Sera from Phylogenetically Related Alligators, Crocodiles and Domestic Chickens Exhibit Comparable Anti-Cancer Activity

Abstract

Background: Crocodilians rarely develop cancer despite long lifespans and continuous exposure to environmental carcinogens, suggesting robust natural anti-tumor defense mechanisms. Methods: We investigated the anti-cancer activity of sera derived from the phylogenetically related species—alligators, crocodiles, and chickens, and studied their underlying immune mechanisms. The anti-tumor activity of alligator serum was tested in murine models of melanoma and lymphoma. Results: Alligator serum (AS) and its (NH₄)₂SO₄-precipitated fraction (ASa) showed rapid and potent cytotoxicity toward multiple murine and human cancer cell lines while sparing non-malignant human cells. Importantly, ASa attenuated melanoma and lymphoma tumor growth in mice. Electrophysiological analyses in PN71 cancer cells treated with ASa revealed rapid membrane depolarization and formation of high-conductance pores consistent with Complement-mediated membrane attack complex (MAC) activity. Proteomic analyses identified the Complement component C5 as a major protein enriched in active fractions, implicating the Complement system in cancer cell killing. Based on phylogenetic similarity of C5, crocodile and chicken sera exhibit alligator-like comparable anti-cancer activity. Mechanistic studies in chicken serum showed that the anti-cancer activity depends on Ca²⁺ and Mg²⁺ ions, terminal Complement components (C5–C8), and IgM antibodies that initiate Complement activation. Immunodepletion of IgM from CSa significantly reduced cytotoxicity, whereas purified chicken IgM activated human Complement to induce cancer cell death. Conclusions: These findings identify a conserved IgM–Complement immune mechanism capable of selectively targeting malignant cells. The evolutionary conservation and cross-species functionality of this pathway highlight its potential as a bio-inspired strategy for developing novel Complement-based cancer immunotherapies.

1. Introduction

A well-known observation is that crocodiles and alligators have been suggested to exhibit a relatively low incidence of cancer based on limited observational reports, offering an opportunity to investigate resistance mechanisms for novel therapies. However, it is important to note that systematic epidemiological data in these species are lacking, and therefore, this notion should be interpreted with caution. Specifically, it remains unclear how crocodiles can live for up to a century without developing malignancies despite constant exposure to harmful factors such as pathogens, decaying food, heavy metals, and radiation, all known carcinogenic factors in humans. Despite these exposures, several reports have proposed that crocodilians may possess robust innate defense mechanisms that contribute to their resilience; however, these observations remain largely descriptive and hypothesis-generating rather than evidence from controlled studies [1,2,3].

Biologically derived molecules have shed light on natural tumor-suppressive mechanisms and have provided a foundation for anti-cancer drug discovery, either as direct therapeutics, as scaffolds for semisynthetic optimization, or as leads that illuminate new mechanistic pathways. A complementary and increasingly active area is the exploration of serum-derived proteins and peptides as sources of cytotoxic or immunomodulatory activities relevant to cancer biology. Indeed, growing evidence supports the central role of biologically derived molecules in shaping modern oncology, both as therapeutic leads and as conceptual frameworks for understanding tumor vulnerability [4]. In addition, serum contains complex mixtures of proteins that participate in host defense, including Complement components, pattern recognition molecules, antimicrobial/host defense peptides, protease inhibitors, and carrier proteins [5,6]. The same biochemical features that enable these molecules to recognize and damage microbial membranes, opsonize targets, or trigger inflammatory cascades may also confer activity against malignant cells.

Crocodilians are of particular interest as sources of potent serum defense activities. Early work demonstrated broad antibacterial activity in American alligator (Alligator mississippiensis) serum compared with human serum and showed that heat treatment under conditions commonly used to inactivate Complement activity abolished the antimicrobial effect, supporting an important role for active serum Complement and/or Complement-associated proteins [2,7,8,9]. More recently, reports testing crocodile sera and organ lysates have described measurable cytotoxicity and growth inhibition using multiple cancer cell lines, with evidence that proteinaceous factors contribute to this activity (e.g., reduced effect following heat treatment or protease digestion in some systems). For example, Siddiqui and colleagues [2] reported that serum and selected organ lysates from Crocodylus palustris triggered substantial prostate cancer cell death in vitro. Similarly, Jeyamogan et al. [10] described the anti-tumor activities of selected pure compounds identified from the serum of Crocodylus porosus, Malayopython reticulatus, Varanus salvator and Cuora kamaroma amboinensis. In addition, crocodile blood extracts were reported to induce apoptosis in A549 lung cancer cells, providing further rationale that crocodilian-blood-derived factors can affect tumor cell survival pathways. Collectively, these and other studies support the broader concept that crocodilian sera contain bioactive molecules with potential relevance to anti-cancer modalities discovery, while also emphasizing the importance of systematic evaluation across species, experimental conditions and non-malignant cells’ controls.

Building on prior observations of crocodilian serum bioactivity and the known abundance of innate and adaptive immune effectors in avian serum, we tested the hypothesis that sera from these phylogenetically close species contain distinct protein/peptide factors capable of reducing cancer cell viability. Specifically, our work aims to decipher whether anti-cancer effects caused by proteinaceous innate immune components, such as Complement activators, provide a foundation for downstream fractionation and identification of the bioactive anti-cancer molecules.

2. Materials and Methods

2.1. Preparation of Alligator, Crocodile and Chicken Sera and 45% (NH₄)₂SO₄ Fractionation

Crocodile and alligator blood samples were drawn from animals residing in farms in Israel (Crocoloco and Hamat Gader) for routine health monitoring, under the veterinary supervision of the animal farms. Chicken blood was collected immediately upon slaughter (25 mL/animal) for commercial purposes at the Milouot Corporation for the Development of Haifa Bay Settlements Ltd. (https://www.milouot.co.il/). Blood samples were stored on ice for 2 h, clots were then punctured, and samples were further spun 2–3 times at 1000 g for 20 min at 4 °C. Supernatants were collected as a clear serum source, aliquoted to 20 mL stocks and stored at −80 °C. A fully saturated (NH₄)₂SO₄ solution kept at 4 °C, featuring the maximal amount of dissolved (NH₄)₂SO₄ (100%) was used to provide a constant dilution of the (NH₄)₂SO₄ salt used in successive processing of different serum sources. Accordingly, the appropriate volume of this saturated (NH₄)₂SO₄ solution was added to the final dilution of 45% salt. After 30 min on ice, the mixture was spun for 15 min at 13,000 rpm and 4 °C. The sediment was subsequently resuspended in 20 mM HEPES + 150 mM NaCl and fractionated again in 45% (NH₄)₂SO₄. The final sediment was collected in half of the original volume of HEPES/NaCl buffer. The precipitate (denoted as ASa) fractions were dialyzed against 1000 volumes of the same HEPES/NaCl buffer for 3 × 1 h in 6.4 cm dialysis membrane tubing (MWCO 12–14 K, Spectrum Medical Industries Inc., Laguna Hills, CA, USA), having a cutoff of 12,000–14,000 Daltons. Finally, ASa fractions were tested for their anti-cancer activity.

2.2. Recording Membrane Potential and Membrane Current from PN71 Cancer Cells

Membrane potential and membrane current were recorded from PN71 cancer cells placed over Matrigel-coated glass coverslips. Following plating, a recovery period of at least two days was allowed before performing electrophysiological experiments. The experiments were conducted in external Tyrode’s solution containing (in mM) 140 NaCl, 5.4 KCl, 1 MgCl₂, 2 sodium pyruvate, 1 CaCl₂, 10 HEPES, and 10 glucose (pH 7.4 adjusted with NaOH). Axopatch 200B, Digidata 1322, and pClamp10 (Molecular Devices, Sunnyvale, CA, USA) were used for data amplification, acquisition and analysis. Signals were digitized at 10 kHz and filtered at 2 kHz. Patch pipettes with resistances of 4–7 MΩ were pulled from borosilicate glass capillaries (Harvard Apparatus, Holliston, MA, USA). Analysis was performed using MATLAB 2016b software (MathWorks, Natick, MA, USA). The patch pipette solution contained (mM): 120 KCl, 1 MgCl₂, 3 Mg-ATP, 10 HEPES, and 10 EGTA titrated to pH 7.2 with KOH and adjusted to 290 mOsm with saccharose.

2.3. Generation of iPSC-CMs

In the current study, we used the previously described human-induced Pluripotent-Stem-Cell-derived Cardiomyocyte (iPSC-CM) clone FSE [11]. iPSC differentiation into cardiomyocytes was performed according to a previously described directed differentiation by modulating Wnt/β-catenin signaling protocol [12,13]. iPSCs were cultured on Matrigel (GFR, BD Biosciences, Franklin Lakes, NJ, USA)-coated 6-well plates in mTeSR1 medium (Stemcell Technologies, Vancouver, BC, Canada) for 5–6 days. To initiate differentiation, cells were incubated with 1 mL/well Versene solution (Invitrogen, Life Technologies, Woburn, MA, USA) at 37 °C for 5 min and seeded on Matrigel-coated 12-well plates in mTeSR1 medium. The medium was replaced daily, and after 2 days, when the monolayer of cells reached 100% confluence, the medium was changed to RPMI supplemented with B27 minus insulin (Invitrogen, Life Technologies, Woburn, MA, USA) containing 10 µM CHIR99021; this day was counted as day 1 of differentiation. On the next day (day 2 of differentiation), the medium was changed to RPMI supplemented with B27 minus insulin. On day 4, the medium was changed to RPMI supplemented with B27 minus insulin, containing 10 µM of IWP-4. On day 6, the medium was changed to RPMI supplemented with B27 minus insulin. Finally, from day 8 onwards, the medium was supplemented with RPMI with B27 complete supplement (Invitrogen, Life Technologies, Woburn, MA, USA). A total of no less than ten different differentiation protocols were utilized throughout this study. To enrich cardiomyocyte fraction in the culture, a MACS magnetic labeling-based cell sorting kit was used (130-110-188, Miltenyi Biotec, Bergisch Gladbach, Germany). To generate beating monolayers, 25–50 K enriched cardiomyocytes were seeded in a ~3 mm diameter circle on glass coverslips, formed by a 40 µL Matrigel droplet.

2.4. Monitoring Cell Viability

2.4.1. MTT Assay

Cancer and normal cells were plated in a 96-well plate (30,000 cells/well) in triplicate and placed in an incubator to adhere. ASa was applied at 1 mg/mL for 18 h, and subsequently, the MTT colorimetric assay determining cell viability was performed by administration of 25 µL of a ready-to-use solution of the MTT reagent (Sigma, St. Louis, MI, USA; M2128) for 2 h at 37 °C. Next, 100 µL/well of dissolving buffer was administered (20 g of SDS dissolved in 100 mL 50% DMF/DDW, and adjusted to pH = 4.7) for an additional 2 h of incubation at 37 °C. The developed color was measured by an ELISA plate reader at 570 nm. The obtained values were processed by blank subtraction, dividing the OD value of each treatment by that of the untreated cells, and finally multiplying by 100 to express cell viability in % change.

2.4.2. [H³]-Thymidine Incorporation

To determine the effects of the anti-cancer fractions on the proliferation activity, cells were incubated for the last 3 h in the presence of 1 mCi/mL [methyl-H³]-thymidine. Cells were washed with PBS and methanol to increase membrane permeability. Soluble thymidine was washed out by rinsing with 1% TCA (tri-chloro-acetic-acid) 3 × 5 min. Cells were then washed with DDW and dissolved with 0.3 M NaOH for 15 min. The level of cell proliferation was expressed in relation to the level of the Control FBS-treated cells.

2.5. Purification/Enrichment of Anti-Cancer Fractions (Methods for Table 2)

Enrichment for serum proteins responsible for the anti-cancer effect was carried out by running independent chromatographic steps using an ÄKTA™ Avant chromatography system (28976337, Cytiva, Malborough, MA, USA) equipped with a Superdex 200 Increase Size Exclusion Chromatography (SEC) 3.2/300 column (28990946, Cytiva), and a RESOURCE™ S Cation Exchange Chromatography (CEX) column (17118001, Cytiva) run either independently or as a stepwise purification process starting with the CEX step followed by the SEC step. Accordingly, the CEX step was processed with Buffer A (HEPES 50 mM pH = 7.4) and Buffer B (HEPES 50 mM pH = 7.4 supplemented with 3 M NaCl). The SEC step was processed with an eluant solution consisting of HEPES 50 mM pH = 7.4 supplemented with 150 mM NaCl. All purification steps were carried out with either pre-fractionated sera ((NH₄)₂SO₄-precipitated) or outcomes of the CEX step once the stepwise purification scheme was implemented. All the resulting protein fractions were tested individually for their anti-cancer effect.

2.6. Assessing the Abundance of C5 Complement System Protein by Mass Spectrometry (MS)

Neighboring fractions (eluted successively) featuring anti-cancer effect (measured by the MTT protocol) were pooled and further subjected to MS analyses at The Smoler Proteomics Center, the Technion-Israel Institute of Technology, Haifa, Israel. All masses obtained were crossed with crocodilian proteins enlisted in the UniProt protein database.

Table 1. Selected peptides, 10–30 amino acids in length, of relevant chicken Complement proteins used to immunize rabbits for raising polyclonal antibodies.

Protein Name	Epitopes Selected
Complement C5	GGKCSEVIRTKRSDFEEQVLKE, RDDGYRGEDGGPLGRLEACAKYRPSRREP
Complement C6	RTVRRNETRKSYRVPAN, PINGGKPCKGEREEEED
Complement C7	EEDGTDEDQCE, NSGHSYSEKKNEQQSR
Complement C8α	LLVILDPVPVGLT, KEEMRRKDIS
Complement C8β	RRRQCNNPTPQNGGS, RKPYNVESYTPETKGKY
Complement C8γ	SLGELVGRWFL

, summarizing the interpretation of all peptides revealed, points to 15 common peptides that appeared in all independent fractionations executed, of which 14 peptides were attributed to crocodile proteins, whereas 12 peptides were annotated to the Complement C5 protein (A0A1U8DWU5 at UniProt).

2.7. Generation of Customized Polyclonal Antibodies Raised Against Chicken C5-C8 Proteins (Methods for Figure 5)

A series of epitopes attributed to chicken Complement proteins was selected by the “Kyte & Doolittle” and the “Hopp & Woods” algorithms, available at https://web.expasy.org/protscale/. These epitopes were used for raising a series of polyclonal antibodies applied to the immuno-depletion studies. Accordingly, peptides were synthesized and further conjugated to KLH at Sigma-Aldrich, Rehovot, Israel. Subsequently, the resulting KLH-conjugated peptides were used to immunize rabbits (2 rabbits/conjugate), and final bleedings were subjected to affinity purifications. Affinity-purified antibodies were employed for the immuno-depletion protocol. The epitopes selected for the chicken Complement C5–C8 proteins are listed in Table 1.

3. Results

3.1. The Anti-Cancer Effect of Alligator Serum

3.1.1. Alligator Serum Kills Mouse and Human Cancer Cell Lines

The fundamental finding of this study is that Alligator mississippiensis serum (AS) kills a broad range of mouse cancer cells. Figure 1A shows the dose-dependent anti-cancer effect measured by the MTT assay (see Materials and Methods for details) for PN71 mouse myeloma cancer cells following 2 h incubation with AS. Increasing AS protein concentration from 0.05 to 5.0 mg/mL reduced cell viability (e.g., fewer live cells stained in black), resulting in complete cell death. This specificity of AS is demonstrated by the observation that, whereas it kills the murine cancer cell lines PN71 and EL4 (a murine T lymphoblast originated from a lymphoma induced in a C57BL mouse by 9,10-dimethyl-1,2-benzanthracene), fetal calf, rabbit, and horse sera are ineffective (Figure 1B). In these experiments, the anti-cancer effect is determined by the ability of AS to inhibit DNA synthesis, as determined by [³H]-Thymidine incorporation into DNA. To partially purify the active anti-cancer component in AS, we performed a calibration protocol aiming at determining the minimal (NH₄)₂SO₄ concentration retaining the anti-cancer activity upon precipitation. As we found that 45% (NH₄)₂SO₄-precipitated serum (ASa) and crude serum (AS) present identical anti-cancer effects, all subsequent experiments were performed using ASa, which rapidly kills PN71 cancer cells (Figure 1C). ASa also kills the human cancer cell lines (Figure 1D) U937 (histiocytic lymphoma), T47D (breast carcinoma) and HeLa (cervical carcinoma), but does not harm human-induced Pluripotent-Stem-Cell-derived Cardiomyocytes (iPSC-CMs) (Figure 1E). The larger sample size for the alligator serum (AS) group reflects the central focus of the study, which was to rigorously characterize its anti-cancer activity across multiple independent preparations and experiments. In contrast, rabbit and horse sera were included as negative biological controls to demonstrate lack of AS cytotoxicity.

As the first step in testing the potential translational anti-cancer applications of ASa, in preliminary experiments we tested the ability of ASa to attenuate melanoma and lymphoma in murine models. Notably, these exploratory experiments were intended as proof-of-concept (POC) preliminary studies, rather than definitive efficacy studies. Murine melanoma was induced by intracutaneous injections of murine B16 (melanoma-derived) cells into the back skin of C57/bl mice; see details in the Supplementary Materials and Methods and Figure S1. Briefly, the morphological appearance of the melanoma shows large, damaged areas in the ASa-treated melanoma, compared to the well-preserved FCS-injected tumor (Figure S1A). As shown in Figure S1B, on day 8 of ASa treatment, it decreased the melanoma tumor volume by ~50% compared to the control and FCS-injected tumors. Next, murine syngeneic peritoneal lymphoma model was generated by injecting 0.5 × 10⁶ EL4 cells intraperitoneally (i.p.) into inbred strain C57Bl/6 mice; see details in Materials and Methods in the Supplementary Materials and Methods and Figure S2. As shown in Figure S2, 4 days after ASa injection, the EL4 cell count decreased by ~80% compared to the cell count in the FCS-injected lymphoma mouse.

3.1.2. The Involvement of the Complement System in the Anti-Cancer Effect of ASa

Previous studies have shown that the membrane attack complex (MAC) of the Complement system causes formation of ion-conducting channels of high conductance in the order of pico-Siemens (pS) [14,15,16]. To test whether the Complement system is involved in ASa-dependent anti-cancer activity, we recorded from ASa-treated PN71 cancer cells the membrane potential (V_M) and membrane current (I_M) using the patch clamp technique. As illustrated in Figure 2A, whereas application of fetal calf serum (FCS) to the recording bath did not affect V_M, a highly diluted ASa caused, within a few minutes, V_M depolarization towards 0 mV, culminating in cell death. Strong evidence for Complement involvement (i.e., MAC) in ASa anti-cancer effects is the induction of perforin-like mega-channels in PN71 cancer cells (Figure 2B). Opening these high-conductance channels rapidly enables large ion influx/efflux, causing cell demise. In support of the notion that the Complement system is involved in the anti-cancer effect of alligator serum, the C5 protein, an important component of the Complement system, was found: (i) in all serum-derived protein fractions resolved via different chromatographic separations (e.g., gel filtration, ion exchange chromatography, hydrophobic interaction; Table 2) enriching for the anti-cancer effect; (ii) C5 was the most abundant Complement component protein identified by MS (more than 100 different peptides collectively covering the entire protein length); and (iii) incubating ASa at 55 °C treatment for 20 min abolished anti-cancer activity.

Table 2. Complement C5 is involved in the anti-cancer activity of crocodilian sera. Pre-fractionation of crocodilian sera by (NH₄)₂SO₄ precipitation protocol was followed by different biochemical purification steps, collectively aimed at enriching for the proteins governing anti-cancer activity. All the resulting protein fractions were tested for their anti-cancer effect against PN71 cells (MTT assay). The neighboring fractions (eluted successively) featuring anti-cancer effects were first pooled and further subjected to mass spec analyses. Eventually, 15 common masses were retrieved in all the purification outcomes exhibiting the anti-cancer effect (red): 14 masses out of those were attributed to specific crocodile proteins, whereas 12 peptides were annotated to the Complement C5 protein (A0A1U8DWU5 at UniProt).

Serum Injected	Biochemical Approaches Aimed at Enriching for the Cell Killing Activity	No. of Peptides Detected in Each Pooled Protein Fractions Featuring the Cell Killing Activity	Common Peptides Detected in All the Protein Fractions	The Protein Identity of the Common Peptides Revealed
Alligator mississippiensis	Affinity Chromatography step executed with a column packed with an immobilized antibody blocking the cell killing activity	1103
	A Size-Exclusion Chromatography (Superdex 200 column)	2370	236
Crocodylus niloticus	A Cation Exchange column followed by a Size-Exclusion step (Superdex 200)	1389	38
	A Size-Exclusion Chromatography (Superdex 200)	2546	15	12 peptides were annotated to the Complement C5 protein

Table 2. Complement C5 is involved in the anti-cancer activity of crocodilian sera. Pre-fractionation of crocodilian sera by (NH₄)₂SO₄ precipitation protocol was followed by different biochemical purification steps, collectively aimed at enriching for the proteins governing anti-cancer activity. All the resulting protein fractions were tested for their anti-cancer effect against PN71 cells (MTT assay). The neighboring fractions (eluted successively) featuring anti-cancer effects were first pooled and further subjected to mass spec analyses. Eventually, 15 common masses were retrieved in all the purification outcomes exhibiting the anti-cancer effect (red): 14 masses out of those were attributed to specific crocodile proteins, whereas 12 peptides were annotated to the Complement C5 protein (A0A1U8DWU5 at UniProt).

Serum Injected	Biochemical Approaches Aimed at Enriching for the Cell Killing Activity	No. of Peptides Detected in Each Pooled Protein Fractions Featuring the Cell Killing Activity	Common Peptides Detected in All the Protein Fractions	The Protein Identity of the Common Peptides Revealed
Alligator mississippiensis	Affinity Chromatography step executed with a column packed with an immobilized antibody blocking the cell killing activity	1103
	A Size-Exclusion Chromatography (Superdex 200 column)	2370	236
Crocodylus niloticus	A Cation Exchange column followed by a Size-Exclusion step (Superdex 200)	1389	38
	A Size-Exclusion Chromatography (Superdex 200)	2546	15	12 peptides were annotated to the Complement C5 protein

3.2. Sera of Crocodiles and Chickens Also Display Anti-Cancer Effect

Based on our findings in alligator serum, we tested the hypothesis that phylogenetically related species also display similar anti-cancer activity. To accomplish this goal, we performed a phylogenetic analysis aimed at identifying species with the closest C5 protein sequence to that of alligators (Figure 3A). Indeed, the first support for this hypothesis emerges from our findings showing that serum from crocodiles, whose C5 sequence is closely related to that of alligators, exhibits alligator-like anti-cancer activity and kills RPMI 8226 cancer cells within 2 h (Figure 3B). Because alligators and crocodiles are protected species, based on the prominent relatedness of the alligator, crocodile and chicken (Gallus gallus) C5 sequence (Figure 3A), we tested whether the chicken serum (CS) exhibits alligator-like anti-cancer activity. Indeed, in support of the C5 hypothesis, CS kills RPMI 8226 cancer cells within 2 h (Figure 3B). Next, we found that the 45% (NH₄)₂SO₄-precipitated fraction of chicken serum (CSa) also kills five myeloma cell lines, exhibiting different sensitivities to CSa, where U266B1 (blue symbols) is the most sensitive (Figure 3C). In addition, the seven leukemic cell lines tested also display different sensitivities to CSa, with the lymphoma cell line Raji being the most sensitive (Figure 3D). In contrast, CSa does not affect the viability of human dermal fibroblasts (HDFs), but does cause dose-dependent killing of the human myeloma cell line RPMI8226 (Figure 3E).

3.3. The Involvement of the Complement System in Chicken Serum Anti-Cancer Activity

As shown for the alligator serum (ASa, Figure 2A), CSa also causes a rapid (~40 s) depolarization of the membrane potential (V_M) toward 0 mV, culminating in cell death, thus supporting the notion that the Complement system contributes to the anti-cancer activity of CSa (Figure 4A). To further support Complement involvement, we tested the dependency of CSa killing on the presence of Mg²⁺ and Ca²⁺ ions which are essential for proper activity of any Complement pathway/arm (classical, alternative and lectin). As shown in Figure 4B, compared to the CaCl₂- and MgCl₂-containing culture medium (green bars), the anti-cancer activity was blocked in the presence of 16 mM EGTA, and partially restored by increasing MgCl₂ and CaCl₂ concentrations. To evaluate the requirement of each Complement protein for CSa anti-cancer effects, we used a series of affinity-purified polyclonal antibodies raised against selected epitopes of MAC-related proteins (C5–C8; see Materials and Methods for further details) for immune depletion assays (IDs) of specific Complement proteins. As illustrated in Figure 5, CSa anti-cancer activity was attenuated to various extents upon depleting different Complement proteins, providing additional proof for Complement-mediated cancer cell death. Regardless of the contribution of any tested Complement protein to the anti-cancer effect, these experiments show that Complement proteins work in concert, while being individually indispensable for executing the cell death program.

Figure 5. Terminal Complement components are required for CSa-mediated cytotoxicity. Immuno-depletion of Complement proteins (C5–C8) using specific polyclonal antibodies significantly reduces the anti-cancer activity of CSa. These findings demonstrate that Complement proteins act cooperatively and are individually required for effective cytotoxicity.

Figure 5. Terminal Complement components are required for CSa-mediated cytotoxicity. Immuno-depletion of Complement proteins (C5–C8) using specific polyclonal antibodies significantly reduces the anti-cancer activity of CSa. These findings demonstrate that Complement proteins act cooperatively and are individually required for effective cytotoxicity.

3.4. IgM Antibodies Participate in the Anti-Cancer Effect of CSa

IgM initiates the classical Complement pathway by recruiting the C1q protein via binding to a certain antigen (Figure 6A). Accordingly, to determine whether the anti-cancer effect of CSa is mediated by IgM molecules, we tested whether immuno-depleting chicken serum IgM by using a commercial anti-chicken Ig-mu antibody (targeting the constant heavy chain of IgM molecules; see details in the Materials and Methods), affects CSa-dependent anti-cancer activity. As illustrated in Figure 6B, the commercial antibody precipitates chicken IgM, as expected. Accordingly, immuno-depleting chicken IgM inhibits CSa anti-cancer activity by 82 ± 24% (Figure 6C), thereby supporting the role of IgM as a mediator of CSa anti-cancer activity, being a potent initiator of the classical pathway.

To decipher whether purified chicken IgM antibodies can trigger human-serum-mediated anti-cancer activity, chicken serum was fractionated using a 10 mL IgM purification column (prepacked with Sepharose high-performance resin), and relevant protein fractions were pooled and further fractionated by a Size Exclusion Chromatography step. Accordingly, IgM-containing fraction (10 µg) was applied to human U266 myeloma cells (30 µL/50,000 U266 cells) in the absence or presence of 40% human serum (HS, v/v) for 3 h at 37 °C. As shown in Figure 6D, the presence of human serum and IgM-containing fraction collectively activated the human Complement system, which resulted in strong anti-cancer activity.

4. Discussion

The present study demonstrates that alligator serum and its 45% (NH4)₂SO₄ fraction (ASa) exhibit potent and selective anti-cancer activity against various murine and human cancer cell lines. Importantly, this killing effect is mediated by the Complement system, as evidenced by both electrophysiological and biochemical analyses. Moreover, the discovery that chicken serum (CS) and its corresponding fraction (CSa) share these anti-cancer properties situates this phenomenon within a broader evolutionary continuum of innate immune defense among reptiles and avians. Collectively, these findings uncover a conserved Complement-mediated mechanism with potential translational applications in anti-cancer immunotherapy. Our findings illustrate that both crude serum (AS) and its 45% (NH₄)₂SO₄-precipitated fraction (ASa) rapidly kill cancer cells (Figure 1A). Across multiple assays, AS/ASa rapidly kills murine cancer lines (PN71 myeloma; EL4 lymphoma) and diverse human cancer lines (e.g., U937, T47D, HeLa), while sparing a non-cancerous human cell type (iPSC-derived cardiomyocyte; Figure 1E) under the tested conditions. Collectively, these findings support the presence of serum factor(s) with preferential cytotoxicity toward malignant cells. Notably, this rapid cytotoxic effect was observed exclusively with crude AS, but not with rabbit, horse or fetal calf sera (Figure 1B).

4.1. In Vivo Anti-Cancer Activity of ASa Supports Potential Translational Outcome/Promise

Injections of ASa to mice bearing B16 melanoma tumors cause pronounced intra-tumoral necrosis (Figure S1A), resulting in a substantial reduction in tumor volume, ~50% compared with FCS-treated controls (Figure S1B). Further, i.p. ASa injection to EL4-cell-induced peritoneal lymphoma caused an 80% decrease in EL4 cell count compared to the cell count in FCS-injected lymphoma mouse (Figure S2). These in vivo anti-tumor effects strengthen the physiological relevance of the in vitro cytotoxicity and argue that Complement-driven cytolysis can operate when the active serum fraction is delivered locally.

4.2. Experimental Evidence Showing That the Complement MAC Underlies ASa Killing Mechanism

Several independent observations converge on a Complement-dependent anti-cancer cytolytic mechanism. First, patch clamp recordings show ASa-induced depolarization of the membrane potential toward ~0 mV within minutes after application, culminating in cell death. Second, ASa triggers very large conductance perforin-like mega-channels in the order of pS consistent with pore formation [16], whereas fetal calf serum does not. These electrophysiological signatures are compatible with membrane perforation by the Complement MAC, which assembles from C5b–C9 and forms pores [17,18,19,20,21]. These large pores disrupt ionic homeostasis, causing rapid depolarization, lysis and extensive leakage of intracellular components, ultimately resulting in cell death (Figure 2A,B). Third, the elimination of the cytotoxic effect by heat treatment (55 °C, 20 min) and the fact that this effect requires the presence of the Mg²⁺ and Ca²⁺ ions, are consistent with heat-labile Complement components being obligatory for cytotoxicity. While this study does not directly quantify Complement regulators’ expression, the observed selectivity (cancer cells killed, whereas iPSC-CMs spared) is consistent with differential susceptibility to Complement-mediated attack. These observations collectively imply a plausible involvement of the Complement system within the serum fractions that trigger the swift cell-death response. We therefore conclude that crude alligator serum harbors potent Complement-mediated activity that alligators exploit to rapidly identify and eliminate cancer cells.

4.3. From Alligators to Crocodiles and Chickens

Because C5 is a key component of the Complement system [22,23,24], we tested the hypothesis that closely related species respecting their C5 sequence (which bears homology to the alligator protein; comparison was carried out using the ‘Maximum Likelihood’ algorithm, Figure 4A) exhibit alligator-like anti-cancer effects; indeed, the two closely related species, crocodile and chicken (Gallus gallus), display alligator-like anti-cancer activity (Figure 4B). Hence, a notable advance here is the extension of the anti-cancer phenomenon beyond alligator to crocodile and chicken sera, guided by phylogenetic relatedness in the Complement C5 sequence. The findings that crocodile and chicken sera kill RPMI 8226 cells rapidly, and that chicken serum fraction (CSa) kills a broad panel of myeloma and leukemia cell lines while sparing human dermal fibroblasts, indicate that the underlying mechanism is not unique to one species. This is consistent with prior work showing strong Complement activity in crocodilian sera [25], supporting the concept that robust innate immune effector mechanisms are a conserved feature in these lineages. Importantly, the chicken results provide a practical and scalable route for future purification and mechanistic dissection without relying on protected crocodilian species.

4.4. Mechanistic Dissection in Chicken Serum Implicates the Involvement of Mg²⁺ and Ca²⁺ Ions, Terminal Complement Proteins and IgM in the Anti-Cancer Activity

Regarding CSa, three mechanistic lines of evidence support Complement dependence: (i) rapid membrane depolarization toward 0 mV; (ii) strong inhibition of killing by EGTA chelation with restoration by Mg²⁺/Ca²⁺, which are required for Complement pathway activity; and (iii) immuno-depletion of terminal Complement proteins (C5–C8) attenuates cytotoxicity. These results strongly favor a model in which Complement activation proceeds to MAC assembly as the proximate executor of tumor cell death (Figure 7). Upstream, IgM appears to participate materially: immuno-depletion of chicken IgM reduced killing by ~82%, and an IgM-enriched chicken fraction can cooperate with human serum to induce activation of the human Complement system, resulting in cell death, consistent with IgM-mediated activation of the classical pathway via C1q binding. This interpretation is well supported by structural and biochemical literature demonstrating how IgM engages C1/C1q to initiate the classical Complement cascade [26,27,28,29]. Accordingly, a plausible unifying mechanism is: (i) chicken (or crocodilian) monomeric IgM (and potentially other “activator” components) binds a cancer-associated target on susceptible cells; (ii) this recruits/activates the Complement (including in heterologous human serum); and (iii) MAC pores drive rapid ionic collapse and lysis.

4.5. Study Limitations and Future Directions

Although this study provides experimental evidence supporting the conclusion of a Complement-mediated mechanism underlying cancer cell killing, several limitations are acknowledged:

(1)

To further support the ASa and CSa selective killing of cancer cells, additional normal cell types should be tested.

(2)

The molecular determinants underlying tumor recognition were not identified. Although our findings provide evidence that IgM antibodies are key initiators of Complement activation, the specific tumor-associated antigens or surface targets triggering this response have not been discovered. Elucidating these targets will be essential for understanding selectivity and for enabling rational therapeutic development.

(3)

The following are the limitations with respect to the melanoma and leukemia murine models: (i) The in vivo sample size was limited and did not enable proper statistical analysis. (ii) The exploratory experiments were intended as proof-of-concept preliminary studies, rather than definitive efficacy studies. (iii) Accordingly, these experiments yielded quantitative data.

(4)

The in vivo melanoma and lymphoma models only demonstrate proof-of-concept efficacy following intratumoral or intraperitoneal administration, and should be further established once the pure anti-cancer molecule is discovered. This approach does not address the challenges associated with systemic delivery; the pharmacokinetics, biodistribution, and stability of serum-derived components remain unknown, and their behavior in more clinically relevant settings requires further investigation. Finally, potential safety concerns related to Complement activation should also be explored.

(5)

While non-malignant human cells were largely spared under the conditions that killed cancer cells, the mechanisms that prevent off-target Complement-mediated damage in vivo should be investigated. Given the potent cytolytic capacity of the Complement system, careful evaluation of toxicity, immunogenicity, and tissue specificity will be critical.

(6)

While the enrichment of the Complement component C5 in the active fractions and depletion studies supports the involvement of the Complement system, it is important to note that C5 is an abundant serum protein, and therefore, its identification by proteomic analysis alone does not establish causality but rather should be interpreted in the context of complementary functional evidence. Therefore, the precise contribution of individual Complement components and regulatory factors should be dissected in future studies.

(7)

Future experiments may include: recombinant protein reconstitution, Complement inhibition assays and targeted depletion and rescue experiments.

(8)

Finally, interspecies differences in immune components may influence translational applicability. While cross-species compatibility was demonstrated in part (e.g., chicken IgM activating human Complement), further work is needed to assess how these findings translate into fully humanized systems.

Importantly, addressing these limitations will be essential to advance this discovery toward clinical application, and to determine whether Complement-based strategies can be safely and effectively harnessed for human cancer therapy.

5. Conclusions

This study reveals an ancient, conserved Complement-dependent immune mechanism in alligator, crocodile and chicken sera that selectively kill cancer cells via IgM-triggered MAC formation. The cross-species functionality and absence of toxicity toward healthy mammalian cells highlight this system’s evolutionary sophistication and potential therapeutic relevance. By harnessing this natural immune mechanism, it may be possible to develop bio-inspired, Complement-driven immuno-therapeutics that emulate the precision and potency of these ancient immune strategies, thereby offering a new frontier in cancer immunotherapy.