytically active C3 convertase C3bBb in the alternative pathway of the complement system. Here we present two crystal structures of FP and two structures of convertase bound FP. A structural core formed by three thrombospondin repeats (TSRs) and a TB domain harbors the convertase binding site in FP that mainly interacts with C3b. Stabilization of the interaction between the C3b C-terminus and the MIDAS bound Mg2+ in the Bb protease by FP TSR5 is proposed to underlie FP convertase stabilization. Intermolecular contacts between FP and the convertase subunits suggested by the structure were confirmed by binding experiments. FP is shown to inhibit C3b degradation by FI due to a direct competition for a common binding site on C3b. FP oligomers are held together by two sets of intermolecular contacts, where the first is formed by the TB domain from one FP molecule and TSR4 from another. The second and largest interface is formed by TSR1 and TSR6 from the same two FP molecules. Flexibility at four hinges between thrombospondin repeats is suggested to enable the oligomeric, polydisperse, and extended architecture of FP. Our structures rationalize the effects of mutations associated with FP deficiencies and provide a structural basis for the analysis of FP function in convertases and its possible role in pattern recognition.

The 54 kDa protein properdin, also known as factor P (FP), plays a major role in the complement system through the stabilization of the alternative pathway convertases. FP circulates in the blood as cyclic dimers, trimers and tetramers, and this heterogeneity challenges detailed structural insight into the mechanism of convertase stabilization by FP. Here, the generation of an intact FP monomer and a variant monomer with the third thrombospondin repeat liberated is described. Both FP monomers were excised from recombinant full-length FP containing internal cleavage sites for TEV protease. These FP monomers could be crystallized, and complete data sets extending to 2.8 Å resolution for the intact FP monomer and to 3.5 Å resolution for the truncated variant were collected. The principle of specific monomer excision and domain removal by the insertion of a protease cleavage site may be broadly applicable to structural studies of oligomeric, flexible and modular proteins.