Membranous biomaterials play a vital role in regenerative medicine by maintaining barrier function and guiding tissue repair, but their clinical efficacy is often limited by premature degradation driven by host immune responses, particularly macrophage fusion. Addressing this challenge, we developed a delivery platform based on mesoporous silica nanoparticles loaded with a cell-permeable calcium chelator (BAPTA), which were uniformly spray-coated onto collagen membranes for precise control of coating thickness. In vitro, these coating ingredients were highly biocompatible, efficiently internalized by macrophages, and effectively attenuated IL4-associated cytosolic Ca2+ elevation and phosphatidylserine externalization, thereby blocking cell fusion initiation. Transcriptomic analysis showed upregulation of cytomembrane-stabilizing and cytoskeletal pathways (Rap1, PI3K-Akt, focal adhesion, ECM-receptor interaction), and downregulation of apoptosis and pro-fusion pathways. In a rat subcutaneous implantation model, BAPTA-coated membranes exhibited a thickness-dependent reduction in multinucleated giant cell formation and delayed membrane degradation, without systemic toxicity. Overall, this study demonstrates that targeted modulation of intracellular calcium signaling and multi-pathway immunoregulation can inhibit macrophage fusion, stabilize cytomembrane-cytoskeleton interactions, and prolong membrane function. This host-targeted approach offers a safe and effective strategy for enhancing the durability of regenerative biomaterials via a designed immunomodulatory interface, and may inform the rational design of next-generation membranes in tissue engineering.

Breast carcinoma with osteoclast-like giant cells (BC-OGC) is a rare morphological variant of invasive breast cancer characterized by scattered benign-appearing giant cells in the stroma. Although these cells are believed to be macrophage-derived, their biological role, polarization state, and clinical significance remain unclear. Recent data suggest that tumor-derived RANKL may drive giant cell formation and influence tumor aggressiveness. A total of 12 BC-OGC cases diagnosed between 2012 and 2024 were retrospectively analyzed, along with 36 matched breast cancer cases without osteoclast-like giant cells (non-BC-OGC). Clinicopathological, immunophenotypic, and stromal features were compared. Immunohistochemistry assessed the expression of macrophage markers (CD68, CD163, CD206) and RANKL. Associations with metastatic outcomes were evaluated. In vitro, the effects of RANKL overexpression and knockdown on breast cancer cell proliferation, migration, and invasion were evaluated using MCF7 cell lines.BC-OGC cases predominantly exhibited the Luminal A-like molecular subtype but showed distinctive loose, vascular-rich stroma compared to controls (p = 0.018). Osteoclast-like giant cells expressed macrophage markers, with widespread CD68 positivity and partial M2 polarization (CD163, CD206). RANKL immunostaining was significantly stronger and more diffuse in BC-OGC than in controls (p = 0.00386), with expression highest in metastatic cases. Metastatic events occurred more frequently in BC-OGC (33.3% vs. 2.8%, CMH p = 0.0126), and within the BC-OGC group, higher RANKL expression was significantly associated with metastasis (p = 0.0123). In vitro, RANKL overexpression promoted, and knockdown suppressed, MCF7 cell proliferation, migration, and invasion.BC-OGC represents a rare but biologically distinctive breast cancer variant with a macrophage-rich, angiogenic stroma and high RANKL expression. RANKL may contribute to both macrophage fusion into giant cells and increased tumor aggressiveness. These findings highlight the potential value of RANKL as a biomarker for metastatic risk and as a therapeutic target in high-risk BC-OGC, warranting further validation in larger cohorts and mechanistic studies.

Single-cell transcriptomic profiling combined with functional and clinical validation identifies fusion of tumor cells and lipid-associated macrophages mediated by the CBX3-SNX10-ANO6 axis as a potentially targetable mechanism driving cancer metastasis.

Precision-targeting and dual silencing osteoclastogenesis and inflammatory pathways for the treatment of radiation-induced bone deterioration.

Chronic hemophilic synovitis (CHS), driven by hemosiderin-laden macrophages from recurrent hemarthrosis, is a major cause of joint damage in hemophilia. Platelet-rich plasma (PRP) is a promising regenerative therapy for joint diseases. This study investigated PRP’s ability to modulate macrophage polarization from a pro-inflammatory (M1) to a pro-resolving, tissue-repairing (M2) phenotype in CHS. We analyzed synovial fluid (SF) from CHS patients (N = 22), both pre- and post-PRP treatment. Ex vivo analysis revealed a predominant M1 profile with an increased proportion of CD11+CD14+CD64hi compared with CD206+ or CD163+ M2 macrophages in CHS SF. In vitro experiments showed that CHS SF skewed monocyte-derived macrophages toward an M1 inflammatory program, evaluated by flow cytometry, qPCR, and ELISA. However, adding PRP significantly modulated the pro-inflammatory macrophage program, promoting an M2 tissue repair profile. Furthermore, a random forest machine learning algorithm, applied to public scRNAseq data, confirmed PRP’s macrophage reprogramming effect. Functional assays also showed increased TGF-β secretion and macrophage fusion when challenged with neutrophil extracellular traps (NETs). A small patient follow-up cohort treated with intra-articular PRP showed similar results, including normalization of cellular content and reduced CD64/CD206 expression. These findings indicate that PRP treatment effectively shifts SF-associated M1 macrophages to an M2-like phenotype, highlighting its potential as a therapeutic strategy for CHS.

The ovary is one of the first organs to exhibit signs of aging, characterized by reduced tissue function, chronic inflammation, and fibrosis. Multinucleated giant cells (MNGCs), formed by macrophage fusion, typically occur in chronic immune pathologies, including infectious and non-infectious granulomas and the foreign body response, but are also observed in the aging ovary. The function and consequence of ovarian MNGCs remain unknown as their biological activity is highly context-dependent, and their large size has limited their isolation and analysis through technologies such as single-cell RNA sequencing. In this study, we define ovarian MNGCs through a deep analysis of their presence across age and species using advanced imaging technologies as well as their unique transcriptome using laser capture microdissection. MNGCs form complex interconnected networks that increase with age in both mouse and nonhuman primate ovaries. MNGCs are characterized by high Gpnmb expression, a putative marker of ovarian and non-ovarian MNGCs. Pathway analysis highlighted functions in apoptotic cell clearance, lipid metabolism, proteolysis, immune processes, and increased oxidative phosphorylation and antioxidant activity. Thus, MNGCs have signatures related to degradative processes, immune function, and high metabolic activity. These processes were enriched in MNGCs compared to primary ovarian macrophages, suggesting discrete functionality. MNGCs express CD4 and colocalize with T-cells, which were enriched in regions of MNGCs, indicative of a close interaction between these immune cell types. These findings implicate MNGCs in modulation of the ovarian immune landscape during aging given their high penetrance and unique molecular signature that supports degradative and immune functions.

3058 Background: Tumor associated macrophages are known to fuse with cancer cells in the blood through a dysfunctional CD47 phagocytic immune pathway resulting in the formation of tumor- macrophage fusion cells (TMFCs) which are observed as heterokaryon (incomplete fusion), synkaryon (full fusion) or hetero-to-synkaryon transition (partial fusion). Previous studies in lung and breast cancer demonstrated that subtypes of TMFCs in blood may correlate with highly aggressive disease unlikely to respond to certain systemic therapies (i.e. chemotherapy). We initiated a prospective study to evaluate the blood of n = 100 metastatic pan-cancer patients (pts) receiving systemic therapy for the presence of full, partial, and incomplete TMFCs to compare their progression-free survival (PFS) and overall survival (OS). Methods: We conducted a prospective pilot study of n = 100 pathologically confirmed metastatic cancer pts with breast (n = 23), prostate (n = 21), pancreas (n = 17), colon (n = 19), or lung (20) with active progressive disease, prior to the induction of new systemic therapies, i.e. chemotherapy (n = 39), PD-L1 immunotherapy (n = 27), hormone therapy (n = 20), or targeted therapy (n = 23). TMFCs were isolated from 7.5ml peripheral blood using CellSieve microfiltration and identified by their enlarged multinucleated structure (> 30 µm), which was categorized into 3 distinct subtypes: full fusion marked by a single multinucleated nuclei, partial fusion marked by 2 contacting nuclei, incomplete fusion marked by 2 distinct non-contacting nuclei. TMFC subtypes were compared to pts’ PFS and OS by cox proportional univariate and multivariate analysis over 24 months. Results: We identified TMFCs in 78% of all pts (n = 78/100), averaging 10 per pt. 37% of pts were found to have more than one TMFC subtype in their sample, with 70 pts having full fusion, 23 partial fusion, and 28 incomplete fusion. At 24 months, pts with incomplete fusion TMFCs had significantly worse PFS (HR, 2.9; 95% CI, 1.5 to 5.7; P = 0.0023) and OS (HR, 2.5; 95% CI, 1.2 to 5.3; P = 0.0202). Interestingly, pts with incomplete fusion and treated with systemic targeted therapy (n = 7) were found to have significantly improved PFS (HR, 4.8; 95% CI, 1.8 to 13.0; p = 0.0052), but not OS (HR, 3.0; 95% CI, 1.0-9.2; p = 0.0999) versus other therapy types. There was no significant PFS differences in pts without incomplete fusion TMFCs being treated with targeted therapies. Conclusions: In a pan metastatic cancer setting, we found that incomplete fusion in circulating TMFCs associates with poorer outcomes at 24 months. Further, it appears that pts with incomplete TMFCs may have had better outcomes when treated with targeted therapies compared to other therapy types. These preliminary findings suggest the need for larger scale prospective studies to further evaluate relationships between TMFCs and therapeutic responses in specific disease populations.

Polygalacturonic acid (PGA), derived from the natural plant polysaccharide, pectin, has been suggested as a biomaterial for implantable medical devices and tissue engineering; particularly in the field of bone implant materials. As a negatively charged polysaccharide, PGA can be considered similar to hyaluronic acid, a component of the extracellular matrix (ECM). PGA-based biomaterials may therefore exhibit favorable biocompatibility with surface chemistry mimicking the natural ECM. In this study, we synthesized semi-interpenetrating polymer networks (SIPNs) incorporating PGA, and conducted physical characterization and in vitro biocompatibility studies. Biocompatibility testing revealed the SIPNs to be cytocompatible, with the PGA component conferring some resistance to the adherence of the macrophage cell line RAW264.7. In addition, SIPNs did not support the fusion of primary murine macrophages into foreign body giant cells (FBGCs). Macrophage adherence and FBGC formation on implanted biomaterial surfaces are important events in the progression of a foreign body response. Our in vitro studies suggest that PGA-based materials may offer desirable biocompatibility profiles, holding promise for future clinical applications.

Abstract Tumor associated macrophages are known to fuse with cancer cells resulting in the formation of a cellular phenomenon called tumor-macrophage fusion cells (TMFCs). TMFCs are a highly aggressive cell subtype that may move into circulation, evade immune surveillance, and possibly aid in seeding of metastatic sites. The formation of TMFCs involves CD47, a ubiquitous surface receptor that acts as a “don’t eat me” signal, interacting with the macrophage protein SIRPa, a down regulator of phagocytosis, and syncytin-1, a mediator of hyperploidy cancer fusion. In this analysis, we isolated circulating TMFCs from the blood of n=39 metastatic cancer patients (pts) to evaluate the expressions of CD47, SIRPa and syncytin-1 as it relates to stable or progressive disease via 30 day imaging by PET scan. We conducted a prospective pilot study of n=39 pts with pathologically confirmed metastatic breast (n=17), prostate (n=7), pancreas (n=7), or colon (n=8) cancer receiving standard of care systemic therapy. We isolated TMFCs from 7.5ml peripheral blood using a CellSieve microfilter, and stained with CD47, SIRPa, & syncytin-1. TMFCs were identified based on large cell size (>30 µm) & multinucleated structure, and were categorized into 4 distinct subtypes: A) >2 distinct non-contacting nuclei within a TMFC, B) 2 distinct non-contacting nuclei, C) 2 contacting nuclei, D) a single multinucleated nuclei. CD47, SIRPa, and syncytin-1 expressions were categorized as negative, low, or high. TMFCs subtypes and marker expressions were compared to pts’ clinical status (i.e. progressive, stable or responding) at first available PET/CT scans after blood draw (∼45 days). TMFCs were identified in 72% of pts (n=28/39), average 8 cells/sample, and ranged 1-77 cells. Of the TMFCs, 12% were subtype A, 11% as B, 11% as C, and 66% as D. CD47 expression was seen in 100% of TMFs, with low expression in 39% of subtype A cells, 58% subtype B, 39% subtype C, & 62% subtype D. Notably, CD47 was significantly lower (p=0.010) in subtype B than other groups. Further, the expression of syncytin-1 in subtype B cells was significantly lower (p=0.02) than in other subtypes, and subtype D cells had 75% higher syncytin-1 localized to the nuclear regions. No correlations were found in the SIRPa expression and subtypes. Interestingly, progressing pts had 200% more TMFCs, more subtype B (83%), and significantly lower (p<0.001) CD47 expressing TMFCs than non-progressing pts. In this pilot study on the expression profiles of circulating TMFCs, it appears that specific subtypes of TMFC with low CD47 expression may be associated with progressive disease. Interestingly, this contrasts existing studies on primary tumors which often correlate higher CD47 with more aggressive cancers. This finding suggests that low CD47 cancer cells circulate blood in some pts with progressing disease and may be pertinent to efforts targeting CD47 in cancer. Citation Format: Sonia Muthuraj, Massimo Cristofanilli, Saranya Chumsri, Susan Tsai, Raymond C. Bergan, Cha-Mei Tang, Daniel L. Adams. Low CD47 expression in specific populations of tumor macrophage fusion cells in blood correlates with progressive disease in metastatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1979.

Langhans giant cells regulate cutaneous immune responses during mycobacterial infection through CXCL1/CXCL2 secretion.

Sarcoidosis, a multisystemic granulomatous disease with unknown etiology, is characterized by formation of noncaseating granulomas, which can affect all organs. Recent studies have made outstanding achievement in understanding the pathology, etiology, genetics, and immune dysregulation involved in granuloma formation of sarcoidosis. Antigen stimulation in genetically predisposed individuals enhances the phagocytic activity of antigen-presenting cells, including macrophages and dendritic cells. CD4 + T cells initiate dysregulated immune responses and secrete significant quantities of inflammatory cytokines, including interleukin (IL)-2 and interferon-gamma (IFN-γ), which play a crucial role in modulating the aggregation and fusion of macrophages to form granulomas. The current therapeutic strategies focus on blocking the formation and spread of granulomas to protect organ function and alleviate symptoms. The efficacy of traditional treatments, such as glucocorticoids and immunosuppressants, has been confirmed in the management of sarcoidosis. Promising therapeutic agents encompass inhibitors of cytokines, like those targeting tumor necrosis factor (TNF)-α, as well as inhibitors of signaling pathways, such as Janus kinase (JAK) inhibitors, which exhibit favorable prospects for application. Although there has been progress in the identification of biomarkers for the diagnosis, prognosis, activity and severity of sarcoidosis, specific and sensitive biomarkers have yet to be identified. This review outlines recent advancements in the molecular mechanisms and therapeutic strategies for the sarcoidosis.

The patients with Arthrogryposis-Renal dysfunction-Cholestasis (ARC) syndrome have genetic susceptibility to the opportunistic infections due to the involvement of VPS33B (vacuolar protein sorting 33 homolog B) in phagolysosome fusion in macrophages. Detailed pathologic studies in ARC patients are missing in literature due to the lack of autopsy. We described the first autopsy case of ARC syndrome in a 2-month-old male infant. His death was due to recurrent sepsis and multiorgan failure despite the appropriate poly-antibiotic therapy and supportive care. The autopsy showed invasive renal candidiasis including bilateral destructive pyelonephritis, pelvic obstructive fungal bezoars, and right large perinephric abscess. The main other findings included severe chronic liver changes and pneumonia. Liver exhibited intrahepatocyte cholestasis, large multinucleated hepatocytes, diffuse portal, bridging and perivenular fibrosis, and interlobular bile duct proliferation. The neuropathologic examination was unremarkable. This case report highlights 3 novel findings. The ARC syndrome-related immunodeficiency may predispose to renal fungal bezoars and perinephric abscess. Cholestatic stress may result in the proliferation of interlobular ducts as an adaptive response. Absence of spinal motor neuron degeneration suggests that the neurogenic amyotrophy is due to the lack of synaptic vesicle trafficking and membrane fusion rather than the defect in cell survival-related autophagosome-lysosome fusion.

Abstract Engineered immune cell therapy has proven to be a transformative cancer treatment despite the challenges of its prohibitive costs and manufacturing complexity. In this study, we propose a concise “lipid droplet fusion” strategy for engineering macrophages. Because of the integration of hydrophobic alkyl chains and π‐conjugated structures, the mildly synthesized sp2C‐conjugated covalent organic framework (COF) UM‐101 induced lipid droplet fusion and metabolic reprogramming of macrophages, thus promoting their antitumor classical activation. Intravenous injection of UM‐101–engineered macrophages effectively inhibited tumor progression. These results represent the first report of room‐temperature synthesis of sp2C‐conjugated COFs for engineered immune cell therapy, providing a new perspective for the development of therapeutic immune cells via organelle manipulation.

Engineered immune cell therapy has proven to be a transformative cancer treatment despite the challenges of its prohibitive costs and manufacturing complexity. In this study, we propose a concise "lipid droplet fusion" strategy for engineering macrophages. Because of the integration of hydrophobic alkyl chains and π-conjugated structures, the mildly synthesized sp2C-conjugated covalent organic framework (COF) UM-101 induced lipid droplet fusion and metabolic reprogramming of macrophages, thus promoting their antitumor classical activation. Intravenous injection of UM-101-engineered macrophages effectively inhibited tumor progression. These results represent the first report of room-temperature synthesis of sp2C-conjugated COFs for engineered immune cell therapy, providing a new perspective for the development of therapeutic immune cells via organelle manipulation.

Even today, the reduction of complications following breast implant surgery together with the enhancement of implant integration and performance through the modulation of the foreign body response (FBR), remains a fundamental challenge in the field of plastic surgery. Therefore, tailoring the material's physical characteristics to modulate FBR can represent an effective approach in implantology. While polydimethylsiloxane (PDMS) patterning on 2D substrates is a relatively established and available procedure, micropatterning multiscaled biointerfaces on a controlled large area has been more challenging. Therefore, in the present work, a specific designed honeycomb-like well biointerface was designed and obtained by replication in PDMS at large scale and its effectiveness towards creating a pro-healing environment was investigated. The grayscale masks assisted laser-based 3D texturing method was used for creating the required moulds in Polycarbonate for large area replication. By comparison to the smooth substrate, the honeycomb topography altered the fibroblasts' behaviour in terms of adhesion and morphology and reduced the macrophages' inflammatory response. Additionally, the microstructured surface effectively inhibited macrophage fusion, significantly limiting the colonization of both Gram-positive and Gram-negative microbial strains on the tested surfaces. Overall, this study introduces an innovative approach to mitigate the in vitro FBR to silicone, achieved through the creation of a honeycomb-inspired topography for prosthetic interfaces.

The ovary is one of the first organs to exhibit signs of aging, characterized by reduced tissue function, chronic inflammation, and fibrosis. Multinucleated giant cells (MNGCs), formed by macrophage fusion, typically occur in chronic immune pathologies, including infectious and non-infectious granulomas and the foreign body response 1 , but are also observed in the aging ovary 2-4 . The function and consequence of ovarian MNGCs remain unknown as their biological activity is highly context-dependent, and their large size has limited their isolation and analysis through technologies such as single-cell RNA sequencing. In this study, we define ovarian MNGCs through a deep analysis of their presence across age and species using advanced imaging technologies as well as their unique transcriptome using laser capture microdissection. MNGCs form complex interconnected networks that increase with age in both mouse and nonhuman primate ovaries. MNGCs are characterized by high Gpnmb expression, a putative marker of ovarian and non-ovarian MNGCs 5,6 . Pathway analysis highlighted functions in apoptotic cell clearance, lipid metabolism, proteolysis, immune processes, and increased oxidative phosphorylation and antioxidant activity. Thus, MNGCs have signatures related to degradative processes, immune function, and high metabolic activity. These processes were enriched in MNGCs compared to primary ovarian macrophages, suggesting discrete functionality. MNGCs express CD4 and colocalize with T-cells, which were enriched in regions of MNGCs, indicative of a close interaction between these immune cell types. These findings implicate MNGCs in modulation of the ovarian immune landscape during aging given their high penetrance and unique molecular signature that supports degradative and immune functions.

Control of Macrophage Fusion By Sensing the Physical Properties of Fibrin(ogen) Matrices

Expression of CD68 and CD163 in malignant epithelial cells of oral squamous cell carcinoma: Phenotypic shift or mere cell fusion

Biomaterial wound dressings, such as hydrogels, interact with host cells to regulate tissue repair. This study investigates how crosslinking of gelatin-based hydrogels influences immune and stromal cell behavior and wound healing in female mice. We observe that softer, lightly crosslinked hydrogels promote greater cellular infiltration and result in smaller scars compared to stiffer, heavily crosslinked hydrogels. Using single-cell RNA sequencing, we further show that heavily crosslinked hydrogels increase inflammation and lead to the formation of a distinct macrophage subpopulation exhibiting signs of oxidative activity and cell fusion. Conversely, lightly crosslinked hydrogels are more readily taken up by macrophages and integrated within the tissue. The physical properties differentially affect macrophage and fibroblast interactions, with heavily crosslinked hydrogels promoting pro-fibrotic fibroblast activity that drives macrophage fusion through RANKL signaling. These findings suggest that tuning the physical properties of hydrogels can guide cellular responses and improve healing, offering insights for designing better biomaterials for wound treatment.

Osteoclasts, the bone resorbing cells of hematopoietic origin formed by macrophage fusion, are essential in bone health and disease. However, in vitro research on osteoclasts remains challenging due to heterogeneous cultures that only contain a few multinucleated osteoclasts. Indeed, a strategy to generate homogeneous populations of multinucleated osteoclasts in a scalable manner has remained elusive. Here, the investigation focuses on whether microencapsulation of human macrophages in microfluidically generated hollow, sacrificial tyramine-conjugated dextran (Dex-TA) microgels could facilitate macrophage precursor aggregation and formation of multinucleated osteoclasts. Therefore, human mononuclear cells are isolated from buffy coats and differentiated toward macrophages. Macrophages are encapsulated in microgels using flow focus microfluidics and outside-in enzymatic oxidative phenolic crosslinking, and differentiated toward osteoclasts. Morphology, viability, and osteoclast fusion of microencapsulated cells are assessed. Furthermore, microgels are degraded to allow cell sorting of released cells based on osteoclastic marker expression. The successful encapsulation and osteoclast formation of human macrophages in Dex-TA microgels are reported for the first time using high-throughput droplet microfluidics. Intriguingly, osteoclast formation within these 3D microenvironments occurs at a significantly higher level compared to the conventional 2D culture system. Furthermore, the feasibility of establishing a pure osteoclast culture from cell transfer and release from degradable microgels is demonstrated.