Metalloimmunotherapy with Rhodium and Ruthenium Complexes: Targeting Tumor-Associated Macrophages

Tumor associated macrophages (TAMs) suppress the cancer immune response and are a key target for immunotherapy. The effects of ruthenium and rhodium complexes on TAMs have not been well characterized. To address this gap in the field, a panel of 22 dirhodium and ruthenium complexes were screened against three subtypes of macrophages, triple-negative breast cancer and normal breast tissue cells. Experiments were carried out in 2D and biomimetic 3D co-culture experiments with and without irradiation with blue light. Leads were identified with cell-type-specific toxicity toward macrophage subtypes, cancer cells, or both. Experiments with 3D spheroids revealed complexes that sensitized the tumor models to the chemotherapeutic doxorubicin. Cell surface exposure of calreticulin, a known facilitator of immunogenic cell death (ICD), was increased upon treatment, along with a concomitant reduction in the M2-subtype classifier arginase. Our findings lay a strong foundation for the future development of ruthenium- and rhodium-based chemotherapies targeting TAMs.     

Natural Phenolic Acid,Product of the Honey Bee,for the Control of Oxidative Stress,Peritoneal Angiogenesis,and Tumor Growth in Mice

Tumor-associated macrophages (TAM) are key regulators of the link between inflammation and cancer, and the interplay between TAM and tumor cells represents a promising target of future therapeutic approaches. We investigated the effect of gallic acid (GA) and caffeic acid (CA) as strong antioxidant and anti-inflammatory agents on tumor growth, angiogenesis, macrophage polarization, and oxidative stress on the angiogenic model caused by the intraperitoneal (ip) inoculation of Ehrlich ascites tumor (EAT) cells (2.5 × 10⁶) in Swiss albino mouse. Treatment with GA or CA at a dose of 40 mg/kg and 80 mg/kg ip was started in exponential tumor growth phase on days 5, 7, 9, and 11. On day 13, the ascites volume and the total number and differential count of the cells present in the peritoneal cavity, the functional activity of macrophages, and the antioxidant and anti-angiogenic parameters were determined. The results show that phenolic acids inhibit the processes of angiogenesis and tumor growth, leading to the increased survival of EAT-bearing mice, through the protection of the tumoricidal efficacy of M1 macrophages and inhibition of proangiogenic factors, particularly VEGF, metalloproteinases -2 and -9, and cyclooxygenase-2 activity.     

In Situ Reprogramming of Tumor-Associated Macrophages with Internally and Externally Engineered Exosomes

The reprogramming of tumor-associated macrophages (TAMs) has emerged as an efficient strategy for immunotherapy. However, most of the approaches did not allow the in situ reprogramming of TAM because their low efficiency, non-specificity, or potential side effects. Herein, we produced exosomes with the clustered regularly interspaced short palindromic repeats interference (CRISPRi) internally engineered and the TAM specific peptide externally engineered onto the exosome membrane. The i nternally and e xternally e ngineered e xosomes ( IEEE , also named as I3E ) allowed the selective homing to tumor tissue and targeted to M2-like TAMs, which nearly repressed the expression of PI-3 kinase gamma (PI3Kγ) completely, and induced the TAMs polarizing to M1 both in vitro and in vivo. The polarized M1 macrophages awakened the “hot” tumor-immunity, causing the increase of T lymphocyte infiltration and the decrease of myeloid-derived suppressor cells, and inhibiting the tumor growth significantly. I3E reprogramed TAMs in situ precisely and efficiently.     

Microfluidic three-dimensional biomimetic tumor model for studying breast cancer cell migration and invasion in the presence of interstitial flow

Cell migration and invasion are critical steps in cancer metastasis, which are the major cause of death in cancer patients. Tumor-associated macrophages(TAMs) and interstitial flow(IF) are two important biochemical and biomechanical cues in tumor microenvironment, play essential roles in tumor progression. However, their combined effects on tumor cell migration and invasion as well as molecular mechanism remains largely unknown. In this work, we developed a microfluidic-based 3 D breast cancer model by co-culturing tumor aggregates, macrophages, monocytes and endothelial cells within 3 D extracellular matrix in the presence of IF to study tumor cell migration and invasion. On the established platform, we can precisely control the parameters related to tumor microenvironment and observe cellular responses and interactions in real-time. When co-culture of U937 with human umbilical vein endothelial cells(HUVECs) or MDA-MB-231 cells and tri-culture of U937 with HUVECs and MDA-MB-231 cells, we found that mesenchymal-like MDA-MB-231 aggregates activated the monocytes to TAM-like phenotype macrophages. MDA-MB-231 cells and IF simultaneously enhanced the macrophages activation by the stimulation of colony-stimulating factor 1(CSF-1). The activated macrophages and IF further promoted vascular sprouting via vascular endothelial growth factor(VEGFα) signal and tumor cell invasion. This is the first attempt to study the interaction between macrophages and breast cancer cells under IF condition. Taken together, our results provide a new insight to reveal the important physiological and pathological processes of macrophages-tumor communication. Moreover, our established platform with a more mimetic 3 D breast cancer model has the potential for drug screening with more accurate results.     

Chirality-Dependent Reprogramming of Macrophages by Chiral Nanozymes

It is known that extracellular free radical reactive oxygen species (ROS) rather than intracellular ROS plays a non-substitutable role in regulation of tumor-suppressing (M1) tumor-associated macrophages (TAMs) polarization. However, most therapeutic nanoplatforms mainly provide intracellular ROS and exhibit insufficient accumulation near TAMs, which strongly limits the macrophage-based immunotherapeutic effects. Here we design and synthesize chiral MoS₂/CoS₂ nanozymes with peroxidase (POD)-like and catalase (CAT)-like activities to efficiently modulate TAMs polarization and reverse tumor immunosuppression by harnessing their chirality-specific interactions with biological systems. MoS₂/CoS₂ nanoparticles coordinated with d -chirality (d -NPs, right-handed) show improved pharmacokinetics with longer circulating half-life and higher tumor accumulation compared with their l ( left-handed)- and dl ( racemate)-counterparts. Further, d -NPs can escape from macrophage uptake in the tumor microenvironment (TME) with the aid of cell-unpreferred opposite chirality and act as extracellular hydroxyl radicals (⋅OH) and oxygen (O₂) generators to efficiently repolarize TAMs into M1 phenotype. On the contrary, l -NPs showed high cellular uptake due to chirality-driven homologous adhesion between l -NPs and macrophage membrane, leading to limited M1 polarization performance. As the first example for developing chiral nanozymes as extracellular-localized ROS generators to reprogram TAMs for cancer immunotherapy, this study opens an avenue for applications of chiral nanozymes in immunomodulation.     

Bimodal Targeting Using Sulfonated,Mannosylated PEI for Combined Gene Delivery and Photodynamic Therapy

Photodynamic therapy (PDT) and gene delivery have both been used to target both cancer cells and tumor‐associated macrophages (TAMs). Given the complex nature of tumor tissue, there could be merit in combining these strategies simultaneously. In this study, we developed a bimodal targeting approach to both cancer cells and macrophages, employing materials conducive to both gene delivery and PDT. Polymers libraries were created that consisted of cationic polyethyleneimine (PEI) conjugated to the photosensitizer pyropheophorbide‐a, with sulfonation (to target selectin‐expressing cells) and mannosylation (to target TAMs). Polyplexes, consisting of these polymers electrostatically bound to DNA, were analyzed for transfection efficacy and cytotoxicity toward epithelial cells and macrophages to assess dual‐targeting. This study provides preliminary proof of principle for using modified PEI for targeted gene delivery and PDT.     

In situ Engineering of Tumor-Associated Macrophages via a Nanodrug-Delivering-Drug (β-Elemene@Stanene) Strategy for Enhanced Cancer Chemo-Immunotherapy

Tumor-associated macrophages (TAMs) play a critical role in the immunosuppressive solid tumor microenvironment (TME), yet in situ engineering of TAMs for enhanced tumor immunotherapy remains a significant challenge in translational immuno-oncology. Here, we report an innovative nanodrug-delivering-drug (STNSP@ELE) strategy that leverages two-dimensional (2D) stanene-based nanosheets (STNSP) and β-Elemene (ELE), a small-molecule anticancer drug, to overcome TAM-mediated immunosuppression and improve chemo-immunotherapy. Our results demonstrate that both STNSP and ELE are capable of polarizing the tumor-supportive M2-like TAMs into a tumor-suppressive M1-like phenotype, which acts with the ELE chemotherapeutic to boost antitumor responses. In vivo mouse studies demonstrate that STNSP@ELE treatment can reprogram the immunosuppressive TME by significantly increasing the intratumoral ratio of M1/M2-like TAMs, enhancing the population of CD4⁺ and CD8⁺ T lymphocytes and mature dendritic cells, and elevating the expression of immunostimulatory cytokines in B16F10 melanomas, thereby promoting a robust antitumor response. Our study not only demonstrates that the STNSP@ELE chemo-immunotherapeutic nanoplatform has immune-modulatory capabilities that can overcome TAM-mediated immunosuppression in solid tumors, but also highlights the promise of this nanodrug-delivering-drug strategy in developing other nano-immunotherapeutics and treating various types of immunosuppressive tumors.     

Pinosylvin Shifts Macrophage Polarization to Support Resolution of Inflammation

Pinosylvin is a natural stilbenoid found particularly in Scots pine. Stilbenoids are a group of phenolic compounds identified as protective agents against pathogens for many plants. Stilbenoids also possess health-promoting properties in humans; for instance, they are anti-inflammatory through their suppressing action on proinflammatory M1-type macrophage activation. Macrophages respond to environmental changes by polarizing towards proinflammatory M1 phenotype in infection and inflammatory diseases, or towards anti-inflammatory M2 phenotype, mediating resolution of inflammation and repair. In the present study, we investigated the effects of pinosylvin on M2-type macrophage activation, aiming to test the hypothesis that pinosylvin could polarize macrophages from M1 to M2 phenotype to support resolution of inflammation. We used lipopolysaccharide (LPS) to induce M1 phenotype and interleukin-4 (IL-4) to induce M2 phenotype in J774 murine and U937 human macrophages, and we measured expression of M1 and M2-markers. Interestingly, along with inhibiting the expression of M1-type markers, pinosylvin had an enhancing effect on the M2-type activation, shown as an increased expression of arginase-1 (Arg-1) and mannose receptor C type 1 (MRC1) in murine macrophages, and C-C motif chemokine ligands 17 and 26 (CCL17 and CCL26) in human macrophages. In IL-4-treated macrophages, pinosylvin enhanced PPAR-γ expression but had no effect on STAT6 phosphorylation. The results show, for the first time, that pinosylvin shifts macrophage polarization from the pro-inflammatory M1 phenotype towards M2 phenotype, supporting resolution of inflammation and repair.     

Co-culture of tumor spheroids and monocytes in a collagen matrix-embedded microfluidic device to study the migration of breast cancer cells

A 3D co-culture microfluidic device was developed to study the effects of ECM stiffness and TAMs on tumor cells migration.     

基于拟靶向液相色谱-质谱联用的胃癌患者血清代谢组分析

胃癌是一种高发的恶性肿瘤,是癌症相关死亡的第二大病因。早期筛查是提高患者生存率的有效手段,但目前临床上尚缺乏实现胃癌无创筛检的可靠标志物。本研究采用了基于液相色谱-质谱联用的拟靶向代谢组学方法分析了20例胃癌患者及40例正常人血清代谢组,以期发现新的潜在代谢标志物。代谢组数据的主成分分析和偏最小二乘法数据分析结果显示,胃癌患者与健康人群的血清代谢组存在明显的差异,结合非参数检验进一步筛选并定性出57个差异代谢物。其中二氢胆固醇经验证组样本验证,具有成为胃癌代谢标志物的潜力。本研究在发现胃癌的潜在代谢标志物的同时,也为胃癌患者代谢分型提供了重要的科学依据。     

Decreased expression of ATF3, orchestrated by β-catenin/TCF3, miR-17-5p and HOXA11-AS,promoted gastric cancer progression via increased β-catenin and CEMIP

ATF3 has been reported to be dysregulated in various cancers and involved in various steps of tumorigenesis. However, the mechanisms underlying the abnormal expression of ATF3 and its biological function in gastric cancer (GC) have not been well investigated. Here, we report ATF3 as one of the key regulators of GC development and progression. Patients with low ATF3 expression had shorter survival and a poorer prognosis. In vitro and in vivo assays investigating ATF3 alterations revealed a complex integrated phenotype that affects cell growth and migration. Strikingly, high-throughput sequencing and microarray analysis of cells with ATF3 silencing or of ATF3-low GC tissues indicated alterations in the Wnt signaling pathway, focal adhesions and adherens junctions. Mechanistically, the expression of β-catenin and cell migration inducing hyaluronidase 1 (CEMIP) was significantly upregulated in GC cells with downregulated ATF3, which was synergistically repressed by the β-catenin/TCF3 signaling axis and noncoding RNA miR-17-5p and HOXA11-AS. In addition, we found that WDR5 expression was promoted by TCF3 and is involved in miR-17-5p and HOXA11-AS activation in GC cells. Taken together, our findings revealed the mechanism of ATF3 downregulation and its biological role in regulating the expression of Wnt signaling-related genes during GC progression, suggesting new informative biomarkers of malignancy and therapeutic directions for GC patients.Subject terms: Gastric cancer, Experimental models of disease     

A Gene-Editable Palladium-Based Bioorthogonal Nanoplatform Facilitates Macrophage Phagocytosis for Tumor Therapy

Macrophage phagocytosis of tumor cells has emerged as an attractive strategy for tumor therapy. Nevertheless, immunosuppressive M2 macrophages in the tumor microenvironment and the high expression of anti-phagocytic signals from tumor cells impede therapeutic efficacy. To address these issues and improve the management of malignant tumors, in this study we developed a gene-editable palladium-based bioorthogonal nanoplatform, consisting of CRISPR/Cas9 gene editing system-linked Pd nanoclusters, and a hyaluronic acid surface layer (HBPdC). This HBPdC nanoplatform exhibited satisfactory tumor-targeting efficiency and triggered Fenton-like reactions in the tumor microenvironment to generate reactive oxygen species for chemodynamic therapy and macrophage M1 polarization, which directly eliminated tumor cells, and stimulated the antitumor response of macrophages. HBPdC could reprogram tumor cells through gene editing to reduce the expression of CD47 and adipocyte plasma membrane-associated protein, thereby promoting their recognition and phagocytosis by macrophages. Moreover, HBPdC induced the activation of sequestered prodrugs via bioorthogonal catalysis, enabling chemotherapy and thereby enhancing tumor cell death. Importantly, the Pd nanoclusters of HBPdC were sufficiently cleared through basic metabolic pathways, confirming their biocompatibility and biosafety. Therefore, by promoting macrophage phagocytosis, the HBPdC system developed herein represents a highly promising antitumor toolset for cancer therapy applications.     

Gene expression profiling of tumor-associated macrophages after exposure to single-dose irradiation

Radiotherapy (RT) is a common cancer treatment approach that accounts for nearly 50% of patient treatment; however, tumor relapse after radiotherapy is still a major issue. To study the crucial role of tumor-associated macrophages (TAMs) in the regulation of tumor progression post-RT, microarray experiments comparing TAM gene expression profiles between unirradiated and irradiated tumors were conducted to discover possible roles of TAMs in initiation or contribution to tumor recurrence following RT, taking into account the relationships among gene expression, tumor microenvironment, and immunology. A single dose of 25 Gy was given to TRAMP C-1 prostate tumors established in C57/B6 mice. CD11b-positive macrophages were extracted from the tumors at one, two and three weeks post-RT. Gene ontology (GO) term analysis using the DAVID database revealed that genes that were differentially expressed at one and two weeks after irradiation were associated with biological processes such as morphogenesis of a branching structure, tube development, and cell proliferation. Analysis using Short Time-Series Expression Miner (STEM) revealed the temporal gene expression profiles and identified 13 significant patterns in four main groups of profiles. The genes in the upregulated temporal profile have diverse functions involved in the intracellular signaling cascade, cell proliferation, and cytokine-mediated signaling pathway. We show that tumor irradiation with a single 25-Gy dose can initiate a time-series of differentially expressed genes in TAMs, which are associated with the immune response, DNA repair, cell cycle arrest, and apoptosis. Our study helps to improve our understanding of the function of the group of genes whose expression changes temporally in an irradiated tumor microenvironment.     

Biofunctional Janus particles promote phagocytosis of tumor cells by macrophages

Herein, a versatile strategy for the construction of biofunctional Janus particles (JPs) through the combination of Pickering emulsion and copper-free click chemistry is developed for the study of particle-mediated cell–cell interactions. A variety of biomolecules including bovine serum albumin (BSA), ferritin, transferrin (Tf), and anti-signal regulatory protein alpha antibodies (aSIRPα), etc., can be incorporated into the Janus platform in a spatially defined manner. JPs consisting of Tf and aSIRPα (Tf–SPA1–aSIRPα JPs) demonstrate a significantly improved binding affinity to either macrophages or tumor cells compared to their uniformly modified counterparts. More importantly, Tf–SPA1–aSIRPα JPs mediate more efficient phagocytosis of tumor cells by macrophages as revealed by real-time high-content confocal microscopy. This study demonstrates the potential advantages of JPs in mediating cell–cell interactions and may contribute to the emerging cancer immunotherapy.

A versatile Janus particle platform modified with biological ligands can facilitate tumor cell phagocytosis by macrophages for promising cancer immunotherapy.     

Anti-inflammatory effect of myristicin on RAW 264.7 macrophages stimulated with polyinosinic-polycytidylic acid

Myristicin (1-allyl-5-methoxy-3,4-methylenedioxybenzene) is an active aromatic compound found in nutmeg (the seed of Myristica fragrans), carrot, basil,cinnamon, and parsley. Myristicin has been known to have anti-cholinergic, antibacterial,and hepatoprotective effects, however, the effects of myristicin on virus-stimulated macrophages are not fully reported. In this study, the anti-inflammatory effect of myristicin on double-stranded RNA (dsRNA)-stimulated macrophages was examined. Myristicin did not reduce the cell viability of RAW 264.7 mouse macrophages at concentrations of up to 50 μM. Myristicin significantly inhibited the production of calcium, nitric oxide (NO),interleukin (IL)-6, IL-10, interferon inducible protein-10, monocyte chemotactic protein(MCP)-1, MCP-3, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein (MIP)-1α, MIP-1β, and leukemia inhibitory factor in dsRNA[polyinosinic-polycytidylic acid]-induced RAW 264.7 cells (P < 0.05). In conclusion,myristicin has anti-inflammatory properties related with its inhibition of NO, cytokines,chemokines, and growth factors in dsRNA-stimulated macrophages via the calcium pathway.     

Diterpenoids Isolated from Podocarpus macrophyllus Inhibited the Inflammatory Mediators in LPS-Induced HT-29 and RAW 264.7 Cells

Species of Podocarpus are used traditionally in their native areas for the treatment of fevers, asthma, coughs, cholera, chest pain, arthritis, rheumatism, and sexually transmitted diseases. To identify natural products having efficacy against inflammatory bowel disease (IBD), we identified a new, 16-hydroxy-4β-carboxy-O-β-D-glucopyranosyl-19-nor-totarol (4) together with three known diterpenoids from P. macrophyllus. Furthermore, all the extracts, fractions, and isolates 1–4 were investigated for their anti-inflammatory effects by assessing the expression on nitric oxide (NO) production and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 and HT-29 cells. Among them, nagilactone B (2) exhibited a potent anti-inflammatory effect against NO production on RAW 264.7 cells; therefore, nagilactone B was further assessed for anti-inflammatory activity. Western blot analysis revealed that nagilactone B significantly decreased the expression of LPS-stimulated protein, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and phosphorylated extracellular regulated kinase (pERK)1/2. In addition, nagilactone B downregulated tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-8 levels in LPS-induced macrophages and colonic epithelial cells. To our best knowledge, this is the first report on the inhibitory effect of nagilactone B (pure state) and rakanmakilactone G against NO production in LPS-stimulated RAW 264.7 cells. Thus, diterpenoids isolated from P. macrophyllus could be employed as potential therapeutic phytochemicals for IBD.     

Metabolomic investigation of gastric cancer tissue using gas chromatography/mass spectrometry

Gastric cancer screening or diagnosis is mainly based on endoscopy and biopsy. The aim of this study was to identify the difference of metabolomic profile between normal and malignant gastric tissue, and to further explore tumor biomarkers. Chemical derivatization together with gas chromatography/mass spectrometry (GC/MS) was utilized to obtain the metabolomic information of the malignant and non-malignant tissues of gastric mucosae in 18 gastric cancer patients. Acquired metabolomic data was analyzed using the Wilcoxon rank sum test to find the tissue metabolic biomarkers for gastric cancer. A diagnostic model for gastric cancer was constructed using principal component analysis (PCA), and was assessed with receiver-operating characteristic (ROC) curves. Results showed that 18 metabolites were detected differently between the malignant tissues and the adjacent non-malignant tissues of gastric mucosa. Five metabolites were also detected differently between the non-invasive tumors and the invasive tumors. The diagnostic model could discriminate tumors from normal mucosae with an area under the curve (AUC) value of 0.9629, and another diagnostic model constructed for clinical staging was assessed with an AUC value of 0.969. We conclude that the metabolomic profile of malignant gastric tissue was different from normal, and that the selected tissue metabolites could probably be applied for clinical diagnosis or staging for gastric cancer.     

18F-FDG PET/CT显像联合肿瘤标志物检查对女性不明原因腹腔积液的诊断价值

本文探讨了18 F-脱氧葡萄糖(18 F-FDG)正电子发射计算机断层显像(PET/CT)联合肿瘤标志物检查对女性不明原因腹腔积液的鉴别诊断价值。选取95例不明原因腹腔积液女性患者的18 F-FDG PET/CT影像资料及肿瘤标志物资料进行回顾性分析,并与病理诊断结果进行对照。结果显示:95例患者中,经病理证实恶性腹腔积液74例,良性腹腔积液21例。PET/CT对良恶性腹腔积液的诊断灵敏度为98.65%,特异度为19.05%,准确度为81.05%。恶性腹腔积液的SUV max与良性腹腔积液比较,差异无统计学意义(P>0.05)。将患者按病因进一步细分后发现,卵巢癌组的SUV max明显高于其他良性原因组(P<0.05),但与结核组和其他恶性肿瘤组比较,差异无统计学意义(P>0.05)。肿瘤标志物中以CA125检测效率最高,其对卵巢癌及结核所致腹腔积液的诊断灵敏度为98.2%,特异度为7.7%,准确度为81.4%。PET/CT和CA125联合诊断对卵巢癌及结核所致腹腔积液的灵敏度、特异度及准确度与单一PET/CT或CA125诊断比较差异均无统计学意义(P>0.05)。本实验表明18 F-FDG PET/CT诊断女性不明原因腹腔积液的良恶性有较高的灵敏度和准确度,但特异性较差,联合肿瘤标志物检查并不能提高对卵巢癌及结核所致腹腔积液的诊断效能。