# How macrophages use extracellular calreticulin to chase their prey

Liwei Zhao, Peng Liu, Oliver Kepp, Guido Kroemer

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Liwei Zhao, Peng Liu, Oliver Kepp, Guido Kroemer. How macrophages use extracellular calreticulin to chase their prey. OncoImmunology, 2025, 14, 10.1080/2162402x.2025.2533494. hal-05208199

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# How macrophages use extracellular calreticulin to chase their prey

Liwei Zhao ID a,b, Peng Liu ID a,b, Oliver Kepp ID a,b, and Guido Kroemer ID a,b,c

ªCentre de Recherche des Cordeliers, Université Paris Cité, Sorbonne Université, Inserm, Equipe labellisée par la Ligue contre le cancer, Institut Universitaire de France, Paris, France; ᵇINSERM US23/CNRS UAR 3655, Université Paris-Saclay, Metabolomics and Cell Biology Platforms, Institut Gustave Roussy, Villejuif, France; ᶜDepartment of Biology, Institut du Cancer Paris CARPEM, Hôpital Européen Georges Pompidou, AP-HP, Paris, France

**ABSTRACT**

Recent findings reveal that macrophages actively control clearance by desialylating target cells via NEU1 and releasing cathepsin-cleaved calreticulin (CALR) to mark them for phagocytosis. This uncovers a dual role for CALR as immune activator or repressor, depending on its form and context, with distinct implications for cancer immunity.

**ARTICLE HISTORY**

Received 6 June 2025
Revised 5 July 2025
Accepted 9 July 2025

**KEYWORDS**

Asialoglycans; cancer; cell stress; immunotherapy

Calreticulin (CALR) is a key chaperone localized in the endoplasmic reticulum (ER), playing essential roles in calcium homeostasis and protein folding.¹,² Beyond these canonical functions within the lumen of the ER, cell surface-exposed CALR serves as an evolutionarily conserved recognition signal, facilitating gamete fusion in both yeast and mammals.³,⁴ Moreover, surface CALR functions as an “eat-me” signal, enabling phagocytes to identify and engulf target cells. During homeostatic clearance, macrophages can detect this signal to silently remove CALR-tagged apoptotic cells, whereas in the course of immunogenic cell death (ICD), ER-stress-activated PERK promotes ERp57-assisted CALR surface exposure dictates antigen uptake by dendritic cells (DC), ultimately triggering T cell-mediated adaptive immune responses⁵,⁶ (Figure 1a). Of note, CD47 expressed on tumors can deliver a ‘don’t-eat-me’ signal that inhibits phagocytosis, allowing malignant cells to evade immune clearance. Moreover, not all forms of CALR support immune activation. Mutations such as CALR del52 and CALR ins5 in myeloproliferative neoplasms, as well as truncations like CALR E405* in solid tumors, that disrupt the C-terminal Lys-Asp-Glu-Leu (KDEL) ER-retention motif, leading to aberrant secretion via Golgi-mediated exocytosis can act as local immunological decoys, saturating receptors like CD91 on antigen-presenting cells.⁷ As a result, such mutant forms of CALR can impair DC phagocytosis, suppressing CD8⁺ T cell responses, thus undermining the efficacy of chemotherapy and immune checkpoint inhibitors (ICI). Conversely it has been shown that macrophages can expose CALR on their surface which acts as a critical signal for guiding the phagocytosis of cancer cells, even when the tumor cells themselves do not express CALR.⁸ Furthermore, macrophages have been shown to actively secrete CALR through cleavage of the KDEL motif which then binds to N-terminal asialoglycans exposed on the surface of target cells tightly regulated by the presence of sialic acid residues, thereby flagging them for phagocytosis.⁹

A recent report from the group of Irving L. Weissman published in *PNAS* now reveals further insights into the molecular mechanisms of programmed cell removal (PrCR) positioning macrophages not only as executors but also as orchestrators of clearance.¹⁰ Here the authors described that macrophages were able to actively secrete the neuraminidase NEU1 to desialylate the cell surface of target cells while simultaneously releasing CALR through cathepsin-mediated cleavage of the KDEL motif. This two-step mechanism, which first unmasks asialoglycans and then overlays them with CALR generated a molecular “eat-me” signal that marked them for PrCR.¹⁰ This shift in perspective invites an illustrative metaphor. In the gladiatorial theaters of ancient Rome, the *retiarius* fought not with brute force but with finesse, using a net and trident to disarm and dispatch his opponent. The macrophage, in the biological arena, performs similarly. Its neuraminidase is the net, stripping protective sialic acids and exposing glycan vulnerabilities; cleaved CALR

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<figure><img src="image_8.png" /><figcaption>**Figure 1.** Multifaceted role of calreticulin (CALR) in immunity and clearance. (a) In the course of immunogenic cell death (ICD) endoplasmic reticulum (ER)-resident calreticulin (CALR) can translocate to the cell surface, where it functions as a conserved "eat-me" signal for antigen presenting dendritic cells (DCs) leading to adaptive immune responses. (b) Under homeostatic conditions surface exposed CALR promotes programmed cell removal (PrCR) by guiding macrophage-mediated phagocytic clearance of target cells. Recent findings further expand the role of macrophages in PrCR revealing that they actively orchestrate clearance by deploying neuraminidase (NEU1) to expose asialoglycans on target cells and releasing cleaved CALR, which binds these sites and marks cells for engulfment, underscoring macrophages as both orchestrators and executors of immune clearance.</figcaption></figure>

is the trident, striking the exposed site with precise, pro-phagocytic intent. The target is marked for phagocytosis and in turn cleared, not through violence, but via controlled unmasking and selective molecular tagging (Figure 1b).

Altogether, these findings underscore the dual and context-dependent role of CALR in immunity, acting either as a prophagocytic "eat-me" signal or as an immunosuppressive decoy depending on its cellular origin, its form and its localization. Clinically, this duality offers both a challenge and an opportunity: while surface-exposed CALR in cancer associates with favorable outcomes, high levels of soluble CALR may serve as a marker of therapeutic resistance. The identification of cathepsin B proteolytically facilitating CALR surface presentation, along with the deployment of the neuraminidase NEU1 by macrophages, now suggests that innate immune cells can actively shape the decision of which cells are cleared and which remain. However, until now the upstream signals governing this selectivity remained elusive. As Banuelos et al. highlighted, immune clearance is a highly orchestrated process requiring both precision and restraint. Harnessing this system for therapeutic benefit will require not only controlling the immunogenic functions of CALR but also avoiding its subversion by tumor intrinsic immunosuppressive signals. Therapeutically, combining agents that enhance CALR surface exposure with CD47 blockade on cancer cells may synergistically promote their selective clearance while minimizing off-target immune activation.

## Acknowledgments

The figure was created in BioRender (www.BioRender.com).
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Conceptualization; Writing – original draft: GK and OK have conceptualized the work and have written the original draft. Writing – review & editing; Visualization: LZ, PL, OK and GK have reviewed and edited the manuscript and have prepared the figure. Supervision GK and OK have jointly supervised the work. All authors have read and approve the final version of the manuscript.

## Author contributions

CRediT: **Liwei Zhao**: Visualization, Writing – review & editing; **Peng Liu**: Visualization, Writing – review & editing;

**Oliver Kepp**: Conceptualization, Supervision, Visualization, Writing – original draft, Writing – review & editing;

**Guido Kroemer**: Conceptualization, Supervision, Visualization, Writing – original draft, Writing – review & editing.

## Disclosure statement

OK is a scientific co-founder of Samsara Therapeutics. GK has been holding research contracts with Daiichi Sankyo, Eleor, Kaleido, Lytix Pharma, PharmaMar, Osasuna Therapeutics, Samsara Therapeutics, Sanofi, Sutro, Tollys, and Vascage. GK is on the Board of Directors of the Bristol Myers Squibb Foundation France. GK is a scientific co-founder of everImmune, Osasuna Therapeutics, Samsara Therapeutics and Therafast Bio. GK is in the scientific advisory boards of Centenara Labs (formerly Rejuveron Life Sciences), Hevolution, and Institut Servier. GK is the inventor of patents covering therapeutic targeting of aging, cancer, cystic fibrosis and metabolic disorders. GK's wife, Laurence Zitvogel, has held research contracts with Glaxo Smyth Kline, Incyte, Lytix, Kaleido, Innovate Pharma, Daiichi Sankyo, Pilege, Merus, Transgene, 9 m, Tusk and Roche, was on the Board of Directors of Transgene, is a cofounder of everImmune, and holds patents covering the treatment of cancer and the therapeutic manipulation of the microbiota. GK's brother, Romano Kroemer, was an employee of Sanofi and now consults for Boehringer-Ingelheim. The funders had no role in the design of the study; in the writing of the manuscript, or in the decision to publish the results.

## Funding

OK receives funding from Institut National du Cancer (INCa), Association pour la recherche sur le cancer (ARC), the European Innovation Council (EIC) and the Fondation Gustave Roussy. GK is supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche [ANR-22-CE14-0066 VIVORUSH, ANR-23-CE44-0030 COPPERMAC, ANR-23-R4HC-0006 Ener-LIGHT]; ARC; Cancéropôle Ile-de-France; Fondation pour la Recherche Médicale (FRM); European Research Council Advanced Investigator Award (ERC-2021-ADG, Grant No.101052444; project acronym: ICD-Cancer, project title: Immunogenic cell death (ICD) in the cancer-immune dialogue); The ERA4 Health Cardinoff Grant Ener-LIGHT; European Union Horizon 2020 research and innovation program Oncobiome (grant agreement number: 825410, Project Acronym: ONCOBIOME, Project title: Gut OncoMicrobiome Signatures [GOMS] associated with cancer incidence, prognosis and prediction of treatment response), Prevalung (grant agreement number 101095604, Project Acronym: PREVALUNG EU, project title: Biomarkers affecting the transition from cardiovascular disease to lung cancer: toward stratified interception), Neutrocure (grant agreement number 861878: Project Acronym: Neutrocure; project title: Development of “smart” amplifiers of reactive oxygen species specific to aberrant polymorphonuclear neutrophils for treatment of inflammatory and autoimmune diseases, cancer and myeloablation); National support managed by the Agence Nationale de la Recherche under the France 2030 program (reference number 21-ESRE-0028, ESR/Equipex+ Onco-Pheno-Screen); Hevolution Network on Senescence in Aging (reference HF-E Einstein Network); INCa; Institut Universitaire de France; PAIR-Obésité INCa_1873, the RHUs Immunolife and LUCA-pi (ANR-21-RHUS-0017 and ANR-23-RHUS-0010, both dedicated to France Relance 2030); Seerave Foundation; SIRIC Cancer Research and Personalized Medicine (CARPEM), SIRIC CARPEM INCa-DGOS-Inserm-ITMO Cancer_18006 supported by Institut National du Cancer, Ministère des Solidarités et de la Santé and INSERM. This study contributes to the IdEx Université de Paris Cité ANR-18-IDEX-0001; Cancéropôle Ile de France; National support managed by the Agence Nationale de la Recherche under the France 2030 program [21-ESRE-0028, ESR/Equipex+ Onco-Pheno-Screen]; SIRIC Cancer Research and Personalized Medicine (CARPEM) supported by Institut National du Cancer, Ministère des Solidarités et de la Santé and INSERM [SIRIC CARPEM INCa-DGOS-Inserm-ITMO Cancer_18006]; RHUs Immunolife and LUCA-pi, both dedicated to France Relance 2030 [ANR-21-RHUS-0017 and ANR-23-RHUS-0010].

## ORCID

Liwei Zhao http://orcid.org/0000-0001-5077-327X

Peng Liu http://orcid.org/0000-0002-1682-9222

Oliver Kepp http://orcid.org/0000-0002-6081-9558

Guido Kroemer http://orcid.org/0000-0002-9334-4405
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**Data availability statement**

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

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