Engineering M1 macrophages with targeting aptamers for enhanced adoptive immunotherapy by modifying the cell surface

Engineering M1 macrophages with targeting aptamers for enhanced adoptive immunotherapy by modifying the cell surface | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Engineering M1 macrophages with targeting aptamers for enhanced adoptive immunotherapy by modifying the cell surface Qian Yang, Shiyi Hu, Yiqiu Wang, Luyi Zhong, Xiaoli Yu, Yifeng Zhang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4250998/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Jul, 2024 Read the published version in Biomedicine & Pharmacotherapy → Version 1 posted You are reading this latest preprint version Abstract Macrophages play a critical role in the body's defense against cancer by phagocytosing tumor cells, presenting antigens, and activating adaptive T cells. However, macrophages are intrinsically incapable of delivering targeted cancer immunotherapies. Engineered adoptive cell therapy introduces new targeting and antitumor capabilities by modifying macrophages to enhance the innate immune response of cells and improve clinical efficacy. In this study, we developed engineered macrophage cholesterol-AS1411-M1 (CAM1) for cellular immunotherapy. To target macrophages, cholesterol-AS1411 aptamers are anchored to the surface of M1 macrophages to produce CAM1 without genetic modification or cell damage. CAM1 induced significantly higher apoptosis/mortality than unmodified M1 macrophages in murine breast cancer cells. Anchoring AS1411 on the surface of macrophages without modifying their original genes and proteins provides a novel approach to tumor immunotherapy. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights 1. M2 phenotype macrophage-targeted Lipo@CpG-FA was used to repolarize TAMs and reverse immunosuppressive TME. 2. The synergic nanoformulation reduced M2 macrophages and caused regression and inhibition of 4T1 breast cancers. Introduction Adoptive cell immunotherapy uses the autoimmune system as an active immune therapy. This approach introduces new antitumor and targeting capabilities by modifying autologous immune cells, thereby enhancing their inherent immune response [1–4] . These cells were isolated from the host, genetically engineered, expanded ex vivo, and reinjected into the patient to eliminate tumor cells [5–8] . Chimeric Antigen Receptor (CAR) technology is a powerful engineering platform in immunotherapy, with CAR-T emerging as a new revolutionary pillar in tumor therapy[ 3 , 9 ]. It is extensively used to treat hematological malignancies and has demonstrated significant clinical efficacy[ 10 – 12 ]. Despite these successes with CAR-T, this genetic engineering strategy may have potential problems and risks[ 13 ]. To date, CAR-T has demonstrated higher clinical efficacy in hematological tumors than in solid tumors. Tumor development restricts T-cell recruitment and infiltration, activates extensive inhibitory pathways to limit T-cell activation, and expresses heterogeneous tumor-associated antigens (TAA), which makes it difficult for CAR-T cells to migrate to solid tumors and survive in the tumor microenvironment (TME)[ 14 – 19 ]. Given these limitations, there is growing interest in exploring novel cell-based immunotherapies that can be universally applied. Macrophages are important innate immune cells. With the increase in tumor volume and development of intratumoral vascular networks, blood monocytes infiltrate into tumor tissues and mature into tumor-associated macrophages (TAMs) [ 20 ]. Compared to T cells, macrophages can influence the surrounding immune cells through pro-inflammatory and anti-inflammatory effects and excel in remodeling the extracellular matrix (ECM) [ 21 , 22 ]. These advantages enable macrophages to easily penetrate solid tumor tissues and interact with tumor cells[ 23 ]. Consequently, engineering macrophages offers new avenues for cancer therapy[ 24 ]. Among the cell types employed in immunotherapy, macrophages have emerged as primary candidates for treating solid tumors. In addition, using CAR-IFN-γ-encoding plasmid DNA nanocomposites in CAR-M development has been observed to enhance antitumor immunoregulatory capabilities of macrophages and inhibit solid tumor growth[ 25 ]. These genetically engineered macrophages hold significant potential for application prospects in treating solid tumors. However, their further utilization is hindered by issues, such as potential target organ toxicity, heterogeneous expression of engineered proteins, and transgene insertional mutagenesis[ 26 , 27 ]. Furthermore, the production of these genetically engineered cells is time-consuming and costly. Consequently, there is a pressing need to develop simpler and safer technologies to overcome the challenges associated with tumor therapy. The precise delivery of anticancer drugs to tumor cells is one of the most important aspects of cancer medicine. This strategy emphasizes not only improving the efficacy of anticancer agents but also reducing nonspecific side effects[ 28 ]. Aptamers are short nucleotide sequences (DNA or RNA) that can potentially form three-dimensional structures that increase the binding affinity and specificity between immune and tumor cells[ 29 ]. Therefore, aptamers enable immune cells to selectively recognize tumor cells and interact with their membrane proteins[ 30 , 31 ]. AS1411 is a 26-mer DNA aptamer with a G-quadruplex structure and a sequence of 5'-GGTGGTGGTGTGTGGTGGTGGT-3'. It is thermostable, resistant to nucleases, and has a low immunogenicity[ 32 ]. This aptamer can be used as a recognition probe to detect the overexpression of nucleolin (NCL) on the tumor cell surface. NCL is primarily present in the nucleolus of normal cells and is commonly identified on the tumor cell surface[ 33 – 36 ]. In this study, cholesterol (Chol), a lipophilic residue that can be conjugated with DNA molecules, was conjugated with the nucleic acid aptamer AS1411 to produce a lipophilic residue-DNA molecule conjugate (Chol-AS1411). After incubation with macrophages, the lipophilic component of Chol-AS1411 entered the phospholipid bilayer of the cell membrane. This process is faster and more convenient than the covalent conjugation. Furthermore, because the insertion does not significantly alter the function of membrane proteins or compromise the integrity of the cell membrane, macrophages are minimally affected. Cellular immunotherapy is a promising approach for cancer treatment. Cell surface engineering was performed in this study. An engineered M1-type macrophage (CAM1) has been developed for cancer immunotherapy. Using the surface-anchored aptamer AS1411, CAM1 cells selectively targeted and efficiently phagocytosed tumor cells, secreted anti-inflammatory cytokines, and remodeled the TME (Fig. 1 A). In addition, compared with the time-consuming and expensive production of genetically engineered cells and complex procedures, the CAM1 produced in this study has a lower cost and shorter cycle time, indicating significant potential in cancer therapy and providing new ideas and approaches for tumor immunotherapy. Materials and methods Materials All aptamer sequences were synthesized and purified by Sangon Biotechnology (Shanghai, China). The details of the aptamer sequences used are listed in Table S1 . Calcein-AM/PI, Double Stain Kit, CellTrace TM Red CMTPX, CellTracker Green CMFDA, DiD Perchlorate, and DiO Perchlorate were all purchased from YEASEN (Shanghai, China). Primers of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) , inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) mRNA were custom synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China). LPS and 4',6-diamidino-2-phenylindole (DAPI) were obtained from Solarbio (Beijing, China). Recombinant murine interferon-γ (IFN-γ) and interleukin-4 (IL-4) were purchased from PERPROTECH (USA). Cell Counting Kit-8 (CCK-8) was obtained from Dalian Meilun Biotechnology Corporation (Dalian, China). CellTracker Green chloromethylfluorescein diacetate (CMFDA), 3,3-Dioctadecyloxacarbocyanine perchlorate (Dio) and 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) were acquired from Yeasen Biotechnology Co., Ltd. (Shanghai, China). TB Green Premix Ex Taq II was procured from Takara (Japan). AxyPrep™ Multisource Total RNA was purchased from AXYGEN (Santa Clara, CA, USA). The antibodies used for immunofluorescence were obtained from Abcam (Cambridge Science Park, UK). Flow-related antibodies were procured from BioLegend (San Diego, CA, USA). All oligonucleotides were dissolved in sterile water and stored at − 20 ℃. Cell culture The mouse macrophage cell line RAW264.7, mouse breast cancer cell line 4T1, and mouse melanoma B16 cell line were obtained from the Chinese Academy of Science Cell Bank for Type Culture Collection (Shanghai, China). RAW264.7 cells were cultured in Dulbecco's modified Eagle medium (DMEM) medium with 10% fetal bovine serum (FBS, ExCell Bio) and 1% penicillin/streptomycin in 5% CO 2 at 37 ℃. 4T1 and B16 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin under the same conditions described above. Preparation of CAM1 RAW264.7 cells were treated with LPS (100 pg/mL) and IFN-γ (20 ng/mL) for 48 h to induce M1 polarization in macrophages. The cells were then incubated with Chol-modified AS1411-FAM (1 µM) aptamers in fresh DMEM medium at 37 ℃ for 1 h. The obtained CAM1 was used directly for in vitro and in vivo studies. To confirm the successful anchoring of the aptamers to the cell surface, the cells were fixed with 4% paraformaldehyde (Biosharp, Hefei, China) for 30 min at room temperature. The cell nuclei were then stained with DAPI for another 30 min. Subsequently, the cells were examined using confocal microscopy (Olympus FV3000RS, Japan) and flow cytometry (Beckmann Coulter CytoFLEX5, USA). Stability analysis of aptamer To assess aptamer stability, RAW264.7 cells were stimulated to M1 polarization in macrophages before being incubated with conjugated Chol-AS1411-FAM aptamers in DMEM medium with 10% FBS at 37 ℃ for varying periods (3, 9, 15, and 48 h) and then analyzed using flow cytometry and confocal microscopy to determine fluorescence intensity. Targeting ability of the aptamer To evaluate the active targeting ability of the aptamer, 4T1, and B16 cells were seeded at a density of 6 × 10 5 cells/mL in a glass-bottom dish (35 mm with 15 mm bottom well) and incubated with AS1411-FAM (1 µM) and C- AS1411-FAM (1 µM) aptamers at 37 ℃ for 24 h. After three washes with phosphate buffer saline (PBS), The cells were observed using confocal microscopy. In addition, fluorescence signals from the cells were quantified using flow cytometry. Cytotoxicity Assay RAW264.7 cells were seeded in a 96-well plate at a density of 2 × 10 4 cells/well and treated with LPS (100 pg/mL), IFN-γ (20 ng/mL) and Chol-AS1411(1 µM) for 24 or 48 h, respectively. After replacing the medium with fresh culture medium, the CCK-8 solution was added and incubated for 2 h. Absorbance was measured at 450 nm using a Spark microplate reader (Tecan, CH). Transwell assays CAM1 cells (1 × 10 5 ) were seeded on the transwell inserts in 500 µL of medium. Medium containing 30% FBS was added to the lower chambers as a chemoattractant. After 24 h, the cells on the upper side of the filter were removed using cotton swabs. Cells permeated to the lower membrane surface were fixed with 4% paraformaldehyde, stained with crystal violet, and counted under an optical microscope (x 100 magnification). Cell counts were expressed as the mean number of cells from 10 random fields per well. Measurement of marker gene expression and cytokine secretion In this study, the CAM1 gene expression was measured using qRT-PCR. RNA was isolated from cells using the Multisource Total RNA kit and reverse-transcribed into cDNA using HiScript Ⅲ qRT SuperMix. The genes were quantified by qRT-PCR using specific primers (Table S2). TNF-α, IL-6, IL-12, and IL-10 cytokines were also detected in the cell supernatant using an ELISA kit (Table S3). Phagocytosis assay CAM1 (1 × 10 5 ) and 4T1/B16 cells (5 × 10 5 ) were individually stained with DiI and DiO, 5- CellTracker Green CMFDA, and anti-F4/80-PE. Briefly, CAM1 (1 × 10 5 cells) and 4T1/B16 cells (5 × 10 5 cells) were incubated with their respective dyes at 37 ℃ for 1 h in the dark. Following this, the cells were rinsed three times with PBS before being combined in a fresh culture medium. After 24 h of incubation, the mixed cells were examined using flow cytometry and confocal microscopy. Animal studies Female BALB/c mice, aged 4–5 weeks, were obtained from the Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). All specific pathogen free (SPF) animals were housed at the Animal Facility Center at Hangzhou Normal University (HZNU). The study was conducted in compliance with the guidelines of the Animal Ethics and Welfare Committee (AEWC) of HZNU (Approval No. HSD20221101). A total of 1 × 10 6 4T1 cells in 100 µL of PBS were subcutaneously injected into the right flank of each mouse. Next, 1×10 5 macrophages (100 µL) were injected into each mouse under the treatment schedule on days 0, 10, 13, 17, and 20. Statistical analysis All statistical analyses were performed using GraphPad Prism 9.0. All quantitative results are presented as the mean ± standard deviation (SD) of at least three independent replicates. Statistical significance between groups was assessed using a one-way analysis of variance (ANOVA). P -values of < 0.05, < 0.01, < 0.001, and < 0.0001 indicated significant differences. Survival analysis was performed using the Kaplan-Meier method and compared using the Log-rank test (Mantel-Cox). Results Construction of CAM1 cells To functionalize M1 macrophages for cancer therapy, we used the lipophilicity of Chol to deliver the targeting molecule Chol-AS1411 to the surface of M1 macrophages, which were called Chol-AS1411-M1 macrophages (CAM1). Confocal microscopy analysis revealed a faint green fluorescent signal (Chol-AS1411-FAM) on the surface of RAW264.7 cells after 10-min incubation with Chol-AS1411-FAM. Furthermore, the intensity of green fluorescence on the surface of RAW264.7 cells increased with increasing incubation time. When the incubation time reached 1 h, a clear and complete circle of green fluorescence was observed on the cell membrane surface, proving that Chol-AS1411 could bind to the cell membrane surface after incubation for 1 h (Figs. 1 B-C). Further determination of green fluorescence by flow cytometry indicated comparable results (Figs. 1 D-E). To confirm the stability of Chol-AS1411 in DMEM medium to ensure the sustained antitumor effect of CAM1 in vivo , M1 cell surfaces coated with Chol-AS1411-FAM inducer were incubated with DMEM for different periods, and the fluorescence signal of the CAM1 surface-modified aptamer was monitored. Confocal microscopy findings (Figs. 1 F-G) indicate that the aptamer signal anchored on the CAM1 surface remained relatively stable for up to 48 h. Over time, the relative fluorescence intensity of the cell surface decreased. This study suggests that this is due to cell division during the proliferation of CAM1 cells, which also leads to a decrease in fluorescence intensity in the field of view. In this study, RAW264.7 cells were induced into M1 macrophages by a specific concentration of Lipopolysaccharides (LPS), which can cause a strong systemic inflammatory response in vivo . Therefore, we quantified the endotoxin content of CAM1 to ensure its biosafety in vivo and in vitro . The results indicated that the concentration of endotoxin in the third wash solution of CAM1 cells was approximately 0.07 EU/mL, a value significantly below the threshold of 0.50 EU/mL established by the Chinese Pharmacopoeia (Fig. S1 ). Alteration of CAM1 Activity in Vitro The viability of CAM1 cells was evaluated using calcein-AM/PI and CCK-8 assays. The calcein-AM/PI assay results revealed no significant difference in the survival rates of M1, M1 (with AS1411), and CAM1 cells (Figs. 2 A-B). CCK-8 assay was used to determine the viability of M1, M1 (with AS1411), and CAM1 cells. There was no significant reduction in the viability of the CAM1 cells (Fig. 2 C). These findings indicate that CAM1 cells have good cellular activity. Moreover, the wound healing results demonstrated that the migration ability of CAM1 cells did not differ significantly from that of M0 and M1 cells in the control group (Figs. 2 D-E), implying that the target molecule Chol-AS1411 did not significantly alter the macrophage migration behavior. Transwell results confirmed this finding (Figs. 2 F-G). To examine alteration of CAM1 properties, we examined the expression of CAM1 marker genes and proteins and cytokine secretion levels to determine whether CAM1 could reprogram the TME in vivo . In this study, qRT-PCR was performed to measure the expression of the CAM1 genes (Fig. 2 H). The results revealed that CAM1 cells significantly expressed iNOS mRNA compared with M0 cells (vs. M0, **** P < 0.0001). This finding suggests that our modified CAM1 cells did not change the M1 polar type of macrophages. In contrast, there was a significant increase in the expression of IL-6 mRNA (vs. M0, **** P < 0.0001), IL-1β (vs. M0, **** P < 0.0001), and TNF-α mRNA (vs. M0, * P < 0.05) in M1 and CAM1 cells compared with M0 cells (Fig. 2 H). ELISA results demonstrated that CAM1 cells secreted significantly more pro-inflammatory factors, such as IL-12 (vs. CAM0, **** P < 0.0001), IL-6 (vs. CAM0, ** P < 0.01), and TNF-α (vs. CAM0, * P < 0.05) compared with CAM0 cells. Concurrently, the secretion of the anti-inflammatory factor IL-10 was inhibited (vs. CAM0, *** P < 0.001) (Fig. 2 I). PE anti-mouse F4/80 was then used to label mature macrophages, and FITC anti-mouse CD86 was used to label the CD86 protein on the surface of macrophages. Flow cytometry results revealed that CAM1 cells could significantly express the CD86 membrane protein compared with other groups (vs. M0, **** P < 0.0001), indicating that the modified CAM1 cells did not change the M1 cell type (Fig. 2 J). Our results demonstrated that CAM1 has excellent cellular activity and the potential to reprogram the TME. Antitumor effects of CAM1 in Vitro AS1411 can specifically bind to NCL overexpression on the surface of tumor cells; therefore, we selected mouse breast cancer 4T1 and mouse melanoma B16 cells with positive expression of NCL for the target assay. Confocal microscopy imaging revealed that the C-AS1411-FAM (random sequence, Table S1 ) incubation group exhibited hardly any green fluorescence. Conversely, noticeable green fluorescence was observed in the AS1411-FAM incubation group (Figs. 3 A and E), and the fluorescence intensity of the AS1411-FAM incubation group differed significantly (**** P < 0.0001 vs . M0) (Figs. 3 B and F), indicating that AS1411 specifically binds to 4T1 tumor cells with positive NCL expression. The fluorescence intensity on B16 tumor cells was further investigated using confocal microscopy and flow cytometry, and the results were consistent (Figs. 3 C and G; D and H). Furthermore, the findings demonstrated that AS1411 had a significant affinity and was specifically bound to NCL on the surface of tumor cells. Therefore, the targeting molecule Chol-AS1411 may facilitate the specific binding of CAM1 cells to NCL-positive tumor cells. The ability of CAM1 cells to phagocytose tumor cells was explored to demonstrate their antitumor effect in vitro . The orange-red fluorescent cell membrane probe DiI and green fluorescent cell membrane probe DiO were used to label the cell membranes of macrophages and tumor cells, respectively. After 2-h co-incubation of macrophages and tumor cells, the phagocytosis of tumor cells by macrophages was observed using laser confocal microscopy. The findings revealed that CAM1 cells had the most robust green fluorescence, indicating that they had the most potent phagocytic activity against B16 and 4T1 cells, followed by M1 cells, with M2 cells demonstrating the least phagocytic activity against tumors (Figs. 3 I-J). Furthermore, CellTracker Red CMTPX and CellTracker Green CMFDA were used to label the cytoplasm of macrophages and tumor cells, respectively, to identify the phagocytosis of CAM1 cells. Flow cytometry was also used to assess the phagocytic ability of CAM1 cells. The results also revealed that CAM1 cells have a strong phagocytic ability against tumors (Fig. S2). Antitumor effects of CAM1 in Vivo Given that CAM1 cells successfully invaded tumors in vitro , we investigated the antitumor effect of CAM1 cells in vivo . First, we established a 4T1 subcutaneous tumor model of breast cancer in female BALB/c mice to evaluate the antitumor efficacy of CAM1 (Fig. 4 A). Next, 1×10 5 macrophages (100 µL) were administered to each mouse following the prescribed treatment regimen via intravenous injection (i.v.) and intratumoral injection (i.t.). The Chol-C-AS1411 (random sequence) modified M1 macrophage (CCAM1) served as the negative control group.Tumor growth in the PBS (i.v.) and CCAM1 (i.v.) groups was almost unrestricted and maintained a high growth rate, indicating that CCAM1 (i.v.) could not inhibit tumor growth. M1 (i.v.) and CAM1 (i.t.) also exhibited modest inhibitory effects on tumor growth. However, tumor growth in the CAM1 (i.v.) group was significantly inhibited, and there was a significant difference ( vs. PBS group, ** P < 0.01), indicating that CAM1 (i.v.) produced an excellent antitumor effect (Fig. 4 B). Moreover, there was no significant variation in the body weight of the mice across all groups, indicating that the engineered macrophage CAM1 had significant antitumor activity without obvious systemic toxicity (Fig. 4 C). Fluorescence imaging, which can accurately reflect the accumulation and metabolism of injected macrophages in mice, is helpful in assessing cancer conditions and cell therapy efficacy during treatment in a timely and accurate manner. The fluorescence of CY5.5 was used in this study to modify the injected engineered macrophage-targeting molecules to obtain Chol-AS1411-CY5.5 and Chol-C-AS1411-CY5.5. By co-incubation with M1 cells, Chol-AS1411-CY5.5-M1 (CAM1-CY5.5) and Chol-C-AS1411-CY5.5-M1 (CCAM1-CY5.5) were obtained. First, we evaluated the distribution of CAM1 cells in tumor-bearing mice and found that all three groups, CCAM1-CY5.5 (i.v.), CAM1-Cy5.5 (i.v.), and CAM1-Cy5.5 (i.t.), effectively emitted fluorescent signals at the tumor site (Figs. 4 D-E). Second, 24 h after the injection of CAM1 cells, we assessed the distribution of CAM1-Cy5.5 (i.v.), CAM1-Cy5.5 (i.v.), and CAM1-Cy5.5 (i.t.) in the major organs (heart, liver, spleen, lung, and kidney) and tumors of tumor-bearing mice. The liver exhibited the highest concentration of fluorescence, followed by the tumor, which is consistent with the fluorescence imaging results within one week. This further indicated that the engineered macrophages could enter the tumor (Fig. 4 D). The spleen, the largest immune organ, is an important site for the body to produce an immune response. The present study revealed that the volume and weight of the spleen in tumor-bearing mice were significantly higher than in normal mice, indicating that the body produces a strong immune response during the growth and treatment of 4T1 tumors, resulting in spleen enlargement. Furthermore, a peak of the fluorescence signal at the tumor site was observed on the fourth day, implying that the injected engineered macrophages successfully infiltrated the tumor (Fig. 4 F). Over time, the injected cells were gradually metabolized by the body, and the fluorescence signal became weaker. However, the fluorescence signal was still detectable one week after infusion, demonstrating that the injected engineered macrophages could effectively remain at the tumor site. To further investigate the antitumor effect of CAM1 cells in vivo , hematoxylin-eosin (HE) staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) were used to detect apoptosis of tumor tissues in tumor-bearing mice in each group after treatment. HE staining demonstrated that the CAM1 (i.v.) group had more pronounced nuclear pyknosis, reduced tumor density, and more severe tumor cell necrosis than the other groups (Fig. 4 G). TUNEL staining revealed that the PBS (i.v.) and CCAM1 (i.v.) groups had almost no green fluorescence. In contrast, the other treatment groups had varying degrees of green fluorescence, with the CAM1 (i.v.) group indicating the strongest green fluorescence (Fig. 4 H). The results of this experiment indicated that CAM1 cell treatment led to a large region of tumor cell necrosis and apoptosis, suggesting that CAM1 cells have potent specific killing effects on tumors, inhibited tumor cell proliferation, and promoted tumor cell apoptosis, which holds promise for developing efficient and safe tumor treatments. Biotoxicity of CAM1 To achieve the subsequent clinical application of CAM1 cells in vivo , the biosafety of each group was assessed following the treatment of tumor-bearing mice with CAM1 cells in organs (heart, liver, spleen, lung, and kidney) using HE staining and routine blood tests. HE staining revealed that the PBS (i.v.) and CCAM1 (i.v.) groups had mild alveolar wall thickening and inflammatory cell infiltration, whereas the other groups had normal lung structures. Other organs in good cellular condition showed no significant pathological changes (Fig. 5 A). CAM1 cells can reduce the severity of organ lesions while being antitumor and demonstrating excellent biological safety in normal tissues. Subsequently, the results of routine blood tests revealed no significant difference in any of the blood indices between the PBS (i.v.) and CAM1 cell groups. This suggests that CAM1 cells are associated with a degree of biological safety (Figs. 5 B–E). In contrast, the index of white blood cells (WBCs) in the PBS group was significantly higher than the normal range, indicating that the number of 4T1 WBCs in the mouse model exceeded the upper limit of the normal reference value. Discussion The diverse functions and high plasticity of macrophages allow them to infiltrate tumor tissues and exert their effects. Therefore, numerous clinical trials have extensively investigated adoptive macrophage transfer therapy as a potential therapeutic approach for cancer treatment[ 24 ]. Considering their critical role in cancer immunity, targeting tumors using macrophage-related therapies has emerged as a promising immunotherapy strategy. In this study, we developed engineered macrophage CAM1 for cellular immunotherapy using aptamer AS1411 specific binding properties to overexpress NCL on the surface of tumor cells. Additionally, we used the antitumor properties of M1 macrophages and exploited the lipophilicity between Chol molecules and the phospholipid bilayer of the membrane. This innovative strategy aims to target tumor cells for phagocytosis while reshaping the TME, thereby providing novel insights into tumor immunotherapy. Immunotherapy is currently focused on improving innate defense mechanisms to eradicate malignant cells. This symbiotic approach exploits the immune system's ability to provide comprehensive cancer treatment while leveraging the innate immune response to optimize therapeutic efficacy. Consequently, these breakthroughs in tumor immunology have energized the field with unparalleled vigor [37] . Resistance of macrophages to genetic engineering techniques is a widely recognized characteristic that limits their therapeutic efficacy in clinical applications[ 38 , 39 ]. In this study, we developed a non-engineered approach, cell surface engineering, to modify macrophages to produce engineered macrophage CAM1. This innovative strategy involves anchoring AS1411 onto the surface of macrophages, enabling it to selectively bind to tumor cells that overexpress NCL on their surfaces. By avoiding any modifications to the original genes and proteins of macrophages, this method has minimal adverse effects on macrophage function while improving their immune response. Therefore, these results provide new insights into tumor immunotherapy. However, CAM1 inevitably encounters certain unfavorable conditions in the TME, such as immune checkpoint ligands and a large population of immunosuppressive cells. Furthermore, variations in pH levels and the potential induction of a "cytokine storm" in mice remain important considerations for CAM1. Considering these challenges, we aimed to further investigate and improve CAM1 in tumor-bearing mice while delving deeper into its immune regulatory mechanisms. Declarations Acknowledgments Funding: This work was supported by the Huadong Medicine Joint Funds of the Zhejiang Provincial Natural Science Foundation of China under Grant No. LHDMZ22H300001, the Joint Funds of the Shandong Provincial Natural Science Foundation of China (ZR202108130026). Declaration of Competing interests No conflict of interest exits in the submission of this manuscript. CRediT Author Statement: Qian Yang: Methodology, Software, Investigation, Formal Analysis, Writing-Original Draft; Shiyi Hu: Data Curation, Software, Writing-Original Draft; Yiqiu Wang: Software, Writing-Original Draft Luyi Zhong: Data Curation, Software; Xiaoli Yu: Writing-Original Draft; Yifeng Zhang: Data Curation, Writing-Original Draft; Yiting Zhang：Data Curation, Writing-Original Draft; Honghua Zhang: Software, Validation; Shuling Wang: Conceptualization, Resources, Supervision. Qingchang Tian: Methodology, Software, Investigation, Formal Analysis, Validation, Funding Acquisition. References Li YR, Zhou Y, Kim YJ, et al. Development of allogeneic HSC-engineered iNKT cells for off-the-shelf cancer immunotherapy. Cell Rep Med. 2021;2:100449. Munshi NC, Anderson LD Jr., Shah N, et al. Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma. N Engl J Med. 2021;384:705–16. Raje N, Berdeja J, Lin Y, et al. Anti-BCMA CAR T-Cell Therapy bb2121 in Relapsed or Refractory Multiple Myeloma. N Engl J Med. 2019;380:1726–37. Wang M, Munoz J, Goy A, et al. KTE-X19 CAR T-Cell Therapy in Relapsed or Refractory Mantle-Cell Lymphoma. N Engl J Med. 2020;382:1331–42. Rapoport AP, Stadtmauer EA, Binder-Scholl GK, et al. NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat Med. 2015;21:914–21. June CH, O'Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science. 2018;359:1361–5. Ma S, Li X, Wang X, et al. Current Progress in CAR-T Cell Therapy for Solid Tumors. Int J Biol Sci. 2019;15:2548–60. Guedan S, Ruella M, June CH. Emerging Cellular Therapies for Cancer. Annu Rev Immunol. 2019;37:145–71. Melenhorst JJ, Chen GM, Wang M, et al. Decade-long leukaemia remissions with persistence of CD4(+) CAR T cells. Nature. 2022;602:503–9. D'Aloia MM, Zizzari IG, Sacchetti B, Pierelli L, Alimandi M. CAR-T cells: the long and winding road to solid tumors. Cell Death Dis. 2018;9:282. Maude SL, Laetsch TW, Buechner J, et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med. 2018;378:439–48. Pang Y, Hou X, Yang C, Liu Y, Jiang G. Advances on chimeric antigen receptor-modified T-cell therapy for oncotherapy. Mol Cancer. 2018;17:91. Laskowski TJ, Biederstädt A, Rezvani K. Natural killer cells in antitumour adoptive cell immunotherapy. Nat Rev Cancer. 2022;22:557–75. Mehrabadi AZ, Ranjbar R, Farzanehpour M, et al. Therapeutic potential of CAR T cell in malignancies: A scoping review. Volume 146. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie; 2022. p. 112512. Chen Y, Yu Z, Tan X, et al. CAR-macrophage: A new immunotherapy candidate against solid tumors. Volume 139. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie; 2021. p. 111605. Wagner J, Wickman E, DeRenzo C, Gottschalk S. CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality? Mol therapy: J Am Soc Gene Therapy. 2020;28:2320–39. Chan JD, Lai J, Slaney CY, Kallies A, Beavis PA, Darcy PK. Cellular networks controlling T cell persistence in adoptive cell therapy. Nat Rev Immunol. 2021;21:769–84. Beatty GL, O'Hara M. Chimeric antigen receptor-modified T cells for the treatment of solid tumors: Defining the challenges and next steps. Pharmacol Ther. 2016;166:30–9. Long KB, Young RM, Boesteanu AC, et al. CAR T Cell Therapy of Non-hematopoietic Malignancies: Detours on the Road to Clinical Success. Front Immunol. 2018;9:2740. Noy R, Pollard JW. Tumor-associated macrophages: from mechanisms to therapy. Immunity. 2014;41:49–61. Franken L, Schiwon M, Kurts C. Macrophages: sentinels and regulators of the immune system. Cell Microbiol. 2016;18:475–87. Yang L, Zhang Y. Tumor-associated macrophages: from basic research to clinical application. J Hematol Oncol. 2017;10:58. Ritchie D, Mileshkin L, Wall D, et al. In vivo tracking of macrophage activated killer cells to sites of metastatic ovarian carcinoma. Cancer Immunol immunotherapy: CII. 2007;56:155–63. Klichinsky M, Ruella M, Shestova O, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol. 2020;38:947–53. Kang M, Lee SH, Kwon M et al. Nanocomplex-Mediated In Vivo Programming to Chimeric Antigen Receptor-M1 Macrophages for Cancer Therapy. Advanced materials (Deerfield Beach, Fla). 2021;33:e2103258. Hao M, Hou S, Li W et al. Combination of metabolic intervention and T cell therapy enhances solid tumor immunotherapy. Sci Transl Med. 2020;12. Zhang D, Zheng Y, Lin Z et al. Equipping Natural Killer Cells with Specific Targeting and Checkpoint Blocking Aptamers for Enhanced Adoptive Immunotherapy in Solid Tumors. Angewandte Chemie (International ed in English). 2020;59:12022–12028. Zhang Y, Hong H, Cai W. Tumor-targeted drug delivery with aptamers. Curr Med Chem. 2011;18:4185–94. Qin SY, Zhang AQ, Cheng SX, Rong L, Zhang XZ. Drug self-delivery systems for cancer therapy. Biomaterials. 2017;112:234–47. Yan J, Gao T, Lu Z, Yin J, Zhang Y, Pei R. Aptamer-Targeted Photodynamic Platforms for Tumor Therapy. ACS Appl Mater Interfaces. 2021;13:27749–73. Keefe AD, Pai S, Ellington A. Aptamers as therapeutics. Nat Rev Drug Discovery. 2010;9:537–50. Bates PJ, Reyes-Reyes EM, Malik MT, Murphy EM, O'Toole MG, Trent JO. G-quadruplex oligonucleotide AS1411 as a cancer-targeting agent: Uses and mechanisms. Biochim et Biophys acta Gen Subj. 2017;1861:1414–28. Bie L, Wang Y, Jiang F, Xiao Z, Zhang L, Wang J. Insights into the binding mode of AS1411 aptamer to nucleolin. Front Mol Biosci. 2022;9:1025313. Miranda A, Santos T, Largy E, Cruz C. Locking up the AS1411 Aptamer with a Flanking Duplex: Towards an Improved Nucleolin-Targeting. Pharmaceuticals (Basel, Switzerland). 2021;14. Moe GR, Steirer LM, Lee JA, Shivakumar A, Bolanos AD. A cancer-unique glycan: de-N-acetyl polysialic acid (dPSA) linked to cell surface nucleolin depends on re-expression of the fetal polysialyltransferase ST8SIA2 gene. J experimental Clin cancer research: CR. 2021;40:293. Vindigni G, Raniolo S, Iacovelli F et al. AS1411 Aptamer Linked to DNA Nanostructures Diverts Its Traffic Inside Cancer Cells and Improves Its Therapeutic Efficacy. Pharmaceutics. 2021;13. Zhang Y, Zhang Z. The history and advances in cancer immunotherapy: understanding the characteristics of tumor-infiltrating immune cells and their therapeutic implications. Cell Mol Immunol. 2020;17:807–21. Sloas C, Gill S, Klichinsky M. Engineered CAR-Macrophages as Adoptive Immunotherapies for Solid Tumors. Front Immunol. 2021;12:783305. Zhang W, Liu L, Su H, et al. Chimeric antigen receptor macrophage therapy for breast tumours mediated by targeting the tumour extracellular matrix. Br J Cancer. 2019;121:837–45. Additional Declarations No competing interests reported. Supplementary Files 2Supporting.docx image6.png Graphical Abstract Cite Share Download PDF Status: Published Journal Publication published 31 Jul, 2024 Read the published version in Biomedicine & Pharmacotherapy → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4250998","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":291172099,"identity":"babf27f0-58d1-420d-b897-d14bd52fe5aa","order_by":0,"name":"Qian Yang","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Yang","suffix":""},{"id":291172100,"identity":"5a01c041-ca66-4693-b4b2-894be9b40e02","order_by":1,"name":"Shiyi Hu","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Shiyi","middleName":"","lastName":"Hu","suffix":""},{"id":291172101,"identity":"3c8f4581-3135-43b7-a21f-137a925390a5","order_by":2,"name":"Yiqiu Wang","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yiqiu","middleName":"","lastName":"Wang","suffix":""},{"id":291172102,"identity":"c45443ba-18cd-4f9f-bdbf-84467834b3b8","order_by":3,"name":"Luyi Zhong","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Luyi","middleName":"","lastName":"Zhong","suffix":""},{"id":291172103,"identity":"e54ff250-23fe-488b-ac3b-32bca10fffc8","order_by":4,"name":"Xiaoli Yu","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoli","middleName":"","lastName":"Yu","suffix":""},{"id":291172104,"identity":"8b91662f-526a-41fb-b36c-f892b5712bfb","order_by":5,"name":"Yifeng Zhang","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yifeng","middleName":"","lastName":"Zhang","suffix":""},{"id":291172105,"identity":"7d534b2e-d25a-4818-b3a6-673a7ec2ce13","order_by":6,"name":"Yiting Zhang","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yiting","middleName":"","lastName":"Zhang","suffix":""},{"id":291172106,"identity":"d3871cd7-2511-45d8-a46a-383faffc404d","order_by":7,"name":"Honghua Zhang","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Honghua","middleName":"","lastName":"Zhang","suffix":""},{"id":291172107,"identity":"302879c0-7dc5-40bc-acf2-ecd8a3203d6f","order_by":8,"name":"Shuling Wang","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Shuling","middleName":"","lastName":"Wang","suffix":""},{"id":291172108,"identity":"c53c4e1a-f54e-45f4-be49-6971b1636ab9","order_by":9,"name":"Qingchang Tian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYDACZjBpkwDhsRGhgweiJY0ULRDqMAla7NmZnz14U3M+T352jwHDh7LDDPyzGwg5jM3ccM6x28WMc84YMM44d5hB4s4BQloYzKR5G24nNkvkGDDzth1mMJBIIKSF/RtQy7nENpCWv8Rp4QHZciCxB6SFkSgth3nKJOccS06cIZFWcLDnXDqPxA0CWtj7j2+TeFNjlzh/RvLGBz/KrOX4ZxDQArEKSh9AYhOpZRSMglEwCkYBVgAAd8k6mOKubk0AAAAASUVORK5CYII=","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":true,"prefix":"","firstName":"Qingchang","middleName":"","lastName":"Tian","suffix":""}],"badges":[],"createdAt":"2024-04-11 08:00:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4250998/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4250998/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.biopha.2024.117064","type":"published","date":"2024-08-01T00:32:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54835012,"identity":"37605d17-b32b-4f06-a206-a67f88964cf3","added_by":"auto","created_at":"2024-04-17 12:20:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":615857,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction of CAM1 with Chol-AS1411 by modifying the cell surface. A. Illustration of engineered M1 macrophages with Chol-AS1411 aptamer for enhanced adoptive immunotherapy by surface modification of M1 macrophages. (B-C) M1 macrophages were incubated with Chol-AS1411-FAM in fresh DMEM medium at 37 °C at different times. Confocal microscopy revealed a green fluorescent signal (Chol-AS1411-FAM) on the surface of RAW264.7 cells (n = 3, mean ± SD). The scale bar is 20 μm. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.5. (D-E) Flow cytometry results of the fluorescence signal (Chol-AS1411-FAM) on CAM1 cells (n = 3, mean ± SD). ****\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001. (F-G) The fluorescence signal of aptamers modified on the surface of CAM1 was observed using confocal microscopy (n = 3, mean ± SD). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ****\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001, and ns for no significant difference.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4250998/v1/773dcf489d54a6850af3ae7d.png"},{"id":54835014,"identity":"a90d4d4f-9381-462f-abf0-e5a3ab76d85d","added_by":"auto","created_at":"2024-04-17 12:20:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":462254,"visible":true,"origin":"","legend":"\u003cp\u003eAlterations in CAM1 activity and properties. (A-B) Survival rate was analyzed by calcein-AM/PI assay (n = 3, mean ± SD); ns, no significant difference (\u003cem\u003evs\u003c/em\u003e. M1). The scale bar is 1000 μm. (C) Results of the CCK-8 assay (n = 3, mean ± SD); ns, no significant difference (\u003cem\u003evs\u003c/em\u003e. M0). (D-E) Wound healing analysis indicated no significant difference in the migration ability of CAM1 cells from that of M0 and M1 cells (n = 3, mean ± SD); ns, no significant difference (\u003cem\u003evs\u003c/em\u003e. M0). The scale bar is 100 μm. (F-G) Migration ability of CAM1, M0, and M1 cells in transwells (n = 3, mean ± SD); ns, no significant difference (\u003cem\u003evs\u003c/em\u003e. M0). The scale bar is 50 μm. (H) Expression of different genes (iNOS, IL-6, IL-1β, and TNF-α mRNA) in M0, M1, and CAM1 cells was measured by qRT-PCR (n = 3, mean ± SD), *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, ns denotes no significant difference (\u003cem\u003evs\u003c/em\u003e. M0). (I) Secreted pro-inflammatory factors (IL-12, IL-6, IL-10, and TNF-α) were measured using ELISA (n = 3, mean ± SD), \u003cem\u003e*P\u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e\u0026lt; 0.01, \u003cem\u003e***P\u003c/em\u003e\u0026lt; 0.001, \u003cem\u003e****P \u003c/em\u003e\u0026lt; 0.0001 (\u003cem\u003evs\u003c/em\u003e. M0). (J) Flow cytometry results revealed that CAM1 cells significantly expressed CD86 membrane protein (n = 3, mean ± SD), \u003cem\u003e****P \u003c/em\u003e\u0026lt; 0.0001 (\u003cem\u003evs\u003c/em\u003e. M0).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4250998/v1/ae29f3360589e1cbdfa0dec3.png"},{"id":54835016,"identity":"43e611ff-2ed3-40d8-ba1d-104c73c93761","added_by":"auto","created_at":"2024-04-17 12:20:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1232653,"visible":true,"origin":"","legend":"\u003cp\u003eAbility of CAM1 cells to phagocytose tumor cells. (A and E) Fluorescence signals from 4T1 cells treated with AS1411 (green) or C-AS1411 (random sequence) at 37 ℃ for 24 h. The scale bar is 20 μm (n = 3, mean ± SD), \u003cem\u003e****P \u003c/em\u003e\u0026lt; 0.0001. (B and F) Fluorescence signals of 4T1 cells treated with AS1411 (green) or C-AS1411(random sequence) determined by flow cytometry (n = 3, mean ± SD), \u003cem\u003e****P \u003c/em\u003e\u0026lt; 0.0001. (C and G) Fluorescence signals from 4T1 cells treated with AS1411 (green) or C-AS1411(random sequence) at 37 ℃ for 24 h. The scale bar is 20 μm (n = 3, mean ± SD), \u003cem\u003e****P \u003c/em\u003e\u0026lt; 0.0001. (D and H) Fluorescence signals of 4T1 cells treated with AS1411 (green) or C-AS1411 (random sequence) determined by flow cytometry (n = 3, mean ± SD), \u003cem\u003e****P \u003c/em\u003e\u0026lt; 0.0001. (I) The cell membranes of macrophages and 4T1 cells were labeled with DiI (orange-red fluorescence) and DiO (green fluorescence). (J) Cell membranes of macrophages and B16 cells were labeled with DiI (orange-red fluorescence) and DiO (green fluorescence).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4250998/v1/b4352bd2480baad9637bbc3e.png"},{"id":54835020,"identity":"e04b1f35-1bc2-4ca5-8480-d4fcc3acb7f4","added_by":"auto","created_at":"2024-04-17 12:20:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":819196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003eantitumor efficacy of CAM1. (A) Treatment schedule used to evaluate CAM1-induced antitumor efficacy ina 4T1 tumor model. (B) Tumor growth curves of 4T1 tumor-bearing mice injected with PBS, M1, CCAM1 (with Chol-C-AS1411), and CAM1 (n = 5, mean ± SD), **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. (C) Body weight of 4T1 tumor-bearing mice from different groups (n = 5, mean ± SD). (D) Fluorescence imaging of major organs and tumor tissues in tumor-bearing mice within 24 h (n = 1). (E) \u003cem\u003eIn vivo\u003c/em\u003e fluorescence imaging of tumor-bearing mice within one week (mean ± SD). (F) Quantitative \u003cem\u003einvivo\u003c/em\u003e fluorescence signal intensity of the tumor tissue in tumor-bearing mice within one week (n = 5, mean ± SD). (G) HE staining diagram of tumor tissue after treatment in each group of tumor-bearing mice. The scale bar is 20 μm. (H) TUNEL staining diagram of tumor tissuesin each group of tumor-bearing mice after treatment. The scale bar is 100 μm.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4250998/v1/869a5a9299f2be41acfcd3d6.png"},{"id":54835022,"identity":"6af620b0-51a4-4cc9-b1ce-179a033300d2","added_by":"auto","created_at":"2024-04-17 12:20:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1208615,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e biological safety evaluation of CAM1 cells. (A) HE staining images of vital organs in each group of tumor-bearing mice after treatment. The scale bar is 20 μm. (B–E) Flow cytometric quantification of conventional cells (RBC, WBC, and PLT) in the blood of tumor-bearing mice after treatment (n = 3, mean ± SD); ns denotes no significant difference.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4250998/v1/4a62a6d580181c3dc7ddf297.png"},{"id":59617289,"identity":"5e28fa39-5306-4bce-b95d-6b7fa6bc9ba6","added_by":"auto","created_at":"2024-07-04 00:32:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4795373,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4250998/v1/5b10280c-bb5b-4104-9d05-b246b9d837a0.pdf"},{"id":54835015,"identity":"f55a9a2a-ba40-4595-9e6a-32adc37a4b72","added_by":"auto","created_at":"2024-04-17 12:20:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":497171,"visible":true,"origin":"","legend":"","description":"","filename":"2Supporting.docx","url":"https://assets-eu.researchsquare.com/files/rs-4250998/v1/9a8f1bab56be0268113c3db9.docx"},{"id":54835017,"identity":"333272bc-e94c-4e38-8181-ce5f103975ab","added_by":"auto","created_at":"2024-04-17 12:20:28","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":212556,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4250998/v1/ff63b97f84b8b76bb475d88e.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Engineering M1 macrophages with targeting aptamers for enhanced adoptive immunotherapy by modifying the cell surface","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1. M2 phenotype macrophage-targeted Lipo@CpG-FA was used to repolarize TAMs and reverse immunosuppressive TME.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2. \u0026nbsp;The synergic nanoformulation reduced M2 macrophages and caused regression and inhibition of 4T1 breast cancers.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eAdoptive cell immunotherapy uses the autoimmune system as an active immune therapy. This approach introduces new antitumor and targeting capabilities by modifying autologous immune cells, thereby enhancing their inherent immune response\u003csup\u003e[1\u0026ndash;4]\u003c/sup\u003e. These cells were isolated from the host, genetically engineered, expanded ex vivo, and reinjected into the patient to eliminate tumor cells\u003csup\u003e[5\u0026ndash;8]\u003c/sup\u003e. Chimeric Antigen Receptor (CAR) technology is a powerful engineering platform in immunotherapy, with CAR-T emerging as a new revolutionary pillar in tumor therapy[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It is extensively used to treat hematological malignancies and has demonstrated significant clinical efficacy[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite these successes with CAR-T, this genetic engineering strategy may have potential problems and risks[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To date, CAR-T has demonstrated higher clinical efficacy in hematological tumors than in solid tumors. Tumor development restricts T-cell recruitment and infiltration, activates extensive inhibitory pathways to limit T-cell activation, and expresses heterogeneous tumor-associated antigens (TAA), which makes it difficult for CAR-T cells to migrate to solid tumors and survive in the tumor microenvironment (TME)[\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Given these limitations, there is growing interest in exploring novel cell-based immunotherapies that can be universally applied.\u003c/p\u003e \u003cp\u003eMacrophages are important innate immune cells. With the increase in tumor volume and development of intratumoral vascular networks, blood monocytes infiltrate into tumor tissues and mature into tumor-associated macrophages (TAMs) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Compared to T cells, macrophages can influence the surrounding immune cells through pro-inflammatory and anti-inflammatory effects and excel in remodeling the extracellular matrix (ECM) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These advantages enable macrophages to easily penetrate solid tumor tissues and interact with tumor cells[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Consequently, engineering macrophages offers new avenues for cancer therapy[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Among the cell types employed in immunotherapy, macrophages have emerged as primary candidates for treating solid tumors.\u003c/p\u003e \u003cp\u003eIn addition, using CAR-IFN-γ-encoding plasmid DNA nanocomposites in CAR-M development has been observed to enhance antitumor immunoregulatory capabilities of macrophages and inhibit solid tumor growth[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These genetically engineered macrophages hold significant potential for application prospects in treating solid tumors. However, their further utilization is hindered by issues, such as potential target organ toxicity, heterogeneous expression of engineered proteins, and transgene insertional mutagenesis[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Furthermore, the production of these genetically engineered cells is time-consuming and costly. Consequently, there is a pressing need to develop simpler and safer technologies to overcome the challenges associated with tumor therapy.\u003c/p\u003e \u003cp\u003eThe precise delivery of anticancer drugs to tumor cells is one of the most important aspects of cancer medicine. This strategy emphasizes not only improving the efficacy of anticancer agents but also reducing nonspecific side effects[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Aptamers are short nucleotide sequences (DNA or RNA) that can potentially form three-dimensional structures that increase the binding affinity and specificity between immune and tumor cells[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, aptamers enable immune cells to selectively recognize tumor cells and interact with their membrane proteins[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAS1411 is a 26-mer DNA aptamer with a G-quadruplex structure and a sequence of 5'-GGTGGTGGTGTGTGGTGGTGGT-3'. It is thermostable, resistant to nucleases, and has a low immunogenicity[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This aptamer can be used as a recognition probe to detect the overexpression of nucleolin (NCL) on the tumor cell surface. NCL is primarily present in the nucleolus of normal cells and is commonly identified on the tumor cell surface[\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, cholesterol (Chol), a lipophilic residue that can be conjugated with DNA molecules, was conjugated with the nucleic acid aptamer AS1411 to produce a lipophilic residue-DNA molecule conjugate (Chol-AS1411). After incubation with macrophages, the lipophilic component of Chol-AS1411 entered the phospholipid bilayer of the cell membrane. This process is faster and more convenient than the covalent conjugation. Furthermore, because the insertion does not significantly alter the function of membrane proteins or compromise the integrity of the cell membrane, macrophages are minimally affected.\u003c/p\u003e \u003cp\u003eCellular immunotherapy is a promising approach for cancer treatment. Cell surface engineering was performed in this study. An engineered M1-type macrophage (CAM1) has been developed for cancer immunotherapy. Using the surface-anchored aptamer AS1411, CAM1 cells selectively targeted and efficiently phagocytosed tumor cells, secreted anti-inflammatory cytokines, and remodeled the TME (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In addition, compared with the time-consuming and expensive production of genetically engineered cells and complex procedures, the CAM1 produced in this study has a lower cost and shorter cycle time, indicating significant potential in cancer therapy and providing new ideas and approaches for tumor immunotherapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eAll aptamer sequences were synthesized and purified by Sangon Biotechnology (Shanghai, China). The details of the aptamer sequences used are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Calcein-AM/PI, Double Stain Kit, CellTrace TM Red CMTPX, CellTracker Green CMFDA, DiD Perchlorate, and DiO Perchlorate were all purchased from YEASEN (Shanghai, China). Primers of \u003cem\u003eglyceraldehyde-3-phosphate dehydrogenase (GAPDH)\u003c/em\u003e, \u003cem\u003einducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α)\u003c/em\u003e mRNA were custom synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China). LPS and 4',6-diamidino-2-phenylindole (DAPI) were obtained from Solarbio (Beijing, China). Recombinant murine interferon-γ (IFN-γ) and interleukin-4 (IL-4) were purchased from PERPROTECH (USA). Cell Counting Kit-8 (CCK-8) was obtained from Dalian Meilun Biotechnology Corporation (Dalian, China). CellTracker Green chloromethylfluorescein diacetate (CMFDA), 3,3-Dioctadecyloxacarbocyanine perchlorate (Dio) and 1,1\u0026prime;-Dioctadecyl-3,3,3\u0026prime;,3\u0026prime;-tetramethylindocarbocyanine perchlorate (Dil) were acquired from Yeasen Biotechnology Co., Ltd. (Shanghai, China). TB Green Premix Ex Taq II was procured from Takara (Japan). AxyPrep\u0026trade; Multisource Total RNA was purchased from AXYGEN (Santa Clara, CA, USA). The antibodies used for immunofluorescence were obtained from Abcam (Cambridge Science Park, UK). Flow-related antibodies were procured from BioLegend (San Diego, CA, USA). All oligonucleotides were dissolved in sterile water and stored at \u0026minus;\u0026thinsp;20 ℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe mouse macrophage cell line RAW264.7, mouse breast cancer cell line 4T1, and mouse melanoma B16 cell line were obtained from the Chinese Academy of Science Cell Bank for Type Culture Collection (Shanghai, China). RAW264.7 cells were cultured in Dulbecco's modified Eagle medium (DMEM) medium with 10% fetal bovine serum (FBS, ExCell Bio) and 1% penicillin/streptomycin in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37 ℃. 4T1 and B16 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin under the same conditions described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of CAM1\u003c/h2\u003e \u003cp\u003eRAW264.7 cells were treated with LPS (100 pg/mL) and IFN-γ (20 ng/mL) for 48 h to induce M1 polarization in macrophages. The cells were then incubated with Chol-modified AS1411-FAM (1 \u0026micro;M) aptamers in fresh DMEM medium at 37 ℃ for 1 h. The obtained CAM1 was used directly for \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies.\u003c/p\u003e \u003cp\u003eTo confirm the successful anchoring of the aptamers to the cell surface, the cells were fixed with 4% paraformaldehyde (Biosharp, Hefei, China) for 30 min at room temperature. The cell nuclei were then stained with DAPI for another 30 min. Subsequently, the cells were examined using confocal microscopy (Olympus FV3000RS, Japan) and flow cytometry (Beckmann Coulter CytoFLEX5, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStability analysis of aptamer\u003c/h2\u003e \u003cp\u003eTo assess aptamer stability, RAW264.7 cells were stimulated to M1 polarization in macrophages before being incubated with conjugated Chol-AS1411-FAM aptamers in DMEM medium with 10% FBS at 37 ℃ for varying periods (3, 9, 15, and 48 h) and then analyzed using flow cytometry and confocal microscopy to determine fluorescence intensity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTargeting ability of the aptamer\u003c/h2\u003e \u003cp\u003eTo evaluate the active targeting ability of the aptamer, 4T1, and B16 cells were seeded at a density of 6 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL in a glass-bottom dish (35 mm with 15 mm bottom well) and incubated with AS1411-FAM (1 \u0026micro;M) and C- AS1411-FAM (1 \u0026micro;M) aptamers at 37 ℃ for 24 h. After three washes with phosphate buffer saline (PBS), The cells were observed using confocal microscopy. In addition, fluorescence signals from the cells were quantified using flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity Assay\u003c/h2\u003e \u003cp\u003eRAW264.7 cells were seeded in a 96-well plate at a density of 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well and treated with LPS (100 pg/mL), IFN-γ (20 ng/mL) and Chol-AS1411(1 \u0026micro;M) for 24 or 48 h, respectively. After replacing the medium with fresh culture medium, the CCK-8 solution was added and incubated for 2 h. Absorbance was measured at 450 nm using a Spark microplate reader (Tecan, CH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eTranswell assays\u003c/h2\u003e \u003cp\u003eCAM1 cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were seeded on the transwell inserts in 500 \u0026micro;L of medium. Medium containing 30% FBS was added to the lower chambers as a chemoattractant. After 24 h, the cells on the upper side of the filter were removed using cotton swabs. Cells permeated to the lower membrane surface were fixed with 4% paraformaldehyde, stained with crystal violet, and counted under an optical microscope (x 100 magnification). Cell counts were expressed as the mean number of cells from 10 random fields per well.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of marker gene expression and cytokine secretion\u003c/h2\u003e \u003cp\u003eIn this study, the CAM1 gene expression was measured using qRT-PCR. RNA was isolated from cells using the Multisource Total RNA kit and reverse-transcribed into cDNA using HiScript Ⅲ qRT SuperMix. The genes were quantified by qRT-PCR using specific primers (Table S2). TNF-α, IL-6, IL-12, and IL-10 cytokines were also detected in the cell supernatant using an ELISA kit (Table S3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhagocytosis assay\u003c/h2\u003e \u003cp\u003eCAM1 (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) and 4T1/B16 cells (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were individually stained with DiI and DiO, 5- CellTracker Green CMFDA, and anti-F4/80-PE. Briefly, CAM1 (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells) and 4T1/B16 cells (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells) were incubated with their respective dyes at 37 ℃ for 1 h in the dark. Following this, the cells were rinsed three times with PBS before being combined in a fresh culture medium. After 24 h of incubation, the mixed cells were examined using flow cytometry and confocal microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnimal studies\u003c/h2\u003e \u003cp\u003eFemale BALB/c mice, aged 4\u0026ndash;5 weeks, were obtained from the Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). All specific pathogen free (SPF) animals were housed at the Animal Facility Center at Hangzhou Normal University (HZNU). The study was conducted in compliance with the guidelines of the Animal Ethics and Welfare Committee (AEWC) of HZNU (Approval No. HSD20221101). A total of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e 4T1 cells in 100 \u0026micro;L of PBS were subcutaneously injected into the right flank of each mouse. Next, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e macrophages (100 \u0026micro;L) were injected into each mouse under the treatment schedule on days 0, 10, 13, 17, and 20.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using GraphPad Prism 9.0. All quantitative results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of at least three independent replicates. Statistical significance between groups was assessed using a one-way analysis of variance (ANOVA). \u003cem\u003eP\u003c/em\u003e-values of \u0026lt;\u0026thinsp;0.05, \u0026lt; 0.01, \u0026lt; 0.001, and \u0026lt;\u0026thinsp;0.0001 indicated significant differences. Survival analysis was performed using the Kaplan-Meier method and compared using the Log-rank test (Mantel-Cox).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of CAM1 cells\u003c/h2\u003e \u003cp\u003eTo functionalize M1 macrophages for cancer therapy, we used the lipophilicity of Chol to deliver the targeting molecule Chol-AS1411 to the surface of M1 macrophages, which were called Chol-AS1411-M1 macrophages (CAM1). Confocal microscopy analysis revealed a faint green fluorescent signal (Chol-AS1411-FAM) on the surface of RAW264.7 cells after 10-min incubation with Chol-AS1411-FAM. Furthermore, the intensity of green fluorescence on the surface of RAW264.7 cells increased with increasing incubation time. When the incubation time reached 1 h, a clear and complete circle of green fluorescence was observed on the cell membrane surface, proving that Chol-AS1411 could bind to the cell membrane surface after incubation for 1 h (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C). Further determination of green fluorescence by flow cytometry indicated comparable results (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E).\u003c/p\u003e \u003cp\u003eTo confirm the stability of Chol-AS1411 in DMEM medium to ensure the sustained antitumor effect of CAM1 \u003cem\u003ein vivo\u003c/em\u003e, M1 cell surfaces coated with Chol-AS1411-FAM inducer were incubated with DMEM for different periods, and the fluorescence signal of the CAM1 surface-modified aptamer was monitored. Confocal microscopy findings (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-G) indicate that the aptamer signal anchored on the CAM1 surface remained relatively stable for up to 48 h. Over time, the relative fluorescence intensity of the cell surface decreased. This study suggests that this is due to cell division during the proliferation of CAM1 cells, which also leads to a decrease in fluorescence intensity in the field of view.\u003c/p\u003e \u003cp\u003eIn this study, RAW264.7 cells were induced into M1 macrophages by a specific concentration of Lipopolysaccharides (LPS), which can cause a strong systemic inflammatory response \u003cem\u003ein vivo\u003c/em\u003e. Therefore, we quantified the endotoxin content of CAM1 to ensure its biosafety \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. The results indicated that the concentration of endotoxin in the third wash solution of CAM1 cells was approximately 0.07 EU/mL, a value significantly below the threshold of 0.50 EU/mL established by the Chinese Pharmacopoeia (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAlteration of CAM1 Activity\u003c/b\u003e \u003cb\u003ein Vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe viability of CAM1 cells was evaluated using calcein-AM/PI and CCK-8 assays. The calcein-AM/PI assay results revealed no significant difference in the survival rates of M1, M1 (with AS1411), and CAM1 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). CCK-8 assay was used to determine the viability of M1, M1 (with AS1411), and CAM1 cells. There was no significant reduction in the viability of the CAM1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These findings indicate that CAM1 cells have good cellular activity. Moreover, the wound healing results demonstrated that the migration ability of CAM1 cells did not differ significantly from that of M0 and M1 cells in the control group (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E), implying that the target molecule Chol-AS1411 did not significantly alter the macrophage migration behavior. Transwell results confirmed this finding (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine alteration of CAM1 properties, we examined the expression of CAM1 marker genes and proteins and cytokine secretion levels to determine whether CAM1 could reprogram the TME \u003cem\u003ein vivo\u003c/em\u003e. In this study, qRT-PCR was performed to measure the expression of the CAM1 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). The results revealed that CAM1 cells significantly expressed iNOS mRNA compared with M0 cells (vs. M0, ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). This finding suggests that our modified CAM1 cells did not change the M1 polar type of macrophages. In contrast, there was a significant increase in the expression of IL-6 mRNA (vs. M0, ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), IL-1β (vs. M0, ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and TNF-α mRNA (vs. M0, *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in M1 and CAM1 cells compared with M0 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). ELISA results demonstrated that CAM1 cells secreted significantly more pro-inflammatory factors, such as IL-12 (vs. CAM0, ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), IL-6 (vs. CAM0, ** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and TNF-α (vs. CAM0, *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared with CAM0 cells. Concurrently, the secretion of the anti-inflammatory factor IL-10 was inhibited (vs. CAM0, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). PE anti-mouse F4/80 was then used to label mature macrophages, and FITC anti-mouse CD86 was used to label the CD86 protein on the surface of macrophages. Flow cytometry results revealed that CAM1 cells could significantly express the CD86 membrane protein compared with other groups (vs. M0, ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), indicating that the modified CAM1 cells did not change the M1 cell type (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Our results demonstrated that CAM1 has excellent cellular activity and the potential to reprogram the TME.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntitumor effects of CAM1\u003c/b\u003e \u003cb\u003ein Vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAS1411 can specifically bind to NCL overexpression on the surface of tumor cells; therefore, we selected mouse breast cancer 4T1 and mouse melanoma B16 cells with positive expression of NCL for the target assay. Confocal microscopy imaging revealed that the C-AS1411-FAM (random sequence, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) incubation group exhibited hardly any green fluorescence. Conversely, noticeable green fluorescence was observed in the AS1411-FAM incubation group (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and E), and the fluorescence intensity of the AS1411-FAM incubation group differed significantly (****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 \u003cem\u003evs\u003c/em\u003e. M0) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and F), indicating that AS1411 specifically binds to 4T1 tumor cells with positive NCL expression. The fluorescence intensity on B16 tumor cells was further investigated using confocal microscopy and flow cytometry, and the results were consistent (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and G; D and H). Furthermore, the findings demonstrated that AS1411 had a significant affinity and was specifically bound to NCL on the surface of tumor cells. Therefore, the targeting molecule Chol-AS1411 may facilitate the specific binding of CAM1 cells to NCL-positive tumor cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe ability of CAM1 cells to phagocytose tumor cells was explored to demonstrate their antitumor effect \u003cem\u003ein vitro\u003c/em\u003e. The orange-red fluorescent cell membrane probe DiI and green fluorescent cell membrane probe DiO were used to label the cell membranes of macrophages and tumor cells, respectively. After 2-h co-incubation of macrophages and tumor cells, the phagocytosis of tumor cells by macrophages was observed using laser confocal microscopy. The findings revealed that CAM1 cells had the most robust green fluorescence, indicating that they had the most potent phagocytic activity against B16 and 4T1 cells, followed by M1 cells, with M2 cells demonstrating the least phagocytic activity against tumors (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-J). Furthermore, CellTracker Red CMTPX and CellTracker Green CMFDA were used to label the cytoplasm of macrophages and tumor cells, respectively, to identify the phagocytosis of CAM1 cells. Flow cytometry was also used to assess the phagocytic ability of CAM1 cells. The results also revealed that CAM1 cells have a strong phagocytic ability against tumors (Fig. S2).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntitumor effects of CAM1\u003c/b\u003e \u003cb\u003ein Vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGiven that CAM1 cells successfully invaded tumors \u003cem\u003ein vitro\u003c/em\u003e, we investigated the antitumor effect of CAM1 cells \u003cem\u003ein vivo\u003c/em\u003e. First, we established a 4T1 subcutaneous tumor model of breast cancer in female BALB/c mice to evaluate the antitumor efficacy of CAM1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Next, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e macrophages (100 \u0026micro;L) were administered to each mouse following the prescribed treatment regimen via intravenous injection (i.v.) and intratumoral injection (i.t.). The Chol-C-AS1411 (random sequence) modified M1 macrophage (CCAM1) served as the negative control group.Tumor growth in the PBS (i.v.) and CCAM1 (i.v.) groups was almost unrestricted and maintained a high growth rate, indicating that CCAM1 (i.v.) could not inhibit tumor growth. M1 (i.v.) and CAM1 (i.t.) also exhibited modest inhibitory effects on tumor growth. However, tumor growth in the CAM1 (i.v.) group was significantly inhibited, and there was a significant difference (\u003cem\u003evs.\u003c/em\u003e PBS group, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that CAM1 (i.v.) produced an excellent antitumor effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Moreover, there was no significant variation in the body weight of the mice across all groups, indicating that the engineered macrophage CAM1 had significant antitumor activity without obvious systemic toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFluorescence imaging, which can accurately reflect the accumulation and metabolism of injected macrophages in mice, is helpful in assessing cancer conditions and cell therapy efficacy during treatment in a timely and accurate manner. The fluorescence of CY5.5 was used in this study to modify the injected engineered macrophage-targeting molecules to obtain Chol-AS1411-CY5.5 and Chol-C-AS1411-CY5.5. By co-incubation with M1 cells, Chol-AS1411-CY5.5-M1 (CAM1-CY5.5) and Chol-C-AS1411-CY5.5-M1 (CCAM1-CY5.5) were obtained. First, we evaluated the distribution of CAM1 cells in tumor-bearing mice and found that all three groups, CCAM1-CY5.5 (i.v.), CAM1-Cy5.5 (i.v.), and CAM1-Cy5.5 (i.t.), effectively emitted fluorescent signals at the tumor site (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E). Second, 24 h after the injection of CAM1 cells, we assessed the distribution of CAM1-Cy5.5 (i.v.), CAM1-Cy5.5 (i.v.), and CAM1-Cy5.5 (i.t.) in the major organs (heart, liver, spleen, lung, and kidney) and tumors of tumor-bearing mice. The liver exhibited the highest concentration of fluorescence, followed by the tumor, which is consistent with the fluorescence imaging results within one week. This further indicated that the engineered macrophages could enter the tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The spleen, the largest immune organ, is an important site for the body to produce an immune response. The present study revealed that the volume and weight of the spleen in tumor-bearing mice were significantly higher than in normal mice, indicating that the body produces a strong immune response during the growth and treatment of 4T1 tumors, resulting in spleen enlargement.\u003c/p\u003e \u003cp\u003eFurthermore, a peak of the fluorescence signal at the tumor site was observed on the fourth day, implying that the injected engineered macrophages successfully infiltrated the tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Over time, the injected cells were gradually metabolized by the body, and the fluorescence signal became weaker. However, the fluorescence signal was still detectable one week after infusion, demonstrating that the injected engineered macrophages could effectively remain at the tumor site.\u003c/p\u003e \u003cp\u003eTo further investigate the antitumor effect of CAM1 cells \u003cem\u003ein vivo\u003c/em\u003e, hematoxylin-eosin (HE) staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) were used to detect apoptosis of tumor tissues in tumor-bearing mice in each group after treatment. HE staining demonstrated that the CAM1 (i.v.) group had more pronounced nuclear pyknosis, reduced tumor density, and more severe tumor cell necrosis than the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). TUNEL staining revealed that the PBS (i.v.) and CCAM1 (i.v.) groups had almost no green fluorescence. In contrast, the other treatment groups had varying degrees of green fluorescence, with the CAM1 (i.v.) group indicating the strongest green fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). The results of this experiment indicated that CAM1 cell treatment led to a large region of tumor cell necrosis and apoptosis, suggesting that CAM1 cells have potent specific killing effects on tumors, inhibited tumor cell proliferation, and promoted tumor cell apoptosis, which holds promise for developing efficient and safe tumor treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBiotoxicity of CAM1\u003c/h2\u003e \u003cp\u003eTo achieve the subsequent clinical application of CAM1 cells \u003cem\u003ein vivo\u003c/em\u003e, the biosafety of each group was assessed following the treatment of tumor-bearing mice with CAM1 cells in organs (heart, liver, spleen, lung, and kidney) using HE staining and routine blood tests. HE staining revealed that the PBS (i.v.) and CCAM1 (i.v.) groups had mild alveolar wall thickening and inflammatory cell infiltration, whereas the other groups had normal lung structures. Other organs in good cellular condition showed no significant pathological changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). CAM1 cells can reduce the severity of organ lesions while being antitumor and demonstrating excellent biological safety in normal tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, the results of routine blood tests revealed no significant difference in any of the blood indices between the PBS (i.v.) and CAM1 cell groups. This suggests that CAM1 cells are associated with a degree of biological safety (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026ndash;E). In contrast, the index of white blood cells (WBCs) in the PBS group was significantly higher than the normal range, indicating that the number of 4T1 WBCs in the mouse model exceeded the upper limit of the normal reference value.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe diverse functions and high plasticity of macrophages allow them to infiltrate tumor tissues and exert their effects. Therefore, numerous clinical trials have extensively investigated adoptive macrophage transfer therapy as a potential therapeutic approach for cancer treatment[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Considering their critical role in cancer immunity, targeting tumors using macrophage-related therapies has emerged as a promising immunotherapy strategy. In this study, we developed engineered macrophage CAM1 for cellular immunotherapy using aptamer AS1411 specific binding properties to overexpress NCL on the surface of tumor cells. Additionally, we used the antitumor properties of M1 macrophages and exploited the lipophilicity between Chol molecules and the phospholipid bilayer of the membrane. This innovative strategy aims to target tumor cells for phagocytosis while reshaping the TME, thereby providing novel insights into tumor immunotherapy.\u003c/p\u003e \u003cp\u003eImmunotherapy is currently focused on improving innate defense mechanisms to eradicate malignant cells. This symbiotic approach exploits the immune system's ability to provide comprehensive cancer treatment while leveraging the innate immune response to optimize therapeutic efficacy. Consequently, these breakthroughs in tumor immunology have energized the field with unparalleled vigor\u003csup\u003e[37]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eResistance of macrophages to genetic engineering techniques is a widely recognized characteristic that limits their therapeutic efficacy in clinical applications[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this study, we developed a non-engineered approach, cell surface engineering, to modify macrophages to produce engineered macrophage CAM1. This innovative strategy involves anchoring AS1411 onto the surface of macrophages, enabling it to selectively bind to tumor cells that overexpress NCL on their surfaces. By avoiding any modifications to the original genes and proteins of macrophages, this method has minimal adverse effects on macrophage function while improving their immune response. Therefore, these results provide new insights into tumor immunotherapy.\u003c/p\u003e \u003cp\u003eHowever, CAM1 inevitably encounters certain unfavorable conditions in the TME, such as immune checkpoint ligands and a large population of immunosuppressive cells. Furthermore, variations in pH levels and the potential induction of a \"cytokine storm\" in mice remain important considerations for CAM1. Considering these challenges, we aimed to further investigate and improve CAM1 in tumor-bearing mice while delving deeper into its immune regulatory mechanisms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding: This work was supported by the Huadong Medicine Joint Funds of the Zhejiang Provincial Natural Science Foundation of China under Grant No. LHDMZ22H300001, the Joint Funds of the Shandong Provincial Natural Science Foundation of China (ZR202108130026).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflict of interest exits in the submission of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT Author Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQian Yang: Methodology, Software, Investigation, Formal Analysis, Writing-Original Draft;\u003c/p\u003e\n\u003cp\u003eShiyi Hu: Data Curation, Software, Writing-Original Draft;\u003c/p\u003e\n\u003cp\u003eYiqiu Wang:\u0026nbsp;Software, Writing-Original Draft\u003c/p\u003e\n\u003cp\u003eLuyi Zhong:\u0026nbsp;Data Curation, Software;\u003c/p\u003e\n\u003cp\u003eXiaoli Yu: Writing-Original Draft;\u003c/p\u003e\n\u003cp\u003eYifeng Zhang:\u0026nbsp;Data Curation, Writing-Original Draft;\u003c/p\u003e\n\u003cp\u003eYiting Zhang：Data Curation, Writing-Original Draft;\u003c/p\u003e\n\u003cp\u003eHonghua Zhang: Software, Validation;\u003c/p\u003e\n\u003cp\u003eShuling Wang: Conceptualization, Resources, Supervision.\u003c/p\u003e\n\u003cp\u003eQingchang Tian: Methodology, Software, Investigation, Formal Analysis, Validation, Funding Acquisition.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi YR, Zhou Y, Kim YJ, et al. Development of allogeneic HSC-engineered iNKT cells for off-the-shelf cancer immunotherapy. Cell Rep Med. 2021;2:100449.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunshi NC, Anderson LD Jr., Shah N, et al. Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma. N Engl J Med. 2021;384:705\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaje N, Berdeja J, Lin Y, et al. Anti-BCMA CAR T-Cell Therapy bb2121 in Relapsed or Refractory Multiple Myeloma. N Engl J Med. 2019;380:1726\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang M, Munoz J, Goy A, et al. KTE-X19 CAR T-Cell Therapy in Relapsed or Refractory Mantle-Cell Lymphoma. N Engl J Med. 2020;382:1331\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRapoport AP, Stadtmauer EA, Binder-Scholl GK, et al. NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat Med. 2015;21:914\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJune CH, O'Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science. 2018;359:1361\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa S, Li X, Wang X, et al. Current Progress in CAR-T Cell Therapy for Solid Tumors. Int J Biol Sci. 2019;15:2548\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuedan S, Ruella M, June CH. Emerging Cellular Therapies for Cancer. Annu Rev Immunol. 2019;37:145\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMelenhorst JJ, Chen GM, Wang M, et al. Decade-long leukaemia remissions with persistence of CD4(+) CAR T cells. Nature. 2022;602:503\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD'Aloia MM, Zizzari IG, Sacchetti B, Pierelli L, Alimandi M. CAR-T cells: the long and winding road to solid tumors. Cell Death Dis. 2018;9:282.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaude SL, Laetsch TW, Buechner J, et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med. 2018;378:439\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePang Y, Hou X, Yang C, Liu Y, Jiang G. Advances on chimeric antigen receptor-modified T-cell therapy for oncotherapy. Mol Cancer. 2018;17:91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaskowski TJ, Biederst\u0026auml;dt A, Rezvani K. Natural killer cells in antitumour adoptive cell immunotherapy. Nat Rev Cancer. 2022;22:557\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMehrabadi AZ, Ranjbar R, Farzanehpour M, et al. Therapeutic potential of CAR T cell in malignancies: A scoping review. Volume 146. Biomedicine \u0026amp; pharmacotherapy\u0026thinsp;=\u0026thinsp;Biomedecine \u0026amp; pharmacotherapie; 2022. p. 112512.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Yu Z, Tan X, et al. CAR-macrophage: A new immunotherapy candidate against solid tumors. Volume 139. Biomedicine \u0026amp; pharmacotherapy\u0026thinsp;=\u0026thinsp;Biomedecine \u0026amp; pharmacotherapie; 2021. p. 111605.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWagner J, Wickman E, DeRenzo C, Gottschalk S. CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality? Mol therapy: J Am Soc Gene Therapy. 2020;28:2320\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChan JD, Lai J, Slaney CY, Kallies A, Beavis PA, Darcy PK. Cellular networks controlling T cell persistence in adoptive cell therapy. Nat Rev Immunol. 2021;21:769\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeatty GL, O'Hara M. Chimeric antigen receptor-modified T cells for the treatment of solid tumors: Defining the challenges and next steps. Pharmacol Ther. 2016;166:30\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLong KB, Young RM, Boesteanu AC, et al. CAR T Cell Therapy of Non-hematopoietic Malignancies: Detours on the Road to Clinical Success. Front Immunol. 2018;9:2740.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNoy R, Pollard JW. Tumor-associated macrophages: from mechanisms to therapy. Immunity. 2014;41:49\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFranken L, Schiwon M, Kurts C. Macrophages: sentinels and regulators of the immune system. Cell Microbiol. 2016;18:475\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang L, Zhang Y. Tumor-associated macrophages: from basic research to clinical application. J Hematol Oncol. 2017;10:58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRitchie D, Mileshkin L, Wall D, et al. In vivo tracking of macrophage activated killer cells to sites of metastatic ovarian carcinoma. Cancer Immunol immunotherapy: CII. 2007;56:155\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlichinsky M, Ruella M, Shestova O, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol. 2020;38:947\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang M, Lee SH, Kwon M et al. Nanocomplex-Mediated In Vivo Programming to Chimeric Antigen Receptor-M1 Macrophages for Cancer Therapy. Advanced materials (Deerfield Beach, Fla). 2021;33:e2103258.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHao M, Hou S, Li W et al. Combination of metabolic intervention and T cell therapy enhances solid tumor immunotherapy. Sci Transl Med. 2020;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang D, Zheng Y, Lin Z et al. Equipping Natural Killer Cells with Specific Targeting and Checkpoint Blocking Aptamers for Enhanced Adoptive Immunotherapy in Solid Tumors. Angewandte Chemie (International ed in English). 2020;59:12022\u0026ndash;12028.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Hong H, Cai W. Tumor-targeted drug delivery with aptamers. Curr Med Chem. 2011;18:4185\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin SY, Zhang AQ, Cheng SX, Rong L, Zhang XZ. Drug self-delivery systems for cancer therapy. Biomaterials. 2017;112:234\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan J, Gao T, Lu Z, Yin J, Zhang Y, Pei R. Aptamer-Targeted Photodynamic Platforms for Tumor Therapy. ACS Appl Mater Interfaces. 2021;13:27749\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeefe AD, Pai S, Ellington A. Aptamers as therapeutics. Nat Rev Drug Discovery. 2010;9:537\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBates PJ, Reyes-Reyes EM, Malik MT, Murphy EM, O'Toole MG, Trent JO. G-quadruplex oligonucleotide AS1411 as a cancer-targeting agent: Uses and mechanisms. Biochim et Biophys acta Gen Subj. 2017;1861:1414\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBie L, Wang Y, Jiang F, Xiao Z, Zhang L, Wang J. Insights into the binding mode of AS1411 aptamer to nucleolin. Front Mol Biosci. 2022;9:1025313.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiranda A, Santos T, Largy E, Cruz C. Locking up the AS1411 Aptamer with a Flanking Duplex: Towards an Improved Nucleolin-Targeting. Pharmaceuticals (Basel, Switzerland). 2021;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoe GR, Steirer LM, Lee JA, Shivakumar A, Bolanos AD. A cancer-unique glycan: de-N-acetyl polysialic acid (dPSA) linked to cell surface nucleolin depends on re-expression of the fetal polysialyltransferase ST8SIA2 gene. J experimental Clin cancer research: CR. 2021;40:293.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVindigni G, Raniolo S, Iacovelli F et al. AS1411 Aptamer Linked to DNA Nanostructures Diverts Its Traffic Inside Cancer Cells and Improves Its Therapeutic Efficacy. Pharmaceutics. 2021;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Zhang Z. The history and advances in cancer immunotherapy: understanding the characteristics of tumor-infiltrating immune cells and their therapeutic implications. Cell Mol Immunol. 2020;17:807\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSloas C, Gill S, Klichinsky M. Engineered CAR-Macrophages as Adoptive Immunotherapies for Solid Tumors. Front Immunol. 2021;12:783305.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang W, Liu L, Su H, et al. Chimeric antigen receptor macrophage therapy for breast tumours mediated by targeting the tumour extracellular matrix. Br J Cancer. 2019;121:837\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4250998/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4250998/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMacrophages play a critical role in the body's defense against cancer by phagocytosing tumor cells, presenting antigens, and activating adaptive T cells. However, macrophages are intrinsically incapable of delivering targeted cancer immunotherapies. Engineered adoptive cell therapy introduces new targeting and antitumor capabilities by modifying macrophages to enhance the innate immune response of cells and improve clinical efficacy. In this study, we developed engineered macrophage cholesterol-AS1411-M1 (CAM1) for cellular immunotherapy. To target macrophages, cholesterol-AS1411 aptamers are anchored to the surface of M1 macrophages to produce CAM1 without genetic modification or cell damage. CAM1 induced significantly higher apoptosis/mortality than unmodified M1 macrophages in murine breast cancer cells. Anchoring AS1411 on the surface of macrophages without modifying their original genes and proteins provides a novel approach to tumor immunotherapy.\u003c/p\u003e","manuscriptTitle":"Engineering M1 macrophages with targeting aptamers for enhanced adoptive immunotherapy by modifying the cell surface","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-17 12:20:23","doi":"10.21203/rs.3.rs-4250998/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"617b16e0-01cf-4680-837d-da68d7c2ec24","owner":[],"postedDate":"April 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-07-04T00:32:03+00:00","versionOfRecord":{"articleIdentity":"rs-4250998","link":"https://doi.org/10.1016/j.biopha.2024.117064","journal":{"identity":"biomedicine-and-pharmacotherapy","isVorOnly":true,"title":"Biomedicine \u0026 Pharmacotherapy"},"publishedOn":"2024-08-01 00:32:03","publishedOnDateReadable":"August 1st, 2024"},"versionCreatedAt":"2024-04-17 12:20:23","video":"","vorDoi":"10.1016/j.biopha.2024.117064","vorDoiUrl":"https://doi.org/10.1016/j.biopha.2024.117064","workflowStages":[]},"version":"v1","identity":"rs-4250998","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4250998","identity":"rs-4250998","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}