β-glucan reprograms macrophages to attenuate efferocytosis of cancer cells

Macrophage phagocytosis has been implicated in regulating anti-tumour immunity. Trained innate immunity (TII), induced via modulation of mature myeloid cells or their bone marrow progenitors, mediates sustained increased responsiveness to secondary challenges. Despite the advances in the study of TII-mediated anti-tumour activity, the impact of TII on the orchestration of phagocytosis in the tumour setting requires further elucidation. Here, we investigated whether macrophage phagocytosis of tumour cells can be modulated through induction of TII. To this end, mice were pre-treated with β -glucan, a fungal-derived agonist of TII, and bone marrow was isolated for macrophage different iation. Macrophages were then co-cultured with tumour cells that were either apoptotic or opsonised with an antibody recognising a tumour antigen, to mimic efferocytosis and antibody-dependent cellular phagocytosis (ADCP), respectively. While TII did not have any impact in the modulation of ADCP, efferocytosis was decreased in trained macrophages. Along the same line, gene expression analysis demonstrated that mRNA levels of molecules promoting e fferocytosis were downregulated in trained macrophages. Trained macrophages exerted decreas ed levels of active caspase-1 and produced decreased levels of interleukin-1 β upon efferocytosis of tumour cells. Our findings reveal a hitherto unknown role of TII in the regulation of anti-tumour immunity and may set the stage for designing new cancer immunotherapeutic approaches targeting macrophage efferocytosis.

Macrophages, phagocytosis, efferocytosis, tr ained innate immunity, trained immunity, inflammasome, interleukin-1β , melanoma, breast cancer. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 3

The identification of novel mechanisms to harness immune responses is essential for bolstering tumour suppression. There is an increasing need to better understand the regulation of macrophage responses to cancer to inform the development of novel therapeutic approaches and to increase the efficacy of current immunotherapies. Macrophages represent a major orchestrator of immune responses in a plethora of pathologies1-3. While residing in the tumour microenvironment, macrophages display remarkable heterogeneity and plasticity turning into essential components that can either promote or suppress tumour growth 4-6. The tightly coordinated recognition and uptake of ‘foreign’ material through the process of phagocytosis is a major macrophage effector function that shapes host i mmune responses to both microbial and sterile stimuli 7,8. In the tumour setting, macrophages can engulf cancer cells, that are opsonised with antibodies recognising surface tumour antigens, through antibody dependent-cellular phagocytosis (ADCP)9 and mediate the clearance of apoptotic canc er cells through the specialised type of phagocytosis called efferocytosis 10. These two different types of phagocytosis, that represent a cardinal function of macrophages, have been linked to distinct effects on tumour growth11. While ADCP promotes anti-tumour effects 9, efferocytosis promotes a tumour- tolerant condition by inducing the release of immunosuppressive mediators such as interleukin 10 10,12. Inhibition of signaling pathways or phagocyte receptors that upregulate efferocytosis, such as Mer proto-oncogene tyrosine kinase (MerTK), has therefore been effective in reducing tumour growth in several experimental cancer models13,14. Trained innate immunity (TII or trained immuni ty) defines long-lasting adaptations of innate immune cells based on transcriptional and epigenetic modifications of myeloid cells and their bone marrow progenitors 15-17. Specifically, TII represents a state of enhanced immune responsiveness of the myeloid compartment to secondary challenges. We have previously shown that TII contributes to tumour suppr ession using experimental models of melanoma and lung cancer18. The anti-tumour effects of TII were attributed at least in part to epigenetic and transcriptomic reprograming in neutrophils and enhanced tumour cell killing by release of reactive oxygen species 18. The tumour suppressive phenotype of TII has been corroborated by several studies employing different tumour models and utilising different agents to induce TII 19-22. While the role of TII in promoting anti-tumour immunity is established, the impact of TII in efferocyto sis or ADCP to shape anti-tumour immunity remains poorly explored. The application of immunotherapies including che ckpoint inhibitors that target the adaptive immune compartment for the treatment of melanoma has significantly improved the clinical management of this malignancy 23. However, current drugs have significant limitations as some patients suffer from side effects or do not benefit from treatment24-27. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 4 Macrophages are abundant in the tumour micr oenvironment of melanoma and their therapeutic targeting may be a promising strategy to complement existing therapies and enhance treatment success 28,29. Of note, macrophages have been shown to contribute to ADCP30 of melanoma cells. In addition, targeted inhibition of efferocytosis incites anti-tumour immunity in melanoma31. Here, we studied the role of TII on macrophage phagocytosis of melanoma cells and the cytokine profile of phagocytic macrophages. Our data demonstrate that ‘trained’ macrophages show decreased efferocytosis of tumour cells accompanied by reduced secretion of IL-1 β . These findings unravel a hither to unrecognized role of TII in the control of macrophage anti- tumour functions. The decreased macrophage effero cytosis of tumour cells has the potential to be harnessed therapeutically. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 5

Mice and mouse experiments Mice C57BL/6 wild type (WT) mice and Fcer1gtm1Rav (Fc γ R-/-; provided from Dr. James Hewitson, University of York) mice32,33 were bred in-house under specific pathogen-free conditions on a standard 12/12 /i2 h light/dark cycle according to the institutional guidelines at the Biological Services Facility (BSF), Universi ty of York. Male and female mice that were 8- 12/i2 weeks old were used in experimental procedures. Food and water were provided ad libitum. Mouse experiments To induce trained innate immunity, mice were pre-treated with a single dose of 1 mg of β - glucan from Trametes versicolor or Saccharomyces cerevisiae (Invivogen) in PBS or with PBS vehicle alone (control) intraperitoneally (i .p.). Seven days after injection, bones were harvested and bone marrow was used for bone marrow-derived macrophage (BMDM) differentiation. All animals were visually inspected daily and were within accepted humane endpoints. Ethics statement All animal experiments were carried out under the authority of a UK Home Office Licence (project licence number PPL PP7424874) that obtained approval by the University of York Animal Welfare and Ethics Review Board. All procedures were performed in compliance with ARRIVE guidelines. No unexpected adverse events were recorded during this study. Generation of mouse bone marrow-derived macrophages (BMDMs) and neutrophils The isolation of bone marrow and differentiation into BMDMs (hereafter mentioned as macrophages) was performed as previously described 34. Briefly, bone marrow was flushed from femurs and tibias of mice and cells were plated and cultured in the presence of recombinant macrophage colony-stimulating factor (M-CSF; 20 ng/ml, PeproTech or Proteintech). Culture medium was replaced every two days, and after seven days differentiated BMDMs were used for further experiments. To isolate neutrophils, bone marrow cells, after erythrocyte lysis, were centrifuged in 62% Percoll gradient (Cytiva) as previously described 34. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 6 Mouse cell lines The B16F10 melanoma cell line (Creative Biog ene) and the breast cancer cell lines PY8119 and PY230 (provided by Will Brackenbury, University of York) were utilised. B16F10 cells were cultured in Dulbecco's Modified Eagl e's Medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco), 100 U/ml penicillin, 100 μ g/ml streptomycin (Gibco) and 2 mM L-Glutamine (Gibco). PY8119 cells were cultured in F-12K Nutrient Mix medium (Gibco) supplemented with 5% FBS and 100 U/ml penicillin, 100 μ g/ml streptomycin. Same medium was used for the culture of PY230 cells supplemented with 0.1% MITO+ serum extender (Corning). Flow cytometric analysis Cell surface staining of MerTK on macrophages was performed in 5% FBS/PBS using 6 μ g/ml of anti-MerTK (PE-conjugated, clone: DSSMMER, Invitrogen) or the IgG2a λ isotype control (PE-conjugated, clone: G013C12, BioLegend) for 25 minutes. Levels of apoptosis were assessed using the annexin V apoptosis detection kit APC (Invitrogen) in combination with propidium iodide as previously described 34. Flow cytometric analysis of stained cells was performed on a CytoFLEX (Beckman Coulter) and data were analysed with CytExpert software. Cell treatments Trained or non-trained macrophages were stimulat ed with 10 ng / ml lipopolysaccharide (LPS, Invivogen) for 16–18 h followed by RNA extraction and assessment of Tnf mRNA levels. To induce apoptosis, cancer cells were treated with 1 μ M of the protein kinase C inhibitor staurosporine35 for 16–18 h (Cell Signalling Technology). To induce apoptosis in bone marrow neutrophils, cells were cultured in HBSS containing 1% FBS for 16 – 18 h34. To inhibit phagocytosis, macrophages were treated with the actin polymerization inhibitor Cytochalasin D (10 - 40 μΜ ; Merck Group or Cayman Chemical Company) for 30 minutes, culture medium was changed and then phagocytic cargo was added. To block signaling through the MerTK receptor, macrophages were treated with the inhibitor UNC2025 (0.1 μΜ , purity ≥ 98%; Cayman Chemical Company) for 1 hour prior to their co- culture with apoptotic cells or prior to RNA isolation. Activity of caspase-1 was measured in macrophages after co-culture by incubating with FAM-YVAD-FMK FLICA (10 μ M, Bio-Rad, as per manufacturer instructions) for 60 minutes with flow cytometry. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 7 Phagocytosis assays Unstained macrophages or macrophages that were stained with either carboxyfluorescein succinimidyl ester (CFSE, 0.75 μ M; Biosciences) or with DiD (3 μ M; Thermo Fisher Scientific) were co-cultured with (i) apoptotic cells pre-labelled with 5 μ M pHrodo Red SE (Invitrogen) in a 1:5 ratio of macrophages to apoptotic cells, (ii) pHrodo-labelled melanoma cells in the presence of 0.5 μ g/ml anti-mouse/human Ab recognising TYRP1/TRP1 (gp75) on melanoma cells (clone: TA99, Bio X Cell) or the mouse IgG2a isotype control (clone: C1.18.4, Bio X Cell) in a 1:10 ratio of macrophages to tumour cells or (iii) pHrodo green E. coli Bioparticles (50 μ g/ml, Invitrogen). Flat bottom 96-well plates that were either non - TC-treated or TC-treated were used for flow cytometry and live cell imaging, respectively. Co-culture plates were spun for 1 minute at 250g prior to the initiation of the phagocytosis assay. Three technical replicates were used for each sample. For live cell imaging, the Livecyte instrument (Phasefocus) with a 10x magnification and a frame size of 1 mm x 1mm was used. At least 50 macrophages per region of interest were evaluated. Quantitative RT-PCR Total RNA was extracted from cultured cells using TRIzol (Ambion Inc) according to manufacturer’s instructions and was quantified by spectrometry at 260 and 280 nm using a Nanodrop instrument (ThermoFischer). Complementary DNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was performed using the Fast SYBR Green Master Mix (Applied Biosystems) and gene-specific primers in a QuantStudio 3 Real time PCR detection system (Thermo Fisher Scientific). 18S was used as an internal control for normalization. Data were analyzed using the comparative ( ΔΔ Ct) method. The following primers were used: 18S-F (GTT CCG ACC ATA AAC GAT GCC), 18S -R (TGG TGC CCT TCC GTC AAT), Tnf-F (AAG CCT GTA GCC CAC GTC GTA), Tnf-R (GGC ACC ACT AGT TGG TTG TCT TTG), Lxra-F (CTC AAT GCC TGA TGT TTC TCC T), L xra-R (TCC AAC CCT ATC CCT AAA GCA A), Abca1-F (GGT TTG GAG ATG GTT ATA CAA TAG TTG T), Abca1-R (CCC GGA AAC GCA AGT CC), Axl-F (ATG GCC GAC ATT GCC AGT G), Axl-R (CGG TAG TAA TCC CCG TTG TAG A), Gas6-F (TGC TGG CTT CCG AGT CTT C), Gas6- R (CGG GGT CGT TCT CGA ACA C). Enzyme-linked immunosorbent assays (ELISAs) Mouse IL-10 and IL-1 β proteins were measured in cell cu lture supernatants using kits from Biolegend and Invitrogen, respectively, according to the instructions of the manufacturer. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 8 Statistical analysis Statistical analyses were carried out with GraphPad Prism 10 software. For infection experiments, researchers were blinded to mous e genotype. This also applies to histology and flow cytometry analyses. Results are presented as mean plus standard error of the mean. Two tailed parametric and non-parametric tests were used as appropriate after testing for normality. Multiple-group comparisons were performed using analysis of variance (ANOVA) and the Tukey’s, Dunnett's or Šídák's multiple comparison tests. A P value of < 0.05 was considered to be statistically significan t. Statistical information is provided in each figure legend. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 9

Efferocytosis of apoptotic tumour cells is decreased by trained macrophages Having previously identified a tumour suppressive role for TII that is mediated by myeloid cells 18, we sought to determine the impact of TII as a regulator of macrophage uptake of apoptotic tumour cells. To mimic efferocytosis conditions, macrophages pre-stained with CFSE were co-cultured with pHrodo red-labelled apoptotic melanoma cells ( supplementary Fig. 1A ). Pre-treatment of macrophages with the actin polymerization inhibitor cytochalasin D prior to their co-culture with tumour cells resulted in dose-dependent blockade of efferocytosis confirming the specificity of the assay ( supplementary Fig. 1B, C). The effect of TII on macrophage efferocyto sis of tumour cells was investigated using bone marrow derived macrophages (BMDMs) from the bone marrow of mice treated with β - glucan (hereafter described as trained macrophages). The increased levels of Tnf in trained macrophages upon their stimulation with lipopolysaccharide (LPS) confirmed the induction of TII36 in our experimental setting ( supplementary Fig. 2). Flow cytometric analysis of the co- cultures revealed that efferocytosis was attenuated in trained macrophages ( Fig. 1B-D). The dynamic behaviour of trained macrophages during efferocytosis of melanoma cells was mapped using real time imaging verifying thei r decreased capacity for tumour cell uptake (Fig. 1E). To determine whether the effect of TII on ma crophage efferocytosis applies to other types of cancer, the two breast cancer cell lines PY230 and PY8119 were used in our co-culture experiments. Consistently with our results on decreased efferocytosis of melanoma cells, we demonstrated that trained macrophages exerted attenuated efferocytic activity upon their co- culture with apoptotic breast cancer cells ( Fig. 2) suggesting a broad role of TII on shaping efferocytosis of cancer cells. TII does not have any effect on macrophage ADCP of melanoma cells We next investigated the role of TII on the macrophage ADCP. To induce ADCP, the co- culture was performed in the presence of the well-characterised anti-mouse/human TA99 Ab that recognises the tumour antigen Tyrp1 37 ( Fig. 3A ). Specificity of ADCP induction was demonstrated using macrophages from mice deficient in Fc γ receptors 32,33 ( Fig. 3B ). Following co-culture of TA99-opsonised melano ma cells with either trained or non-trained macrophages, levels of ADCP were comparable between the two groups ( Fig. 3C, D ), suggesting that TII does not play any role in macrophage ADCP of melanoma cells under these conditions. Given that macrophage phagocytosis can be differ entially modulated by different types of phagocytic cargo 38,39, we sought to investigate the behaviour of trained macrophages .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 10 towards different key phagocytic cargos. To this end, trained or non-trained macrophages were cultured together with microbial bioparticles or apoptotic neutrophils to mimic phagocytosis during infection or resolution phase of sterile inflammatory responses, respectively. The uptake of both E. coli particles (supplementary Fig. 3A-D) and apoptotic neutrophils (supplementary Fig. 3E, F ) was elevated in trained macrophages compared to the phagocytic activity shown in control macr ophages. These findings underline the ability of trained macrophages to shape differential responses to distinct phagocytic cues. Induction of TII combined with MerTK inhibition further downregulate efferocytosis of tumour cells. MerTK functions as an efferocytic receptor recognising the ‘eat-me’ signal PS on the outer surface membrane of apoptotic cells with the help of bridging molecules such as Gas6. As MerTK blockade has already been shown to promote tumour suppression 31, we investigated the consequences of MerTK inhibition on trained macrophage efferocytosis. To this end, trained or control macrophages were cultured with apoptotic melanoma cells in the presence of the small-molecule MerTK inhibitor UNC2025 40. In trained macrophages, the presence of UNC2025 resulted in decreased efferocytosis compared to that one in control macrophages treated with the inhibitor ( Fig. 4A ). Gene expression analysis of molecules downstream of MerTK signaling 14 in macrophages treated with the UNC2025 verified the downregulation of Liver X receptor alpha ( Lxra) and ATP-binding cassette transporter (Abca1) ( supplementary Fig. 4 ). We then assessed the impact of TII in the expression levels of molecules implicated in the MerTK pathway and found that the components of MerTK-dependent signaling Gas6 , Lxra and Abca1 were downregulated in trained macrophages ( Fig. 4B, C ). MerTK protein levels were downregulated in trained macrophages ( Fig. 4D ). Therefore, decreased efferocytosis of tumour cells by trained macrophages may be attributed at least in part to the downregulated molecular machinery orchestrating MerTK-dependent efferocytosis. Secretion of IL-1 β by trained macrophages upon efferocytosis of tumour cells is blunted. Macrophage efferocytosis not only paves the way towards the clearance of apoptotic cells, but also promotes metabolic alterations in macrophages 41-43. At the same time, metabolic alterations in efferocytic macrophages induced by digested apoptotic material substantially regulates the cytokine secretion by efferocytic macrophages 44. Since upregulation of interleukin-1β (IL-1 β ) has been linked to both enhanced efferocytosis 45 and tumour growth46,47, we determined the levels of IL-1β production in our co-culture setting. Production of IL-1β was induced as a result of efferocytosis, as IL-1 β was minimally detected in either .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 11 macrophage or apoptotic melanoma cell monocultures ( Fig. 5A ). Trained macrophages displayed lower levels of IL-1 β production as shown in co-culture supernatants compared to those from control macrophages ( Fig. 5A ), further underscoring the tumour suppressive effect of TII through inhibition of efferocytosis. Consistently, we demonstrated that levels of active caspase-1 were decreased in trained macrophages after efferocytosis of apoptotic tumour cells (Fig. 5B, C) thereby explaining at least in part the decreased levels of active IL- 1 β in co-culture supernatants. We also determined the levels of immunosuppressive cytokine interleukin 10 (IL-10), previously described to be upregulated after efferocytosis 12, in the supernatants of efferocytosis co-cultures. Levels of IL-10 in the co-culture supernatant were comparable between the trained and control macrophages (Fig. 5D). Together, these data unveil a novel role for TII in shaping macrophage-cancer cell interactions, providing a better understanding on mechanisms that may be targeted to block efferocytosis-dependent tumour growth. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 12

The orchestration of host immune responses against cancer involves several immune cell types and events including monocyte infiltration, macrophage activation, cytokine release and phagocytosis of cancer cells, all of whic h aim to contribute to tumour suppression 48. Although immune cell interactions play important roles in the immunopathology of cancer, the network of molecules and mechanisms that coordinate this complex process warrants further elucidation. Therefore, identification of immune regulatory networks that aim to harness cancer-associated immunosuppression are required. In an experimental model of melanoma, induction of TII has been shown to promote myeloid cell – dependent tumour suppression through repr ograming the phenotype of neutrophils to inhibit tumour growth 18. Here, we investigated whether TII regulates macrophage phagocytosis of tumour cells. We show for first time that trained macrophages have decreased phagocytic activity of apoptotic tumour cells and that is accompanied by lower levels of secreted IL-1 β . We also found that Gas6, Lxra, Abca1 and MerTK, all previously linked to enhanced efferocytosis 34,49, were downregulated by trained macrophages. Under the tested conditions, levels of ADCP were comparable between trained and control macrophages. Use of antibodies recognising alternative melanoma surface antigens may promote ADCP by trained macrophages. Further studies would be useful to addres s why trained macrophages demonstrate a cargo- dependent phagocytic behaviour, i.e. decreased effe rocytosis of tumour cells, unaltered ADCP and enhanced phagocytosis of apoptotic neutrophils and E. coli particles. In agreement with the literature, although using a different approach to induce TII, enhanced phagocytosis of pathogen particles 36 and apoptotic neutrophils 50 by trained phagocytes has been demonstrated. Our findings on trained macrophage efferocytosis of different apoptotic cargos further support previous reports suggesting that the cellular identity of the engulfed apoptotic cell instruct distinct macrophage phenotypes 51 that may be potentially linked to enhanced effectiveness of macrophage-based cell therapies. Targeting efferocytosis to bolster anti-tumour immunity has recently attracted attention 10,13. Of note, efferocytosis has been linked to cancer therapeutic resistance among several types of cancer 52,53. The role of TII on attenuating macrophage efferocytosis in the tumour setting was corroborated by showing that inhibition of the efferocytosis receptor MerTK further decreased the uptake of apoptotic tumour cells in co-cultures with trained macrophages. As blockade of MerTK may have a direct impact in cancer cells 54, further studies are required to address whether MerTK inhibition affects solely efferocytosis in our experimental setting. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 13 The decreased caspase-1 activity in trained efferocytic macrophages was reflected by lower levels of IL-1 β levels. Efferocytosis has been shown to induce the activation of NLRP3 inflammasome signaling in myeloid cells thereby promoting IL-1 β secretion and tumour growth45. Further focus on the identification of specific inflammasomes that contribute to the attenuated efferocytosis by trained macrophages may support the development of novel cancer immunotherapeutic targets. In addition, fu ture studies will provide further insight into the potential role of MerTK inhibition on macrophage IL-1β secretion during efferocytosis. Given the macrophage heterogeneity 55 and the different types of central and peripheral trained innate immunity 56, the use of BMDMs as the only source of macrophages may represent a limitation of this study. However, BMDMs have been utilised extensively for the elucidation of mechanisms orches trating cargo-dependent macrophage phagocytosis 51. Future validation experiments using different types of macrophages will strengthen the impact of this study. Lack of in vivo data on macrophage efferocytosis is another limitation of our study. Further analyses are required to address the physiological relevance of trained macrophages in efferocytosis of tumour cells in vivo. Cancer combination therapies, including the administration of immunotherapy, have contributed to the improvement of the clinic al management of cancer particularly in aggressive types such as melanoma 57,58 and also breast cancer59,60. The anti-tumour effect of β -glucan and the vaccine Bacillus Calmette-Guerin, both inducers of TII, is being tested in clinicals trials 61-63. TII - dependent decrease of efferocytosis may be a promising adjuvant immunotherapeutic strategy to complement existing cancer therapies and enhance treatment success. In summary, the present study pinpoints a novel role for trained innate immunity in attenuating macrophage efferocytosis of tumour cells and demonstrates that the decreased efferocytosis by trained macrophages is associated with lower levels of active caspase-1 and IL-1 β secretion. This novel role of trai ned innate immunity could be therapeutically exploited in pre-clinical models to ex pand our current mechanistic and translational understanding of the anti-tumour potential of trained immunity. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 14 Author contributions AC contributed to experimental design, performed experiments, analysed and interpreted data; JL performed experiments; DL contributed to experimental design and interpreted data; DB contributed to experimental design, interpreted data and supervised research; IK conceived and designed the study, performed experiments, supervised research, interpreted data, and wrote the paper. All authors critiqued and edited the manuscript. Funding Statement This work was funded by the Hull York Medical School (PhD studentship to AC) and was funded by the Academy of Medical Sciences Springboard grant (SBF007\100172), the Royal Society grant (RGS\R2\202032) and the Rosetrees Trust grant (Seedcorn2021 100043) awarded to I.K.. D.B. is funded by the Academy of Medical Sciences Springboard Award (SBF006\1025) and a Medical Research Council New Investigator grant (MR/Z504221/1).

We would like to thank staff at the Imaging and Cytometry Lab in the University of York Bioscience Technology. We thank Prof. Will Br ackenbury (University of York) for providing the PY8119 and PY230 cell lines and Dr. James Hewitson (University of York) for providing the Fcer1gtm1Rav mice. Competing Interest Statement The authors have declared no competing interest. Author Declarations We confirm all relevant ethical guidelines have been followed, and any necessary ethics committee approvals have been obtained. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 15 Figure 1. The effect of trained innate immunity on macrophage efferocytosis of melanoma cells. (A) Schematic diagram of experimental layout. (B-D) Trained or control macrophages were pre-stained with CFSE and were then co- cultured with pHrodo-labelled apoptotic B16F10 melanoma cells for 4 h. (B) Efferocytosis, (C) Representative flow cytometric plot; Numbers in outlined areas indicate the percentage of macrophages that is pHrodo + red), (D) Ratio of mean fluorescence intensity (MFI) are shown and were calculated as the percentage of pHrodo + macrophages and pHrodo MFI in macrophages, respectively. (B, D) Each paired line represents average values from nine different experiments ( n/i2 =/i2 44 separate cell isolations per group). (E) Live cell imaging was performed in co-cultures as shown in (A). Levels of efferocytosis are shown over time (left, n/i2 =/i2 8-10 separate cell isolations per group). Representative images in selected time points are shown (right). Arrows denote phagocytic macrophages. *P<0.05, **P<0.01, ****P<0.0001. Paired (B, D) two-tailed Student’s t-test, Two-way ANOVA test (E). Data are presented as mean and mean ± s.e.m. Figure 2. The impact of trained macrophages in efferocytosis of breast cancer cells. Trained or control macrophages were pre-stained with CFSE and were then co-cultured with pHrodo-labelled apoptotic PY230 (A-C) and PY8119 (D-F) breast cancer cells for 4 h. (A, D) Relative efferocytosis and (B, E) relative ratio of mean fluorescence intensity (MFI) are shown and were calculated as the percentage of pHrodo + macrophages and pHrodo MFI in macrophages, respectively. Data are expressed relative to the control group, set as 1 (n=8-9 (A-C) and n/i2 =/i2 9-10 (D-F) separate cell isolations per group). (C, F) Representative flow cytometric plots ar e shown. Numbers in outlined areas indicate the percentage of macrophages that is pHrodo+ red. *P<0.05, ** P<0.01, two-tailed Student’s t-test (A-F). Data are presented as mean ± s.e.m. and are pooled from two experiments (A-F). Figure 3. Trained innate immunity does not impact macrophage ADCP of melanoma cells. (A) Schematic diagram of experimental layout. (B) Macrophages derived from WT or Fc γ R-/- mice were pre-stained with CFSE and were then co-cultured with pHrodo-labelled B16F10 cells in the presence of 0.5 μ g/ml TA99 Ab that recognises the tumour antigen tyrosine related protein-1 (TYRP1 or gp75) or isotype control for 4 h. Relative ADCP is shown and is calculated as the percentage of pHrodo + .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 16 macrophages. Data are expressed relative to the untreated control group, set as 1 ( n/i2 =/i2 3 separate cell isolations per group). (C, D) Trained or control macrophages were pre-stained with CFSE and were then co- cultured with pHrodo-labelled B16F10 cells as in (B). (C) ADCP and (D) relative ADCP are shown and calculated as the percentage of pHrodo + macrophages. Data are expressed relative to the control group, set as 1 ( n/i2 =/i2 6 separate cell isolations per group). *P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, n.s., non-significant. One-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± s.e.m. and are pooled from three independent experiments (B-D). Figure 4. The role of MerTK pathway on the phenotype of trained macrophages. (A) Trained or control macrophages were pre-stained with CFSE, were treated with 0.1 μ M of the MerTK inhibitor UNC2025 or control vehicle for 1h and were then co-cultured with pHrodo-labelled apoptotic melanoma cells for 4 h. Relative efferocytosis using flow cytometry is shown and was calculated as the percentage of pHrodo + macrophages. Data are expressed relative to the control group, set as 1 ( n=7-8 separate cell isolations per group). (B, C) Relative mRNA expression of Gas6, Lxra, Abca1, Axl from control or trained macrophages. Relative mRNA expression was normalized against 18S rRNA and was set as 1 in the control macrophages (n=15 separate cell isolations per group). (C) Each paired line represents average values from different experiments shown in (B). (D) The relative MerTK MFI in control or tr ained macrophages is shown. Relative MFI ratio was set as 1 in the control macrophages (n=17-18 separate cell isolations per group). *P<0.05, **P<0.01, ***P<0.001, n.s., non-significant. One-way ANOVA with Tukey’s multiple comparisons test (A), two-tailed Student’s t-test (B, D). Data are presented as mean and mean ± s.e.m. and are pooled from two (A), three (B, C), four experiments (D). Figure 5. The levels of immune mediators and active caspase-1 during macrophage efferocytosis of melanoma cells. Trained or control macrophages were co-cultur ed with apoptotic melanoma cells for 4 or 20 h. Protein concentrations of ( Α ) IL-1 β and (D) IL-10 were measured in efferocytosis co- culture supernatants. Monocultures of trained or control macrophages and apoptotic melanoma cells were used as controls. N=9-10 separate cell isolations per group for macrophages and n=2 for B16F10 cells. (B, C) Trained or control macrophages , pre-stained with the DiD, were co-cultured with apoptotic melanoma cells for 4 h. The probe FAM-YVAD- FMK FLICA (10 μ M) was added 1 h prior to the end of the co-culture. Relative caspase-1 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 17 activity is shown and was calculated as the percentage of positive macrophages that are labelled with the FLICA ( n=8-9 separate cell isolations per group). Data are expressed relative to the control group, set as 1. (C) Representative flow cytometric plots are shown. Numbers in outlined areas indicate the percentage of macrophages that is FAM-FLICA+. *P<0.05, *** P<0.001, **** P<0.0001, n.s., non-significant. One-way ANOVA with Šídák's multiple comparisons test (A, D), two-tailed Student’s t-test (B). Data are presented as mean ± s.e.m. and are pooled from two experiments (A-D). Supplementary Figure 1. The inhibitory effect of cytochalasin D on macrophage efferocytosis. (A) Schematic diagram of experimental layout. (B) CFSE-labelled macrophages were treated with the actin polymerization inhibitor cytochalasin D for 30 min prior to their co-culture with pHrodo-labelled apoptotic melanoma cells for 4 h (n=3 separate cell isolations). Samples in which apoptotic cells were added to the macrophage cultures just before the flow cytometric analysis, without any incubation, se rved as negative control. Efferocytosis is shown as the percentage of pHrodo+ macrophages. (C) Representative flow cytometric plots are shown. Numbers in outlined areas indicate percentage of macrophages that is pHrodo + red. *P<0.05, **P<0.01, One-way ANOVA with Dunnett's multiple comparisons test. Data ar e presented as mean ± s.e.m. and are derived from one experiment. Supplementary Figure 2. The effect of LPS on Tnf mRNA expression levels in trained macrophages. Relative mRNA expression of Tnf in trained and control macrophages that were cultured in the presence or absence of 10 ng/ml LPS fo r 16-18 h. Relative mRNA expression was normalized against 18S rRNA and was set as 1 in macrophages that were not treated with LPS (n=10 separate cell isolations for control and 9 for trained macrophages). *** P<0.001, n.s., non-significant. Mann-Whitney test. Data are presented as mean ± s.e.m. and are pooled from three experiments. Supplementary Figure 3. The role of trained innate immunity on macrophage phagocytosis of microbes and apoptotic neutrophils. (A-D) Trained or control macrophages were treated with 50 μ g/ml of pHrodo green E. coli particles for 1 h and 3 h. Percent age of macrophages that have engulfed E. coli particles is shown (A, C: n/i2 =/i2 8-10 separate cell isolations per group). .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 18 (E, F) Trained or control macrophages, pre-stained with CFSE, were co-cultured with pHrodo red – labelled apoptotic neutrophils for 30 min. Percentage of macrophages that have engulfed apoptotic neutrophils is shown (n=8 separate cell isolations per group). (B, D, F) Representative fluorescence-activated cell sorting plots for E. coli particle phagocytosis (B, D) and neutrophil efferocytosis (F) are shown. Numbers in outlined areas indicate the percentage of macrophages that is pHrodo+ green (B, D) and pHrodo+ red (F). *P<0.05, ** P<0.01. Two-tailed Student’s t-test (A, C, E). Data are presented as mean ± s.e.m. and are pooled from two experiments (A, C) or derived from one experiment (E). Supplementary Figure 4. The effect of MerTK blockade on macrophage gene expression. Relative mRNA expression of Lxra and Abca1 in macrophages that were treated or not with 0.1 μΜ of the MerTK inhibitor UNC2025 for 60 minutes. Relative mRNA expression was normalized against 18S rRNA and was set as 1 in the untreated macrophages (n=9-10 separate cell isolations per group). ** P<0.01, *** P<0.001. Two-tailed unpaired t-test test. Data are presented as mean ± s.e.m. and are pooled from two experiments. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 19

1 Kour tzelis, I. , Hewitson, J. & Roger, T. Editorial: Macrophage Plasticit y in St erile and Patho g en -Induced Inflammation . Frontiers in immunology 12, 823023, doi:10.3389/fimmu .2021. 8230 23 (20 21). 2 Laz arov , T. , Jua r ez - Carreno, S ., Cox , N. & Geissmann, F . Phy s iology and di seas es of tiss ue-resident macropha ge s. Nat ure 618, 69 8-707, do i: 10. 1038 / s 4158 6-02 3-0600 2- x (2023). 3 Loc at i, M., Cur tale, G . & Mantov ani, A. Div er s it y, Mechanis ms , and Significance of Macro phage Pla sticit y. Annual review of pathology 15, 123- 147, doi:10.1146/annur ev - pathmechdis-0 12418-012 718 (2 02 0). 4 Coult on, A. et al. U sing a pan-cancer atlas t o inv estigate t umour assoc iat e d macrophage s a s r eg u lat ors of immun ot herapy response. Nature communications 15, 5665, doi:10. 1038 / s 4 146 7-024-4988 5 -8 ( 2024). 5 De Zuani, M. et al. S ingle-cell and s pat ial t ransc r iptomics a na ly si s of non-s mall cell lung can c er . Nature communications 15, 438 8, d oi:10.1 038/s4 1467-02 4- 48 700-8 (2024) . 6 Habib, S. et al. Tumo r as so ciated mac rophages a s key c ont ribut ors a n d targets in curr ent and fut ure ther apies for mela noma. Expert Rev Clin Immunol 20, 895- 911, doi:10.1080/ 1744 666X .2024 .2326 62 6 ( 2024). 7 Kour tzelis, I. , Hajishengallis , G. & Cha vakis, T. Phagoc y t osis of Apoptotic Cells in Resolution of Inflammation. Frontiers in immunology 11, 553, doi:10.3389/fimmu .2020. 0055 3 (202 0). 8 Uribe-Quero l, E. & Rosale s, C. Pha goc ytosis: Our Cur rent Understanding of a Univ er s al Biological Process . Frontiers in immunology 11, 1066, doi:10.3389/fimmu .2020. 0106 6 (202 0). 9 Cao, X. et al. Pr omoting antibody - dependent cellular phagocytosi s for effective macrophage- bas ed can cer immunot h erapy. Sci Adv 8 , eabl9171, doi:10.1126/ sciadv.abl917 1 (2022). 10 Astut i, Y. et al. Ef ferocytosi s r eprograms the t umor micr oenvir onm ent to p r omote pancreatic c anc er liver metastas is. Nat Cancer 5 , 774-790, doi:10. 1038 / s 4 30 18-024- 00731-2 (2 024). 11 Zhou, X. , Liu, X. & Huang, L . Ma c r ophage-Mediat ed Tumor Cell Phagocytos is : Opport unity for Nanomedicine Inter v ention. Adv Funct Mater 31, doi:10.1002/adfm.20 200 6 220 (202 1). 12 Zhang , S. et al. Efferocytosis Fuels Re quirements of F at ty Acid O xidation and t he Electron Tr ans port Chain t o Polariz e Mac r ophage s f or Tissue Repair. Cell metabolism 29, 443-45 6 e445, doi:10 .1 01 6/ j. cmet.2018.1 2 .004 (2 019). 13 Zhou, Y ., Yao, Y. , Deng, Y. & S hao, A. Regulation of efferocytosis a s a novel canc er ther apy. Cell Commun Signal 18 , 71 , do i: 10.1186/s 12964-02 0- 00542-9 (20 20). 14 Myer s, K. V. , Amend, S. R. & Pient a, K. J. Targeting Tyro3, Ax l and MerTK (TAM recepto rs): implic at ions f or macrophages in the tumor microenviro nmen t. Molecular cancer 18, 94 , doi:10.11 86/s12 943-0 19- 1022-2 (2019) . 15 Mit roulis, I. et al. Modulation of Myelopoiesis Pr og enit ors Is an Int e gral Component of Tr a ined Immunity. Cell 172, 147-16 1 e112, doi:10.1 016 / j .cell.201 7.11. 03 4 (2018). 16 Divangahi, M. et al. Tr a ined immun ity, toler anc e, pr iming and diff er ent iatio n: distinct immunological proces s e s. Nature immunology 22, 2-6, doi:10.10 38 /s4 1590- 020-00845- 6 (202 1). .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 20 17 Netea, M. G. et al. Defining tr ained immunity and its role in health and di sea se. Nature reviews. Immunology 20, 375- 388, doi:10.1 038/ s4157 7-02 0-0285-6 (20 20). 18 Kalafati, L. et al. Innate Im mune T r ain ing of G r anulopoiesis Pr omotes Anti- t umor Activity. Cell 183, doi:10.101 6/j. c ell.2 020.09. 058 ( 202 0). 19 Priem, B. et al. Trained Immun it y - Pro moting N ano biologic Ther a p y Suppr esse s Tumor G r owth and Potent iates C heck point Inhibition. Cell 183, 786-801 e719, doi:10.1016/j .cell.20 20 .09 .059 (20 20) . 20 Ding, C. et al. Ind uc in g tr ained immun it y in pr o-me t a stat ic macrop hages to cont rol tumor me tastasi s . Nature immunology 24, 239-2 54, doi:10. 1038/ s41590 -0 22-01388- 8 (2023). 21 Geller , A. E. et al. The induction of pe r ipheral tr aine d immunity in the pancreas incites ant i-tumor ac t iv it y to contr ol pa nc r eatic c ancer pr ogre s s ion. Nature communications 13, 759, doi:10 .103 8 / s4 1467-02 2- 28407-4 (202 2). 22 Liu, G . et al. Bacter ia- derived nanove s icle s en han c e t umour vacc inat ion by trained immunit y. Nat Nanotechnol 19, 387-398, doi:10 .10 38/s4 1565-023-0 15 53-6 (2024). 23 Hu a n g , A. C. & Zappa sodi, R. A decad e of chec kpo in t bloc k ade immunothe r apy in melanoma: und erstanding the mo lecular basis fo r immune sensit iv it y and r es istan ce. Nature immunology 23, 660- 670, doi: 10.1038 /s415 90-022 -0114 1-1 (2022) . 24 Scotte, F., Ratt a, R. & Beuzeboc, P. Si de effects of immuno t herapy: a cons t ant challenge f or oncologis t s . Curr Opin Oncol 31, 280-285, doi:10.1097/ CC O. 00000 0000 00005 41 (2019) . 25 Lefler, D. S., Manobianc o, S . A. & Ba s hir, B. Immunothe r apy r es istance in solid tumors: mechanis ms and potent ial solutions . Cancer Biol Ther 25, 231565 5, doi:10.1080/ 1538 4047. 2024. 23156 5 5 (2 024). 26 Schoenfeld, A. J. & Hellmann, M. D. Ac quir ed Res istance to Immune Checkpoint Inhibitors. Cancer cell 37, 443-455 , doi:10.1016 /j. cc ell.2020.03 .017 ( 20 20). 27 Kichloo, A. et al. Sy s t emic adver s e effects and toxicities a s s ociated with immuno the rapy: A r eview. World J Clin Oncol 12 , 15 0-163, doi:10.5306/ wj c o .v 12.i3.1 50 ( 2021). 28 Mod ak , M. et al. CD206+ t umor- a s so c iat ed mac r ophage s cro s s-present t umor antigen and dr iv e antit umo r immunity. JCI Insight 7 , doi:10.117 2 / j ci.ins ight .15 5022 (2022) . 29 Xiong, K. , Qi, M., Stoeger, T. , Zhang, J. & Chen, S. The role of tum or-assoc iat ed macrophage s and s o lu ble media t ors in pulmonary met as t a t ic melano ma. F rontiers in immunology 13, 1000927, doi:1 0.33 8 9/ fimmu.2022.1000 927 (202 2). 30 Adams, R. et al. Influencing tum or-ass ociated ma cr ophages in malignant melanoma with mono c lonal antibodies . Oncoimmunology 11, 2127284, doi:10.1080/ 2162 402X .2022 .2127 28 4 ( 2022). 31 Ho lt z hausen, A. et al. TAM Family R ec ept or Kinase Inhibition Reverses MDS C- Mediat ed S u ppress ion and Augments Anti- PD-1 Ther apy in Melanoma. Cancer Immunol Res 7 , 1 672-1686 , doi:10.11 5 8/ 232 6-606 6. CIR-19-0008 ( 20 19). 32 Takai, T., Li, M., S ylv estr e, D., Clynes , R. & Ravetch, J. V. FcR gamma chain deletion

in pleiotroph ic eff ec t or cell de fects . Ce ll 76, 519-529 , do i: 10.10 16/ 0 092- 8674(94) 90 115-5 (1994). 33 Takai, T. Multiple los s of eff ec t or cell f unctions in FcR gamma-def ic ient mic e. Int Rev Immunol 13, 369- 381 , doi:10.31 09/ 0 8 83018 96090 61759 (1 996). .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 21 34 Kour tzelis, I. et al. DEL-1 pr omotes macrophage effer ocy t o s is and clear ance of inflamma t ion. Nature immunology 20, 40-49, doi:1 0. 1038/s 41590-0 18-024 9 -1 (2019) . 35 Zhang , X. D. , Gillespie, S. K. & Hersey, P. St a urosporine induce s a poptosis of melanoma by bot h c aspa s e- dependent and -ind ep endent apop t otic pathw ay s . Mol Cancer Ther 3 , 1 87-1 97 ( 200 4). 36 Jent ho, E. et al. Trained inna te im mu nity, long-las t ing epig enetic modulation, and s k ewed myelopoiesis b y heme. Proceedings of the National Academy of Sci ences of the United States of America 118, doi: 10.107 3/ pna s.21 0269 8118 (20 21) . 37 Take c hi, Y ., Hara, I., N aft zger , C. , Xu, Y. & Houghton, A. N. A melanosomal membr ane protein is a cell sur fac e t arget for melanoma ther apy. Clin Cancer Res 2 , 18 37-1842 (1996) . 38 Gordon, S. Phagoc ytosis: An Immunobiologic Proce ss. Immunity 44, 463- 4 75, doi:10.1016/j .immuni.2016.02 .0 26 (2 016). 39 Pauwels, A. M., Trost, M., Be y aer t, R. & Hoff mann, E. Patter ns, Rec ept ors, and Signals: Regulation o f Phagos ome Ma tur ation. Trends in immunology 38, 407-422, doi:10.1016/j .it.2017.0 3.006 ( 2 017). 40 DeR y c kere, D. et al. U NC202 5, a MERTK Small-Molec ule Inhibit or, Is Therapeutically Effective Alone and in Comb ination w ith Metho t rexate in L eukemia Mode ls . Clin Cancer Res 23 , 14 81-1492 , doi:10.115 8/1078-04 32.C C R-16-1330 (2 01 7). 41 Green, D. R., Oguin, T. H. & Mar tinez , J . The clearance of dying cells : table f or two. Cell Death Differ 23, 915 -926, doi:10. 1038/ c dd .20 15.1 72 (2016). 42 Hoc hr eiter - Hu ff ord, A. & Ravichandran, K. S. Clearing the dead: apoptot ic c ell sens ing, re cognition, eng ulf ment, and digestion. Cold Spring Harbor perspectives in biology 5 , a008748 , doi:10.11 01/ cshp erspect.a0 08748 (2 013) . 43 Stunault , M. I., Bories, G ., Guinamard, R. R. & Ivanov, S. Metabolism Pla y s a Key Role dur in g Mac r ophage Activation. Medi ators Inflamm 2018 , 24 2613 8, doi:10.1155/ 2018 /2426 138 (201 8). 44 Schilp eroor t, M. et al. The rol e of e ffe roc ytosis-fueled macropha ge met abolis m in the r es olut ion of in flamma t ion. Immunol Rev 319, 65-80, doi:10. 1111/imr. 13 214 (2023) . 45 Lang, C. et al. Efferocytosi s dr ives my eloid NL RP3 d ependent infla mma some signa ling se c r etion of IL-1be t a t o pro mote tu mor growt h. Frontiers in immunology 13, 993 771, doi:10 .3389 /fimmu. 2022 .993771 (2 022). 46 Kaplanov, I. et al. Blockin g IL-1bet a r everses th e immunosuppres s ion in mous e breast cancer and syner gi z es w it h anti-PD-1 for t umor abrogation. Proceedings of the National Academy of Sciences of the United States of America 116, 1361-1 369, doi:10.1073/pna s .1812 2661 15 (2019) . 47 Ga r landa, C. & Mantovani, A. Int erleu kin-1 in tumor p rogr es sion, ther apy , and prevent io n. Cance r cell 39, 1023-102 7 , doi:10.101 6/ j. c cell. 2021.0 4.011 (2 021). 48 van Vlerken-Ysla, L . , Tyur ina , Y. Y., Kagan, V. E. & G abrilovich, D. I. Functional states of myeloid cells in cancer. Cancer cell 41, 490-50 4, do i: 10.1 016/ j .c cell.2 023 .02.009 (2023) . 49 Choi, J. Y. et al. Mer signaling increa s es the abundanc e of the tr a ns cript ion factor LXR to pr omo t e t he resolution o f acut e ster ile inf la mmation. Science signaling 8 , r a21, doi:10.1126/ sci sig n al.20 05 86 4 ( 2015) . .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint 22 50 Kang, Y. Y . et al. Innate Immune Tr ain ing Initiates Efferocytosi s t o Prot ec t a gainst Lung Injury. Adv Sci (Weinh) 11, e230 8978, doi:10 .1 002 / advs .2023 0897 8 (2024). 51 Liebold, I. et al. Apopt o tic c ell identity induc e s di s t inc t functional response s t o IL - 4 in effer ocy t ic macro phage s . Science 384, eabo7027, doi:10. 1126 / s cien ce.abo 7027 (2024) . 52 Jiang, Z. et al. T YRO3 induc e s anti-PD-1/PD-L1 ther apy resistance by limitin g innate immunit y and t umo r al ferrop tosis . The Journal of clinical investigation 131, doi:10.1172/J CI139 434 ( 202 1). 53 Qiu, H. et al. Effe r ocy t osis: An ac complic e of c ancer immune es cape. Biomed Pharmacother 167, 115 540 , doi:10.1 016/j.biopha.20 23.11 554 0 ( 20 23). 54 DeRy c kere, D., Huelse, J. M. , Earp, H. S. & Graham, D. K. TAM family kinas es as ther apeut ic target s at t he inter f a ce of cancer and immunit y. Nat Rev Clin Oncol 20, 755-779, do i: 10. 1038 /s4157 1-023-00 81 3-7 (2023). 55 Mas s , E., Nimmerjahn, F., Kier dorf, K. & S chlit z er , A. Tissue-spe cif ic macrop hag es : how they develop and choreogr aph tis s ue biology. Nature reviews. Immunology 23 , 563-579, do i: 10. 1038 /s4157 7-023-00 84 8-y (202 3). 56 Vus can, P., Kischkel, B., Joosten, L. A. B. & Netea, M. G . Trained immunity: G ener al and emer g ing concept s. Immunol Rev 323, 16 4-185, doi:10. 111 1/ imr .13 32 6 (2024). 57 Smalley, K. S., Er oglu, Z . & Sonda k, V. K. Combinat ion Therapies for Melanoma: A New St a n dard of Car e? Am J Clin Dermatol 17, 99-105, doi:10.1 0 07/s4 025 7-016- 0174-8 (2 016). 58 Ho eller , C. The futur e of combina tion t herapies in advanced melanoma. memo - Magazine of European Medical Oncology 13 , 30 9-313 , doi:10.10 07/ s 12 254 - 020- 00640-x (2020) . 59 Jaak s , P. et al. Ef fec t iv e dr ug c ombinations in breast, colon and pancreatic c ancer cells. Nature 603 , 166- 173 , doi:10.10 38/s4158 6-022- 0443 7-2 (2022). 60 Fis usi, F. A. & A ka la, E. O. Dr ug Combinations in Breast Cancer Ther a py. Pharm Nanotechnol 7 , 3-23, doi:10.217 4/22 117385 07666 19012 2111 224 ( 201 9). 61 Vet v icka, V., Vannuc ci, L ., Sima, P. & Ric ht er, J. Bet a Glucan: Supplement o r Dr ug? From Labor a t ory to Clin ical Trials. Molecules 24 , doi:10.339 0/molecules240 71251 (2019) . 62 Cognigni, V., Ranallo, N., Tronconi, F., Morgese, F . & Ber ardi, R. Pot ential b enefit of beta-glucans a s adjuvant therapy in immuno-onc ology: a review. Explor Target Antitumor Ther 2 , 122-138, doi:10 .37 349/etat.20 21. 00036 (2 021). 63 Kremenovic, M., Schenk , M. & Lee, D. J. Clinical and molec ular ins ights into BCG immuno the rapy for melan oma. J Inte rn Med 288 , 625-6 40 , d oi:10.1 111/ joi m.1 3037 (2020) . .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 12, 2024. ; https://doi.org/10.1101/2024.11.11.622862doi: bioRxiv preprint