Immunogenic cell death (ICD) has emerged as a paradigm-shifting concept in the field of immunology and cancer therapeutics. It combines the ability to kill cancer cells and restore the lost immunological ability to identify and interact with dying cancer cells. This leads to the stimulation of innate and adaptive immune responses, and thereby establishment of long-term immunological memory (1). ICD is an overarching term that includes cell death modalities such as apoptosis (2), necroptosis (3–5), and ferroptosis (6–8). It can be induced by specific stimuli, including oncolytic viruses (9), conventional chemotherapeutics such as mitoxantrone (MTX) (10), and physical interventions such as radiotherapy, including heavy ions (11) or X-rays radiotherapy (12), or photodynamic therapy (13). Perturbation of endoplasmic reticulum (ER) homeostasis and activation of ER stress pathways, also known as the unfolded protein response (14), together with the generation of reactive oxygen species, are essential components of nearly (15) all scenarios in which ICD occurs (16–18). For cell death to possess immunogenic properties also requires both antigenicity and adjuvanticity (1, 19, 20). Antigenicity refers to the availability of either tumour-specific antigens, tumour-associated antigens, or neo-antigens, which enable the specific recognition and killing of the tumour cells by the immune system. Adjuvanticity involves the spatio-temporal release of damage-associated molecular patterns (DAMPs) and pro-inflammatory cytokines/chemokines from the dying cancer cells. This triggers the recruitment, activation and maturation of antigen-presenting cells (APCs), such as dendritic cells, via their respective pattern recognition receptors (PRRs). Several crucial DAMPs and cytokines have been discovered, including but not limited to high-mobility group B1, surface-exposed calreticulin, extracellular secretion of adenosine triphosphate, annexin A1 and several members of the type 1 interferon family (1, 21). Upon engulfment of dying tumour cells, APCs undergo activation and maturation characterised by upregulation of major histocompatibility complex class II molecules and costimulatory markers, such as CD80 and CD86, while migrating to the tumour-draining lymph nodes to present cancer antigens to CD8⁺ T cells. The activated cytotoxic CD8⁺ T lymphocytes then relocate to the tumour site, releasing interferon gamma, promoting eradication of the neoplastic lesion, and stimulating the formation of a long-lasting immunological memory against the tumour (1, 22).

CX3C chemokine ligand 1 (CX3CL1), originally named neurotactin (23) and later fractalkine (24), is a chemokine intricately involved in cell signalling and in immune cell recruitment and activation (25). CX3CL1 can exist as a membrane-bound variant (mCX3CL1) serving as an adhesion protein for cells expressing the CX3CL1 receptor (CX3CR1), including various immune cell types such as NK cells, monocytes, dendritic cells, granulocytes, and CD3⁺ T cells (24, 26, 27). Conversely, the soluble form of CX3CL1 (sCX3CL1), which is released upon proteolytic cleavage of mCX3CL1, predominantly exerts a potent chemoattractant function (24, 25, 27–30). Importantly, CX3CL1 has been identified as a “find me” signal that attracts immune cells towards dying cells, facilitating efferocytosis (31–35). However, in the context of ICD, it remains uncertain whether CX3CL1 is an important mediator of the effect of ICD or, due to its potential pro-tumourigenic features (36–42), might act as a “keep out” signal, hindering efferocytosis and the initiation of an effective ICD immune cycle (43, 44).

In this study, we assessed the secretion of CX3CL1 from MTX-treated murine fibrosarcoma MC205 and melanoma B16-F10 cells undergoing immunogenic apoptosis (i.e., apoptotic cell death exhibiting ICD characteristics (10, 45)), and explored its potential as a mediator of anti-tumour immunity during ICD. Our results demonstrate that CX3CL1 is released specifically during apoptotic cell death regardless of the cell type. Furthermore, the addition of 1 ng or 10 ng of recombinant CX3CL1 (rCX3CL1) to a non-immunogenic dose of dying/dead cancer cells for prophylactic vaccination of mice significantly increased the tumour-free survival of mice and restored immunogenicity of dying cancer cells. In addition, analysis of publicly available human database (The Cancer Genome Atlas-Skin Cutaneous Melanoma (TCGA-SKCM)) containing data from 98 melanoma patients revealed a correlation between high levels of CX3CR1 expression and higher overall survival probability. Moreover, CX3CR1 was predominantly associated with increased presence of CD8⁺ T cells, and a high level of CX3CR1 expression was correlated with increased expression of CD8⁺ T cell signature. These findings identify CX3CL1 as an effective mediator of an adaptive immune response during immunogenic apoptosis and position it as a promising therapeutic adjuvant in ICD-based treatment.

We examined the secretion of CX3CL1 from MTX-treated cancer cells undergoing immunogenic apoptosis and its potential role as a mediator of anti-tumour immunity during ICD. In our study, CX3CL1 was released exclusively during apoptotic cell death induced by MTX in murine fibrosarcoma MCA205 and melanoma B16-F10 cells. Moreover, in prophylactic vaccination of mice, the addition of 1 ng or 10 ng of rCX3CL1 to non-immunogenic doses of dying/dead cancer cells significantly enhanced tumour-free survival and restored immunogenicity of the dying cancer cells. Furthermore, analysis of the TCGA-SKCM database of data from 98 melanoma patients revealed a significant correlation between increased CX3CR1 expression and the patients’ improved overall survival probability. In addition, CX3CR1 was predominantly associated with increased abundance of CD8⁺ T cells, with high CX3CR1 expression levels correlating with increased CD8⁺ T cell signature.

Cytokines and chemokines are recognised as crucial regulators in cancer development, cell dynamics within the tumour microenvironment, and intercellular signalling processes. These molecular mediators are also an integral part of ICD, acting as signals for cellular recruitment (“find me” and “eat me” signals), as well as for immune evasion (“keep out” and “don’t eat me” signals) (1). The concept of ICD has gained significant attention as a novel immunotherapeutic strategy and is characterised by the release of DAMPs and pro-inflammatory cytokines/chemokines that can be detected by APCs via their corresponding PRRs (21). This drives the recruitment of APCs to the tumour site, facilitates recognition, engulfment, and subsequent processing and presentation of tumour antigens by APCs, and provides guidance for cytotoxic lymphocytes. CX3CL1 has been described as an important chemokine that can be implicated in the context of cancer, although controversies persist regarding the properties and activities of this chemokine due to its pro- and anti-cancer characteristics (25). But what is the role of CX3CL1 during ICD, in particular during immunogenic apoptosis?

In our experimental findings, we demonstrated that CX3CL1 was only detected in the supernatants of MTX-treated immunogenic apoptotic fibrosarcoma MCA205 and melanoma B16-F10 cells, and to a significantly lesser extent in the supernatants of viable cancer cells, RSL3-treated non-immunogenic late ferroptotic (6) cancer cells and non-immunogenic F/T accidentally necrotic cancer cells ( Figures 1C, F ). This indicates the exclusivity of CX3CL1 release during immunogenic apoptosis, suggesting its potential role in mediating immunogenicity of cancer cells undergoing apoptotic ICD. The receptor for CX3CL1, CX3CR1, is expressed on various immune cells, including NK cells, monocytes, dendritic cells, granulocytes, and CD3⁺ T cells, stimulating their adhesion, retention, and transendothelial migration to sites characterised by strong inflammatory reactions (24, 57). It has been shown that sCX3CL1 also serves as a potent chemoattractant for these CX3CR1-expressing immune cells, enabling their chemotaxis towards the cancer niche and activation of their anti-cancer functions (24, 30, 58–61). Moreover, CX3CL1 expression is crucial for dendritic cell migration, maturation, and adhesion to T cells (58), while the presence of CX3CL1 on mature dendritic cells also activates resting NK cells (62). Hence, the release of CX3CL1 by the MTX-induced dying cancer cells ( Figures 1C, F ) may establish a gradient directed towards the tumour, presumably augmenting CX3CR1⁺ immune cell migration along the gradient and activation of an anti-tumour immune cycle. Nevertheless, it is important to note that the upregulation of CX3CR1 expression may be associated with increased expression of this protein on the tumour cells, which may result in metastasis when the cancer cells enter the bloodstream and bind CX3CR1 on endothelial cells (25). However, due to the elevated concentration of CX3CL1, cancer cells expressing CX3CR1 might also be retained within the tumour and become the target of immune cells.

Furthermore, we demonstrated that the addition of CX3CL1 to a non-immunogenic dose of MTX-treated dying cancer cells is sufficient to elicit an immunogenic response in a tumour prophylactic mouse model, thereby revealing CX3CL1 as a power switch of immunogenic apoptosis induced by MTX ( Figure 2D ). Since it has been shown that CX3CL1 enhances efferocytosis of apoptotic thymocytes (34), it is conceivable that the presence of CX3CL1 in the prophylactic vaccine may lead to increased recruitment of phagocytes and clearance of dying cancer cells, consequently culminating in an overall improved anti-tumour immunity. However, the presence of CX3CL1 can also be associated with tolerogenic apoptosis (63). This underscores the nuanced and context-dependent role of CX3CL1 during cell death, where different factors intricately interact to determine the ultimate outcome. During immunogenic apoptosis, the secretion of other DAMPs fosters the anti-tumour effect, whereas during tolerogenic apoptosis DAMPs release is minimal, thus constraining the activation of an adaptive immune response. Therefore, it will be interesting to determine the differences in CX3CL1 secretion between tolerogenic apoptosis and immunogenic apoptosis, as the quantity of CX3CL1 secretion might be the key to shifting tolerogenic cell death towards an immunogenic form. In addition, it is important to note that prophylactic vaccination of mice with CX3CL1 alone (i.e., without any MTX-treated cancer cells) had no effect on the tumour-free survival of the mice ( Supplementary Figure S1 ), logically due to the absence of any antigens (antigenicity) or other DAMPs (adjuvanticity) during vaccination.

Remarkably, our findings from the tumour prophylactic vaccination experiments ( Figure 2D ) demonstrate that only lower doses (1 ng or 10 ng) of CX3CL1 exhibit an enhanced anti-tumour effect of ICD, whereas use of a high dose (100 ng) leads to almost complete absence of the anti-tumour effect ( Figure 2D ). This observation might indicate that the inherently inflammatory nature of CX3CL1 could contribute to hyperinflammation at higher doses, potentially promoting a pro-tumourigenic environment. Moreover, lack of sufficient DAMPs (due to a non-immunogenic dose of MTX-treated cancer cells) and excessive influx of immune cells (30, 58, 59) (due to a high CX3CL1 dose) might diminish activation of infiltrating immune cells, leading to an immunosuppressive phenotype and thus loss of the anti-tumour effect in the presence of a high dose of CX3CL1. Although gene therapy involving the transfer of CX3CL1 to cancer cells was demonstrated to induce a robust anti-cancer effect (64–66), we did not see a significant reduction in tumour size following co-treatment with CX3CL1 and MTX in the therapeutic mouse model ( Supplementary Figure S2 ). It is conceivable that the supplementary CX3CL1 introduced alongside the already secreted CX3CL1 by the MTX-treated tumour cells resulted in an excessively high concentration of CX3CL1, which ceases to elicit an additive effect.

Of interest, analysis of RNA-seq data and corresponding patient clinical information of the TCGA-SKCM of 98 melanoma patients demonstrated a positive correlation between increased CX3CR1 levels and increased overall survival probability in the melanoma patients, along with a discernible CD8⁺ T cell signature ( Figure 3 ). CX3CR1 has been reported to be associated with CD8⁺ T cells that respond to PD1 therapy while resisting cell death during chemotherapy (67). Moreover, CX3CR1-deficient mice injected with melanoma cells had increased tumour burden, cachexia, and defective anti-tumour responses (68). It has also been shown that an increase in CX3CL1 expression in the tumour is linked to improved prognosis of many cancer patients with breast carcinoma (69), colorectal cancer (70, 71) or lung adenocarcinoma (72), among other cancers (70–75).

In summary, our study indicates that CX3CL1 serves as a potent mediator of immunogenicity during immunogenic apoptosis induced by MTX. CX3CL1 is released by immunogenic apoptotic cancer cells regardless of the cancer cell type. Moreover, the addition of CX3CL1 to non-immunogenic doses of MTX-treated dying cancer cells in mouse prophylactic tumour vaccination models resulted in the activation of an adaptive immune response and effectively lengthened survival. In addition, an increase in CX3CR1 expression was correlated with increased overall survival probability of melanoma patients and increased CD8⁺ T cell signature. Our data provide a rationale for exploiting CX3CL1 as a future adjuvant to render therapy-induced cell death immunogenic. The addition of CX3CL1 to other treatments could affect their immunogenicity, unleashing their full immunogenic potential during cell death in anti-cancer therapy. However, to achieve an overall favourable immunogenic outcome, accurate dosing of CX3CL1 might be of paramount importance.

The original contributions presented in the study are included in the article/ Supplementary Material , further inquiries can be directed to the corresponding author/s.

Ethical approval was not required for the study involving humans in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and the institutional requirements. The animal study was approved by Ethics Committee of Ghent University. The study was conducted in accordance with the local legislation and institutional requirements.

FN: Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – original draft, Writing – review & editing. RD: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. OV: Formal analysis, Software, Writing – review & editing. IE: Writing – review & editing. MS: Writing – review & editing. GD: Data curation, Writing – review & editing. TM: Writing – review & editing. MV: Writing – review & editing. OK: Writing – review & editing. EC: Writing – review & editing. DK: Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing.