Microglial MERTK eliminates phosphatidylserine-displaying inhibitory post-synapses.

Glia contribute to synapse elimination through phagocytosis in the central nervous system. Despite the important roles of this process in development and neurological disorders, the identity and regulation of the "eat-me" signal that initiates glia-mediated phagocytosis of synapses has remained incompletely understood. Here, we generated conditional knockout mice with neuronal-specific deletion of the flippase chaperone Cdc50a, to induce stable exposure of phosphatidylserine, a well-known "eat-me" signal for apoptotic cells, on the neuronal outer membrane. Surprisingly, acute Cdc50a deletion in mature neurons causes preferential phosphatidylserine exposure in neuronal somas and specific loss of inhibitory post-synapses without effects on other synapses, resulting in abnormal excitability and seizures. Ablation of microglia or the deletion of microglial phagocytic receptor Mertk prevents the loss of inhibitory post-synapses and the seizure phenotype, indicating that microglial phagocytosis is responsible for inhibitory post-synapse elimination. Moreover, we found that phosphatidylserine is used for microglia-mediated pruning of inhibitory post-synapses in normal brains, suggesting that phosphatidylserine serves as a general "eat-me" signal for inhibitory post-synapse elimination.

Molecular Force Imaging Reveals That Integrin-Dependent Mechanical Checkpoint Regulates Fcγ-Receptor-Mediated Phagocytosis in Macrophages.

Macrophages are a type of immune cell that helps eliminate pathogens and diseased cells. Recent research has shown that macrophages can sense mechanical cues from potential targets to perform effective phagocytosis, but the mechanisms behind it remain unclear. In this study, we used DNA-based tension probes to study the role of integrin-mediated forces in FcγR-mediated phagocytosis. The results showed that when the phagocytic receptor FcγR is activated, the force-bearing integrins create a "mechanical barrier" that physically excludes the phosphatase CD45 and facilitates phagocytosis. However, if the integrin-mediated forces are physically restricted at lower levels or if the macrophage is on a soft matrix, CD45 exclusion is significantly reduced. Moreover, CD47-SIRPα "don't eat me" signaling can reduce CD45 segregation by inhibiting the mechanical stability of the integrin barrier. These findings demonstrate how macrophages use molecular forces to identify physical properties and combine them with biochemical signals from phagocytic receptors to guide phagocytosis.

Effects of blocking CD24 and CD47 'don't eat me' signals in combination with rituximab in mantle-cell lymphoma and chronic lymphocytic leukaemia.

Mantle-cell lymphoma (MCL) is a B-cell non-Hodgkin Lymphoma (NHL) with a poor prognosis, at high risk of relapse after conventional treatment. MCL-associated tumour microenvironment (TME) is characterized by M2-like tumour-associated macrophages (TAMs), able to interact with cancer cells, providing tumour survival and resistance to immuno-chemotherapy. Likewise, monocyte-derived nurse-like cells (NLCs) present M2-like profile and provide proliferation signals to chronic lymphocytic leukaemia (CLL), a B-cell malignancy sharing with MCL some biological and phenotypic features. Antibodies against TAMs targeted CD47, a 'don't eat me' signal (DEMs) able to quench phagocytosis by TAMs within TME, with clinical effectiveness when combined with Rituximab in pretreated NHL. Recently, CD24 was found as valid DEMs in solid cancer. Since CD24 is expressed during B-cell differentiation, we investigated and identified consistent CD24 in MCL, CLL and primary human samples. Phagocytosis increased when M2-like macrophages were co-cultured with cancer cells, particularly in the case of paired DEMs blockade (i.e. anti-CD24 + anti-CD47) combined with Rituximab. Similarly, unstimulated CLL patients-derived NLCs provided increased phagocytosis when DEMs blockade occurred. Since high levels of CD24 were associated with worse survival in both MCL and CLL, anti-CD24-induced phagocytosis could be considered for future clinical use, particularly in association with other agents such as Rituximab.

Prognostic role of the innate immune signature CD163 and "eat me" signal calreticulin in clear cell renal cell carcinoma.

The effects of the innate immune status on patients with clear cell renal cell carcinoma (ccRCC) currently remain unknown. We herein provided more extensive information about the inner landscape of immunobiology of ccRCC. In total, 260 ccRCC samples from three different cohorts consisting of 213 primary tumors and 47 metastases were obtained. We focused on five representative innate immune signatures, CD68, CD163, the "eat me" signal calreticulin, the "don't eat me" signal CD47, and signal regulatory protein α, and examined the role of each signature by quantitative immunohistochemistry. We then conducted an integrated genome mutation analysis by next-generation sequencing. Among the five markers, high CD163 and low calreticulin expression levels were prognostic in ccRCC. The application of a new risk model based on CD163 and calreticulin levels, named the innate immune risk group (high risk: high-CD163/low calreticulin, intermediate risk: high-CD163/high calreticulin or low CD163/low calreticulin, low risk: low-CD163/high calreticulin), enabled the sequential stratification of patient prognosis and malignancy. Although organ-specific differences were observed, metastases appeared to have a higher innate immune risk, particularly in the lungs, with 50% of ccRCC metastases being classified into the high-risk group according to our risk score. An analysis of genomic alterations based on the innate immune risk group revealed that alterations in the TP53/Cell cycle pathway were highly prevalent in high-risk ccRCC patients according to two innate immune signatures CD163 and calreticulin. The present results provide insights into the immune-genomic biology of ccRCC tumors for innate immunity and will contribute to future therapies focused on the innate immune system in solid cancers.

Breaking the 'don't eat me' signal: in silico design of CD47-directed peptides for cancer immunotherapy.

The leading cause of death worldwide is cancer. Although there are various therapies available to treat cancer, finding a successful one can be like searching for a needle in a haystack. Immunotherapy appears to be one of those needles in the haystack of cancer treatment. Immunotherapeutic agents enhance the immune response of the patient's body to tumor cells. One of the immunotherapeutic targets, Cluster of Differentiation 47 (CD47), releases the "don't eat me" signal when it binds to its receptor, Signal Regulatory Protein (SIRPα). Tumor cells use this signal to circumvent the immune system, rendering it ineffective. To stop tumor cells from releasing the "don't eat me" signal, the CD47-SIRPα interaction is specifically targeted in this study. To do so, in silico peptides were designed based on the structural analysis of the interaction between two proteins using point mutations on the interacting residues with the other amino acids. The peptide library was designed and docked on SIRPα using computational tools. Later on, after analyzing the docked complex, the best of them was selected for MD simulation studies of 100 ns. Further analysis after MD studies was carried out to determine the possible potential anti-SIRPα peptides.

Non-coding RNAs and lipids mediate the function of extracellular vesicles in cancer cross-talk.

Research on extracellular vesicles (EVs) has been expanded, especially in the field of cancer. The cargoes in EVs, especially those in small EVs such as exosomes include microRNAs (miRNAs), mRNA, proteins, and lipids, are assumed to work cooperatively in the tumor microenvironment. In 2007, it was reported that miRNAs were abundant among the non-coding RNAs present in exosomes. Since then, many studies have investigated the functions of miRNAs and have tried to apply these molecules to aid in the diagnosis of cancer. Accordingly, many reviews of non-coding RNAs in EVs have been published for miRNAs. This review focuses on relatively new cargoes, covering long noncoding (lnc) RNAs, circular RNAs, and repeat RNAs, among non-coding RNAs. These RNAs, regardless of EV or cell type, have newly emerged due to the innovation of sequencing technology. The poor conservation, low quantity, and technical difficulty in detecting these RNA types have made it difficult to elucidate their functions and expression patterns. We herein summarize a limited number of studies. Although lipids are major components of EVs, current research on EVs focuses on miRNA and protein biology, while the roles of lipids in exosomes have not drawn attention. However, several recent studies revealed that phospholipids, which are components of the EV membrane, play important roles in the intercommunication between cells and in the generation of lipid mediators. Here, we review the reported roles of these molecules, and describe their potential in cancer biology.

Multifunctional Immunoliposomes Enhance the Immunotherapeutic Effects of PD-L1 Antibodies against Melanoma by Reprogramming Immunosuppressive Tumor Microenvironment.

The immunosuppressive tumor microenvironment (TME) can significantly limit the immunotherapeutic effects of the PD-L1 antibody (aPDL1) by inhibiting the infiltration of CD8+ cytotoxic T cells (CTLs) into the tumor tissues. However, how to reprogram the immunosuppressive TME and promote the infiltration of CTLs remains a huge challenge for aPDL1 to achieve the maximum benefits. Herein, the authors design a multifunctional immunoliposome that encapsulates the adrenergic receptor blocker carvedilol (CAR) and connects the "don't eat me" signal antibody (aCD47) and aPDL1 in series via a reactive oxygen species (ROS)-sensitive linker on the surface. In ROS-enriched immunosuppressive TME, the multifunctional immunoliposome (CAR@aCD47/aPDL1-SSL) can first release the outer aCD47 to block the "do not eat me" pathway, promote the phagocytosis of tumor cells by phagocytic cells, and activate CTLs. Then, the aPDL1 on the liposome surface is exposed to block the PD-1/PD-L1 signaling pathway, thereby inducing CTLs to kill tumor cells. CAR encapsulated in CAR@aCD47/aPDL1-SSL can block the adrenergic nerves in the tumor tissues and reduce their densities, thereby inhibiting angiogenesis in the tumor tissues and reprogramming the immunosuppressive TME. According to the results, CAR@aCD47/aPDL1-SSL holds an effective way to reprogram the immunosuppressive TME and significantly enhance immunotherapeutic efficiency of aPDL1 against the primary cancer and metastasis.

The expression profiles of CD47 in the tumor microenvironment of salivary gland cancers: a next step in histology-driven immunotherapy.

BACKGROUND: Salivary gland carcinomas (SGC) are extremely rare malignancies with only limited treatment options for the metastatic phase of the disease. Treatment with anti-CD47 antibodies could represent a potent therapy for SGCs by promoting the phagocytic clearance of tumor cells through various mechanisms. However, the efficacy of anti-CD47 therapy is largely dependent on the expression of CD47 within the tumor microenvironment (TME). MATERIALS AND METHODS: In 43 patients with SGC, we were the first to investigate the CD47 expression in both tumor cells and tumor-infiltrating immune cells (TIIC) in the center and periphery of primary tumors. We also correlated the data with the clinicopathological variables of the patients and offered novel insights into the potential effectiveness of anti-CD47 therapy in SGCs. RESULTS: We observed that the CD47+ tumor cells are outnumbered by CD47+ TIICs in mucoepidermoid carcinoma. In the tumor center, the proportion of CD47+ tumor cells was comparable to the proportion of CD47+ TIICs in most histological subtypes. In low-grade tumors, significantly higher expression of CD47 was observed in TIICs in the periphery of the tumor as compared to the center of the tumor. CONCLUSION: The reason for a high expression of 'don't eat me' signals in TIICs in the tumor periphery is unclear. However, we hypothesize that in the tumor periphery, upregulation of CD47 in TIICs could be a mechanism to protect newly recruited leukocytes from macrophage-mediated phagocytosis, while also allowing the removal of old or exhausted leukocytes in the tumor center.

Phagocytic elimination of synapses by microglia during sleep.

Synaptic strength reduces during sleep, but the underlying mechanisms of this process are unclear. This study showed reduction of synaptic proteins in rat prefrontal cortex (PFC) at AM7 or Zeitgeber Time (ZT0), when the light phase or sleeping period for rats started. At this time point, microglia were weakly activated, displaying larger and more granular somata with increased CD11b expression compared with those at ZT12, as revealed by flow cytometry. Expression of opsonins, such as complements or MFG-E8, matrix metalloproteinases, and microglial markers at ZT0 were increased compared with that at ZT12. Microglia at ZT0 phagocytosed synapses, as revealed by immunohistochemical staining. Immunoblotting detected more synapsin I in the isolated microglia at ZT0 than at ZT12. Complement C3- or MFG-E8-bound synapses were the most abundant at ZT0, some of which were phagocytosed by microglia. Systemic administration of synthetic glucocorticoid dexamethasone reduced microglial size, granularity and CD11b expression at ZT0, resembling microglia at ZT12, and increased synaptic proteins and decreased the sleeping period. Noradrenaline (NA) suppressed glutamate-induced phagocytosis in primary cultured microglia. Systemic administration of the brain monoamine-depleting agent reserpine decreased NA content and synapsin I expression in PFC, and increased expression of microglia markers, C3 and MFG-E8, while increasing the sleeping period. A NA precursor l-threo-dihydroxyphenylserine abolished the reserpine-induced changes. These results suggest that microglia may eliminate presumably weak synapses during every sleep phase. The circadian changes in concentrations of circulating glucocorticoids and brain NA might be correlated with the circadian changes of microglial phenotypes and synaptic strength.

HIF-1 regulates CD47 expression in breast cancer cells to promote evasion of phagocytosis and maintenance of cancer stem cells.

Increased expression of CD47 has been reported to enable cancer cells to evade phagocytosis by macrophages and to promote the cancer stem cell phenotype, but the molecular mechanisms regulating CD47 expression have not been determined. Here we report that hypoxia-inducible factor 1 (HIF-1) directly activates transcription of the CD47 gene in hypoxic breast cancer cells. Knockdown of HIF activity or CD47 expression increased the phagocytosis of breast cancer cells by bone marrow-derived macrophages. CD47 expression was increased in mammosphere cultures, which are enriched for cancer stem cells, and CD47 deficiency led to cancer stem cell depletion. Analysis of datasets derived from thousands of patients with breast cancer revealed that CD47 expression was correlated with HIF target gene expression and with patient mortality. Thus, CD47 expression contributes to the lethal breast cancer phenotype that is mediated by HIF-1.

Macrophages eat cancer cells using their own calreticulin as a guide: roles of TLR and Btk.

Macrophage-mediated programmed cell removal (PrCR) is an important mechanism of eliminating diseased and damaged cells before programmed cell death. The induction of PrCR by eat-me signals on tumor cells is countered by don't-eat-me signals such as CD47, which binds macrophage signal-regulatory protein α to inhibit phagocytosis. Blockade of CD47 on tumor cells leads to phagocytosis by macrophages. Here we demonstrate that the activation of Toll-like receptor (TLR) signaling pathways in macrophages synergizes with blocking CD47 on tumor cells to enhance PrCR. Bruton's tyrosine kinase (Btk) mediates TLR signaling in macrophages. Calreticulin, previously shown to be an eat-me signal on cancer cells, is activated in macrophages for secretion and cell-surface exposure by TLR and Btk to target cancer cells for phagocytosis, even if the cancer cells themselves do not express calreticulin.

"Velcro" engineering of high affinity CD47 ectodomain as signal regulatory protein α (SIRPα) antagonists that enhance antibody-dependent cellular phagocytosis.

CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy.

Pyroptotic cells externalize eat-me and release find-me signals and are efficiently engulfed by macrophages.

Pathogenic intracellular bacteria often hijack macrophages for their propagation. The infected macrophages release IL-1ß and IL-18 and simultaneously commit suicide, which is called pyroptosis; both responses require caspase-1. Here, we found that pyroptotic cells induced by microbial infection were efficiently engulfed by human monocytic THP-1-cell-derived macrophages or mouse peritoneal macrophages. This engulfment was inhibited by the D89E mutant of milk fat globule (MFG) epidermal growth factor (EGF) factor 8 (MFG-E8; a phosphatidylserine-binding protein) that has been shown previously to inhibit phosphatidylserine-dependent engulfment of apoptotic cells by macrophages, suggesting that the engulfment of pyroptotic cells by macrophages was also phosphatidylserine dependent. Using a pair of cell lines that respectively exhibited pyroptosis or apoptosis after muramyl dipeptide treatment, we showed that both pyroptotic and apoptotic cells bound to a T-cell immunoglobulin and mucin domain-containing 4 (Tim4; another phosphatidylserine-binding protein)-coated plate, whereas heat-killed necrotic cells did not, indicating that phosphatidylserine was externalized in pyroptosis and apoptosis but not in accidental necrosis. Macrophages engulfed apoptotic cells most efficiently, followed by pyroptotic and then heat-killed necrotic cells. Pyroptotic cells also released a macrophage attractant(s), 'find-me' signal, whose activity was diminished by apyrase that degrades nucleoside triphosphate to nucleoside monophosphate. Heat-killed necrotic cells and pyroptotic cells released ATP much more efficiently than apoptotic cells. These results suggest that pyroptotic cells, like apoptotic cells, actively induce phagocytosis by macrophages using 'eat-me' and find-me signals. Based on these results, a possible role of coordinated induction of pyroptosis and inflammatory cytokine production is discussed.

YB-1-based oncolytic virotherapy in combination with CD47 blockade enhances phagocytosis of pediatric sarcoma cells.

Oncolytic viruses (OVs) selectively replicate in tumor cells resulting in lysis, spreading of new infectious units and induction of antitumor immune responses through abrogating an immunosuppressive tumor microenvironment (TME). Due to their mode of action, OVs are ideal combination partners with targeted immunotherapies. One highly attractive combination is the inhibition of the 'don't-eat-me'-signal CD47, which is known to increase the phagocytic potential of tumor-associated macrophages. In this work, we analyzed the combination approach consisting of the YB-1-based oncolytic adenovirus XVir-N-31 (XVir) and the CD47 inhibitor (CD47i) B6.H12.2 concerning its phagocytic potential. We investigate phagocytosis of XVir-, adenovirus wildtype (AdWT)-, and non-infected established pediatric sarcoma cell lines by different monocytic cells. Phagocytes (immature dendritic cells and macrophages) were derived from THP-1 cells and healthy human donors. Phagocytosis of tumor cells was assessed via FACS analysis in the presence and absence of CD47i. Additional characterization of T cell-stimulatory surface receptors as well as chemo-/cytokine analyses were performed. Furthermore, tumor cells were infected and studied for the surface expression of the 'eat-me'-signal calreticulin (CALR) and the 'don't-eat-me'-signal CD47. We herein demonstrate that (1) XVir-infected tumor cells upregulate both CALR and CD47. XVir induces higher upregulation of CD47 than AdWT. (2) XVir-infection enhances phagocytosis in general and (3) the combination of XVir and CD47i compared to controls showed by far superior enhancement of phagocytosis, tumor cell killing and innate immune activation. In conclusion, the combination of CD47i and XVir causes a significant increase in phagocytosis exceeding the monotherapies considerably accompanied by upregulation of T cell-stimulatory receptor expression and inflammatory chemo/-cytokine secretion.

Septin 9 expression regulates 'don't eat me' signals and identifies an immune-epithelial class of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is a highly heterogeneous and aggressive liver cancer with limited therapeutic options. Precise classification and immunotherapy are perspectives to improve the treatments. We reported the role of septin 9 in apico-basal polarity and epithelial-to-mesenchymal transition (EMT). Here, we aim to elucidate its role in iCCA. We analyzed single-cell transcriptomes from human iCCA tumor cells based on phenotype and cell state. Knockdown of the septin 9 gene (SEPT9) was done using small interfering RNA (siRNA); interferon-γ (IFN-γ) stimulation was performed using different CCA cells; gene expressions were analyzed by reverse transcription and real-time PCR analysis (RT-qPCR); and immunofluorescence, immunoblotting, and flow cytometry were performed to assess the expression of proteins. The differential distributions of SEPT9 and vimentin (VIM) gene expressions allowed us to define specific cellular trajectories of malignant cells and thus identified distinct clusters of iCCA cells. One cluster was enriched in VIM and extracellular-matrix (ECM) remodeling molecules, and another had high expression of SEPT9 and genes from the 'don't eat me' signal involved in immune escape. This antagonism between SEPT9 and VIM was confirmed by in vitro experiments. Notably, SEPT9 and 'don't eat me' gene expressions were inversely correlated to those of vimentin and the EMT markers. SEPT9 expression was upregulated by IFN-γ and SEPT9 knockdown decreased expression of 'don't eat me' signal genes and increased expression of mesenchymal markers. Cancer Cell Line Encyclopedia (CCLE) transcriptome database analyses confirmed that iCCA cells enriched in septin 9 exhibit epithelial-like features. This study revealed septin 9 as a cytoskeleton element of iCCA epithelial-like cells and a regulator of the immune system response. It also brings new insights into the enigmatic relationship between EMT and immune response. Notably, we decoded a potential mechanism that could sensitize patients to immunotherapies.

Enhanced treatment of breast cancer brain metastases with oncolytic virus expressing anti-CD47 antibody and temozolomide.

Limited therapeutic options are available for patients with breast cancer brain metastases (BCBM), and thus there is an urgent need for novel treatment approaches. We previously engineered an effective oncolytic herpes simplex virus 1 (oHSV) expressing a full-length anti-CD47 monoclonal antibody (mAb) with a human IgG1 scaffold (OV-αCD47-G1) that was used to treat both ovarian cancer and glioblastoma. Here, we demonstrate that the combination of OV-αCD47-G1 and temozolomide (TMZ) improve outcomes in preclinical models of BCBM. The combination of TMZ with OV-αCD47-G1 synergistically increased macrophage phagocytosis against breast tumor cells and led to greater activation of NK cell cytotoxicity. In addition, the combination of OV-αCD47-G1 with TMZ significantly prolonged the survival of tumor-bearing mice when compared with TMZ or OV-αCD47-G1 alone. Combination treatment with the mouse counterpart of OV-αCD47-G1, termed OV-A4-IgG2b, also enhanced mouse macrophage phagocytosis, NK cell cytotoxicity, and survival in an immunocompetent model of mice bearing BCBM compared with TMZ or OV-A4-IgG2b alone. Collectively, these results suggest that OV-αCD47-G1 combined with TMZ should be explored in patients with BCBM.

"Find Me" and "Eat Me" signals: tools to drive phagocytic processes for modulating antitumor immunity.

Phagocytosis, a vital defense mechanism, involves the recognition and elimination of foreign substances by cells. Phagocytes, such as neutrophils and macrophages, rapidly respond to invaders; macrophages are especially important in later stages of the immune response. They detect "find me" signals to locate apoptotic cells and migrate toward them. Apoptotic cells then send "eat me" signals that are recognized by phagocytes via specific receptors. "Find me" and "eat me" signals can be strategically harnessed to modulate antitumor immunity in support of cancer therapy. These signals, such as calreticulin and phosphatidylserine, mediate potent pro-phagocytic effects, thereby promoting the engulfment of dying cells or their remnants by macrophages, neutrophils, and dendritic cells and inducing tumor cell death. This review summarizes the phagocytic "find me" and "eat me" signals, including their concepts, signaling mechanisms, involved ligands, and functions. Furthermore, we delineate the relationships between "find me" and "eat me" signaling molecules and tumors, especially the roles of these molecules in tumor initiation, progression, diagnosis, and patient prognosis. The interplay of these signals with tumor biology is elucidated, and specific approaches to modulate "find me" and "eat me" signals and enhance antitumor immunity are explored. Additionally, novel therapeutic strategies that combine "find me" and "eat me" signals to better bridge innate and adaptive immunity in the treatment of cancer patients are discussed.

Don't eat me/eat me signals as a novel strategy in cancer immunotherapy.

Cancer stands as one of the prominent global causes of death, with its incidence burden continuously increasing, leading to a substantial rise in mortality rates. Cancer treatment has seen the development of various strategies, each carrying its drawbacks that can negatively impact the quality of life for cancer patients. The challenge remains significant within the medical field to establish a definitive cancer treatment that minimizes complications and limitations. In the forthcoming years, exploring new strategies to surmount the failures in cancer treatment appears to be an unavoidable pursuit. Among these strategies, immunology-based ones hold substantial promise in combatting cancer and immune-related disorders. A particular subset of this approach identifies "eat me" and "Don't eat me" signals in cancer cells, contrasting them with their counterparts in non-cancerous cells. This distinction could potentially mark a significant breakthrough in treating diverse cancers. By delving into signal transduction and engineering novel technologies that utilize distinct "eat me" and "Don't eat me" signals, a valuable avenue may emerge for advancing cancer treatment methodologies. Macrophages, functioning as vital components of the immune system, regulate metabolic equilibrium, manage inflammatory disorders, oversee fibrosis, and aid in the repair of injuries. However, in the context of tumor cells, the overexpression of "Don't eat me" signals like CD47, PD-L1, and beta-2 microglobulin (B2M), an anti-phagocytic subunit of the primary histocompatibility complex class I, enables these cells to evade macrophages and proliferate uncontrollably. Conversely, the presentation of an "eat me" signal, such as Phosphatidylserine (PS), along with alterations in charge and glycosylation patterns on the cellular surface, modifications in intercellular adhesion molecule-1 (ICAM-1) epitopes, and the exposure of Calreticulin and PS on the outer layer of the plasma membrane represent universally observed changes on the surface of apoptotic cells, preventing phagocytosis from causing harm to adjacent non-tumoral cells. The current review provides insight into how signaling pathways and immune cells either stimulate or obstruct these signals, aiming to address challenges that may arise in future immunotherapy research. A potential solution lies in combination therapies targeting the "eat me" and "Don't eat me" signals in conjunction with other targeted therapeutic approaches. This innovative strategy holds promise as a novel avenue for the future treatment of cancer.

A novel anti-CD47-targeted blockade promotes immune activation in human soft tissue sarcoma but does not potentiate anti-PD-1 blockade.

PURPOSE: The treatment options for metastatic soft tissue sarcomas (STSs) are limited. In most cases, immunotherapy with immune checkpoint inhibitors has not been successful so far. Macrophages dominate the immune landscape of STSs; thus, combinatorial strategies aiming at both tumor-infiltrating lymphocytes and macrophages may represent a particularly relevant treatment approach for metastatic or recurrent STSs. METHODS: In this cohort study, 66 patients who underwent surgery for STSs were enrolled. Tumor cells and tumor-infiltrating immune cells were analyzed using flow cytometry and immunohistochemistry. In cell suspensions obtained from surgical resections, human T cells were activated by superparamagnetic polymer beads and cultured at a concentration of 0.3 × 106/µl in the absence or presence of therapeutic monoclonal antibodies (anti-PD-1, anti-CD47, and anti-PD-1 + anti-CD47). Supernatants from cell suspensions were analyzed using multiplex Luminex cytokine bead-based immunoassays. RESULTS: The most profound response to anti-CD47 therapy was observed in an undifferentiated pleiomorphic sarcoma which also displayed high expression of CD47 in the tumor microenvironment. Both anti-PD-1 and anti-CD47 therapies drastically increased the production of pro-inflammatory cytokines in the tumor microenvironment of STSs, but co-administration of both agents did not further increase cytokine secretion. Furthermore, all patient samples treated with a combination of both anti-PD-1 and anti-CD47 antibodies showed a dramatic reduction in cytokine secretion. CONCLUSION: Our findings suggest that anti-PD-1 and anti-CD47 therapies do not enhance each other, and the combined application of anti-PD-1 and anti-CD47 agents in vitro limits rather than potentiates their efficacy.

Monocytes engineered with iSNAP inhibit human B-lymphoma progression.

Monocytes are important regulators for the maintenance of homeostasis in innate and adaptive immune system and have been reported to play important role in cancer progression. CD47-SIRPα recognition is a coinhibitory immune signal to inhibit phagocytosis in monocytes and macrophages and has been well-known as the "Don't eat me" signal. By using an approach of integrated sensing and activating proteins (iSNAPs), we have rewired the CD47-SIRPα axis to create iSNAP-M which activates pathways in engineered human monocytes (iSNAP-MC). The mRNA expression levels of the monocyte/macrophage markers CD11b, CD14, and CD31 are upregulated in iSNAP-monocytes (iSNAP-MC). With PMA induction, the iSNAP-MC-derived macrophages (iSNAP-MΦ) showed upregelation in CD86 and CD80, but not CD206. TNFα expression and secretion were also increased in iSNAP-MΦ. Furthermore, the injection of iSNAP-MC into mice bearing human B-lymphoma tumors led to the suppression of tumor progression. Therefore, the engineered monocytes, via blockage of coinhibitory immune signals by rewiring CD47-SIRPα axis, can be applied to suppress target tumors for cancer immunotherapy.