Stromal Macrophages Research Articles

Rare case of microglandular-like adenocarcinoma of endometrium

This paper focuses on the microglandular-like pattern, a rare pattern of endometrial carcinoma whose appearance resembles microglandular hyperplasia of the endocervix. Its similarity with benign lesions makes diagnosis of this pattern of carcinoma difficult, particularly in minute specimens. Moreover, it raises a question of whether the tumor originates from the uterine corpus or the cervix. Microglandular-like adenocarcinoma of the endometrium may be an individual type of carcinoma or combined with endometrioid or mucinous carcinoma. The immunohistochemical evaluation is recommended for precise diagnosis: microglandular-like adenocarcinoma expresses markers typical for cervical carcinomas (p16, СК17, and carcinoembryonic antigen) and endometrial carcinomas (estrogen, vimentin, and CD10). The presence of foamy macrophages in the stroma of the tumor and intraglandular foci of squamous differentiation help in the differential diagnosis of microglandular hyperplasia of the endocervix. Immunohistochemical evaluation also facilitates differential diagnosis: a negative PAX2 expression and a high mitotic index (Ki-67 10%) indicate a microglandular-like adenocarcinoma.

Deciphering a critical role of uterine epithelial SHP2 in parturition initiation at single cell resolution

The timely onset of female parturition is a critical determinant for pregnancy success. The highly heterogenous maternal decidua has been increasingly recognized as a vital factor in setting the timing of labor. Despite the cell type specific roles in parturition, the role of the uterine epithelium in the decidua remains poorly understood. This study uncovers the critical role of epithelial SHP2 in parturition initiation via COX1 and COX2 derived PGF2α leveraging epithelial specific Shp2 knockout mice, whose disruption contributes to delayed parturition initiation, dystocia and fetal deaths. Additionally, we also show that there are distinct types of epithelium in the decidua approaching parturition at single cell resolution accompanied with profound epithelium reformation via proliferation. Meanwhile, the epithelium maintains the microenvironment by communicating with stromal cells and macrophages. The epithelial microenvironment is maintained by a close interaction among epithelial, stromal and macrophage cells of uterine stromal cells. In brief, this study provides a previously unappreciated role of the epithelium in parturition preparation and sheds lights on the prevention of preterm birth.

The possible transport and exclusion mode of lipofuscin in rat myocardium under electron microscopy

Lipofuscin accumulation has been observed in human coronary arteries but whether or not myocardial tissue can release lipofuscin generated within cardiomyocytes must be clarified, as this may provide indicators for future anti-ageing research. The hearts of Sprague Dawley rats, aged 6–24 months, were embedded in resin and ultrathin sections cut for electron microscopy. Lipofuscin granules were abundant in cardiomyocytes. Cardiomyocytes were seen to release lipofuscin granules into the myocardial interstitium as cytoplasmic fragments with irregular protrusions on the sarcolemma surface. The cytoplasmic fragments entering the stroma fused directly with the endothelial cells of adjacent capillaries, delivering lipofuscin to the vessel wall. These fragments were also seen to be engulfed by stromal macrophages or fused with fibroblasts, which then combined with capillary endothelial cells to deliver lipofuscin to the vessel wall. Some cytoplasmic fragments disaggregated and formed membrane-like waste, which travelled to the vessel wall from the myocardial stroma as soluble fine particles via diffusion or pinocytosis of capillary endothelial cells. Lipofuscin entered the vascular wall and endothelial cells, forming large and small protrusions or folds that transported the lipofuscin to the vascular lumen and bloodstream.

Interaction between mesenchymal stromal cells and tuberculous mycobacteria in vitro

The objective: in an in vitro experiment, we compared phagocytic parameters of mesenchymal stromal cells (MSCs) and macrophages to tuberculous mycobacteria, assessed the ability of MSCs and macrophages to lyse mycobacteria or maintain their intracellular growth, their effect on formation of phenotypic drug resistance of mycobacteria, as well as the effect of tuberculous mycobacteria on the type of MSCs cell death.Subjects and Methods. Balb/c male mice, aged 6 to 8 weeks, were used in the experiment. Bone marrow MSCs were obtained from femurs and tibias by further cultivation, peritoneal macrophages were elicited with 4% alpha-glucan. The intracellular content of mycobacteria was counted using a confocal microscope with x 400 magnification. Susceptibility of mycobacteria to isoniazid and development of phenotypic drug resistance after culturing MSCs and macrophages with MTB on Lowenstein–Jensen medium was assessed by counting CFU. In 5 days after the infection, the number of apoptotic and necrotic MSCs and macrophages was determined by a flow cytometer.Results. On Day 1, the total number of phagocytosed MTB, as well as the number of phagocytic-active macrophages, exceeds the corresponding figures for MSCs more than twice. MSCs phagocytize tuberculous mycobacteria in a smaller amount, but MTB reproduces in them more actively: the number of CFU after 7 days of cell cultivation with MTB exceeded the corresponding parameter by almost 50 times after 24 hours of cultivation. In cultures of infected MSCs cultivated for 7 days, regardless of the presence of isoniazid, there was a rapid growth of tuberculous mycobacteria. On Day 5 after infection of macrophage culture with tuberculous mycobacteria, the number of necrotic cells was 2.7 times greater than that of uninfected necrotic macrophages, but the number of apototic cells in these groups differed slightly. In the culture of MSCs, there were 8.5 times more infected nectrotic cells versus uninfected necrotic MSCs, and the number of necrotic MSCs was 4.5 times higher than the number of MSCs with apoptosis, while in the culture of infected macrophages, the number of necrotic cells was the same as number of apoptotic cells. Unlike macrophages, treatment of MSCs with isoniazid did not inhibit the intracellular proliferation of MTB.Conclusion. MSCs have the ability to phagocytose mycobacteria, but they do it less actively than macrophages and, unlike macrophages, they are not able to restrain the reproduction of tuberculous mycobacteria. Mycobacteria have phenotypic drug resistance in MSCs. In MSCs, when infected with tuberculous mycobacteria, there is a pronounced shift towards necrosis in the type of cell death, which can lead to dissemination of MTB and development of local destructive changes.

TIM-3 Expression and M2 Polarization of Macrophages in the TGFβ-Activated Tumor Microenvironment in Colorectal Cancer.

TGFβ signaling in the tumor microenvironment (TME) drives immune evasion and is a negative predictor of immune checkpoint inhibitor (ICI) efficacy in colorectal cancer (CRC). TIM-3, an inhibitory receptor implicated in anti-tumor immune responses and ICI resistance, has emerged as an immunotherapeutic target. This study investigated TIM-3, M2 macrophages and the TGFβ-activated TME, in association with microsatellite instability (MSI) status and consensus molecular subtypes (CMSs). Transcriptomic cohorts of CRC tissues, organoids and xenografts were examined (n = 2240). TIM-3 and a TGFβ-inducible stromal protein, VCAN, were evaluated in CRC specimens using immunohistochemistry (n = 45). TIM-3 expression on monocytes and generated M2 macrophages was examined by flow cytometry. We found that the expression of HAVCR2 (TIM-3) significantly correlated with the transcriptional signatures of TGFβ, TGFβ-dependent stromal activation and M2 macrophage, each of which were co-upregulated in CMS4, CMS1 and MSI CRCs across all datasets. Tumor-infiltrating TIM-3+ immune cells accumulated in TGFβ-responsive cancer stroma. TIM-3 was increased on M2-polarized macrophages, and on monocytes in response to TGFβ treatment. In conclusion, we identified a close association between TIM-3 and M2-like polarization of macrophages in the TGFβ-rich TME. Our findings provide new insights into personalized immunotherapeutic strategies based on the TME for CRCs.

Pubertal exposure to dietary advanced glycation end products disrupts ductal morphogenesis and induces atypical hyperplasia in the mammary gland

BackgroundAdvanced glycation end products (AGEs) are reactive metabolites intrinsically linked with modern dietary patterns. Processed foods, and those high in sugar, protein and fat, often contain high levels of AGEs. Increased AGE levels are associated with increased breast cancer risk, however their significance has been largely overlooked due to a lack of direct cause-and-effect relationship.MethodsTo address this knowledge gap, FVB/n mice were fed regular, low AGE, and high AGE diets from 3 weeks of age and mammary glands harvested during puberty (7 weeks) or adulthood (12 weeks and 7 months) to determine the effects upon mammary gland development. At endpoint mammary glands were harvested and assessed histologically (n ≥ 4). Immunohistochemistry and immunofluorescence were used to assess cellular proliferation and stromal fibroblast and macrophage recruitment. The Kruskal–Wallis test were used to compare continuous outcomes among groups. Mammary epithelial cell migration and invasion in response to AGE-mediated fibroblast activation was determined in two-compartment co-culture models. In vitro experiments were performed in triplicate. The nonparametric Wilcoxon rank sum test was used to compare differences between groups.ResultsHistological analysis revealed the high AGE diet delayed ductal elongation, increased primary branching, as well as increased terminal end bud number and size. The high AGE diet also led to increased recruitment and proliferation of stromal cells to abnormal structures that persisted into adulthood. Atypical hyperplasia was observed in the high AGE fed mice. Ex vivo fibroblasts from mice fed dietary-AGEs retain an activated phenotype and promoted epithelial migration and invasion of non-transformed immortalized and tumor-derived mammary epithelial cells. Mechanistically, we found that the receptor for AGE (RAGE) is required for AGE-mediated increases in epithelial cell migration and invasion.ConclusionsWe observed a disruption in mammary gland development when mice were fed a diet high in AGEs. Further, both epithelial and stromal cell populations were impacted by the high AGE diet in the mammary gland. Educational, interventional, and pharmacological strategies to reduce AGEs associated with diet may be viewed as novel disease preventive and/or therapeutic initiatives during puberty.

Cytomorphologic panorama of giant cell tumour of tendon sheath.

Giant Cell Tumour of Tendon Sheath (GCTTS) is a slow growing benign soft tissue tumour arising from synovium of tendon sheath or joint. These tumours occur more frequently in upper limbs, especially hands. In the present study, we aimed to evaluate the cytomorphological spectrum of GCTTS. This retrospective study includes a total of 56 cases of GCTTS diagnosed over a period of 8 years. The clinical and radiological details of these cases were retrieved from the cytopathology records and detailed cytomorphological features were studied and analysed. Histopathological correlation was done in 16/56 cases, where follow-up was available. The mean age of patients at the time of presentation was 32 years and were predominantly females (68%). The most common site of GCTTS was fingers (76%), followed by foot, wrist and toes. The most consistent finding on cytology was stromal cells (100%) of polygonal, spindle and plasmacytoid morphology with interspersed multinucleated osteoclastic giant cells (100%), followed by binucleated stromal cells (75%), xanthoma cells (61%) and hemosiderin laden macrophages (52%). Presence of proteinaceous fluid background was also observed in 50% of the cases. GCTTS can be diagnosed with certainty on FNAC based on characteristic cytomorphological features in an appropriate clinical and radiological setting. FNAC plays a pivotal role in diagnosing GCTTS and differentiating it from other giant cell rich lesions, thus obviating the need of tissue biopsy for diagnosis, which in turn helps the clinician in timely and adequate management of the patient.

Assessment of tumor microenvironment in gastric adenocarcinoma.

Gastric cancer (GC), despite the current possibilities of early diagnosis and curative treatment, remains a major public health problem, being one of the main causes of cancer, due to its detection in advanced stages. Screening programs applied in Western countries led to low incidence rates in these countries. Helicobacter pylori bacterial infection is considered to be the highest risk factor for the onset of GC because it causes chronic inflammation of the gastric mucosa and damages hydrochloric acid secretory glands, eventually leading to atrophic gastritis, which has a potential to progress to GC. In our study, we aimed at assessing the tumor microenvironment in gastric adenocarcinomas as approximately 90% of GCs are adenocarcinomas. Our study showed that the tumor microenvironment has an extremely complex morphological structure, totally different from the microscopic structure of the gastric wall, consisting of stromal cells, lymphocytes, plasma cells, macrophages, blood vessels, collagen fibers, extracellular connective matrix, other cells. The tumor microenvironment presents phenotypic, cellular and molecular heterogeneity; therefore, the microscopic aspect differs from one tumor to another and even from one region to another in the same tumor. Poorly or moderately differentiated adenocarcinomas show a more intense desmoplastic reaction than well-differentiated ones. Alpha-smooth muscle actin (α-SMA)-positive stromal cells (tumor-associated fibroblasts) and tumor macrophages were the most numerous cells of the tumor microenvironment. The tumor microenvironment is the result of cooperation between tumor cells, cancer-associated fibroblasts, immune system cells and blood vessels. It allows tumor cells to multiply, grow and metastasize.

Multifunctional hydrogel enhances bone regeneration through sustained release of Stromal Cell-Derived Factor-1α and exosomes.

Fracture nonunion remains a great challenge for orthopedic surgeons. Fracture repair comprises of three phases, the inflammatory, repair and remodeling stage. Extensive advancements have been made in the field of bone repair, including development of strategies to balance the M1/M2 macrophage populations, and to improve osteogenesis and angiogenesis. However, such developments focused on only one or the latter two phases, while ignoring the inflammatory phase during which cell recruitment occurs. In this study, we combined Stromal Cell-Derived Factor-1α (SDF-1α) and M2 macrophage derived exosomes (M2D-Exos) with a hyaluronic acid (HA)-based hydrogel precursor solution to synthesize an injectable, self-healing, adhesive HA@SDF-1α/M2D-Exos hydrogel. The HA hydrogel demonstrated good biocompatibility and hemostatic ability, with the 4% HA hydrogels displaying great antibacterial activity against gram-negative E. coli and gram-positive S. aureus and Methicillin-resistant Staphylococcus aureus (MRSA). Synchronously and sustainably released SDF-1α and M2D-Exos from the HA@SDF-1α/M2D-Exos hydrogel enhanced proliferation and migration of human bone marrow mesenchymal stem cell (HMSCs) and Human Umbilical Vein Endothelial Cells (HUVECs), promoting osteogenesis and angiogenesis both in vivo and in vitro. Overall, the developed HA@ SDF-1α/M2D-Exos hydrogel was compatible with the natural healing process of fractures and provides a new modality for accelerating bone repair by coupling osteogenesis, angiogenesis, and resisting infection at all stages.

MiRNA 24-3p-rich exosomes functionalized DEGMA-modified hyaluronic acid hydrogels for corneal epithelial healing.

The damage of corneal epithelium may lead to the formation of irreversible corneal opacities and even blindness. The migration rate of corneal epithelial cells directly affects corneal repair. Here, we explored ocu-microRNA 24-3p (miRNA 24-3p) that can promote rabbit corneal epithelial cells migration and cornea repair. Exosomes, an excellent transport carrier, were exacted from adipose derived mesenchymal stem cells for loading with miRNA 24-3p to prepare miRNA 24-3p-rich exosomes (Exos-miRNA 24-3p). It can accelerate corneal epithelial migration in vitro and in vivo. For application in cornea alkali burns, we further modified hyaluronic acid with di(ethylene glycol) monomethyl ether methacrylate (DEGMA) to obtain a thermosensitive hydrogel, also reported a thermosensitive DEGMA-modified hyaluronic acid hydrogel (THH) for the controlled release of Exos-miRNA 24-3p. It formed a highly uniform and clear thin layer on the ocular surface to resist clearance from blinking and extended the drug-ocular-epithelium contact time. The use of THH-3/Exos-miRNA 24-3p for 28 days after alkali burn injury accelerated corneal epithelial defect healing and epithelial maturation. It also reduced corneal stromal fibrosis and macrophage activation. MiRNA 24-3p-rich exosomes functionalized DEGMA-modified hyaluronic acid hydrogel as a multilevel delivery strategy has a potential use for cell-free therapy of corneal epithelial regeneration.

Hyperglycemia exerts disruptive effects on the secretion of TGF-β1 and its matrix ligands, decorin and biglycan, by mesenchymal sub-populations and macrophages during bone repair

IntroductionBone has a high capacity for repair, but for patients with uncontrolled type 2 diabetes mellitus (T2DM), the associated hyperglycemia can significantly delay osteogenic processes. These patients respond poorly to fracture repair and bone grafts, leading to lengthy care plans due to arising complications. Mesenchymal stromal cells (MSCs) and M2 macrophages are both major sources of transforming growth factor-β1 (TGF-β1), a recognized mediator for osteogenesis and whose bioavailability and activities are further regulated by matrix small leucine-rich proteoglycans (SLRPs), decorin and biglycan. The aim of this study was to investigate how in vivo and in vitro hyperglycemic (HGly) environments can influence levels of TGF-β1, decorin, and biglycan during bone repair, with additional consideration for how long-term glucose exposure and cell aging can also influence this process.ResultsFollowing bone healing within a T2DM in vivo model, histological and immuno-labeling analyses of bone tissue sections confirmed delayed healing, which was associated with significantly elevated TGF-β1 levels within the bone matrices of young diabetic rats, compared with normoglycemic (Norm) and aged counterparts. Studies continued to assess in vitro effects of normal (5.5 mM) and high (25 mM) glucose exposure on the osteogenic differentiation of compact bone derived mesenchymal stromal cells (CB-MSCs) at population doubling (PD)15, characterized to contain populations of lineage committed osteoblasts, and at PD150, where transit-amplifying cells predominate. Short-term glucose exposure increased TGF-β1 and decorin secretion by committed osteoblasts but had a lesser effect on transit-amplifying cells. In contrast, the long-term exposure of CB-MSCs to high glucose was associated with decreased TGF-β1 and increased decorin secretion. Similar assessments on macrophage populations indicated high glucose inhibited TGF-β1 secretion, preventing M2 formation.DiscussionCollectively, these findings highlight how hyperglycemia associated with T2DM can perturb TGF-β1 and decorin secretion by MSCs and macrophages, thereby potentially influencing TGF-β1 bioavailability and signaling during bone repair.

Extracellular vesicles budding from stromal macrophages from the blood circulating in patients with metastatic non-small cell lung cancer to predict clinical outcomes.

e14547 Background: Extracellular vesicles (EVs), which include exosomes, microvesicles, and apoptotic bodies, are involved in cellular communication, aiding tumor growth, and in metastasis of cancer. Recently, extracellular budding structures on Cancer Associated Macrophage-Like Cells (CAMLs), a subtype of phagocytic circulating stromal cells found in circulation, was observed in patients with metastatic non-small cell lung carcinoma (mNSCLC). In this prospective analysis of n=104 metastatic NSCLC (mNSCLC) patients, we enumerated EV budding on CAMLs to determine their clinical significance on Progression Free Survival (PFS) & Overall Survival (OS), further subtyping based on treatment with or without PD-L1 Immunotherapy (IMT) based on standard of care treatment. These preliminary data suggests that EV positive (EV+) CAMLs prognosticates for worse PFS & OS in mNSCLC. Methods: We initiated a single blind multi-year prospective study to investigate the relationship between EV budding in CAMLs to PFS & OS prior to start of new treatment lines for mNSCLC. Anonymized blood was procured from n=104 pathologically confirmed mNSCLC patients and filtered to isolate CAMLs to measure EV budding using tumor/EV markers (i.e. cytokeratin, CD63, or CD81) and an immune specific marker (PD-L1). Blood was filtered by CellSieve microfiltration and EV budding was characterized as small (≤1 µm) bulbous protrusions from the cell cytoplasm. EVs were quantified by presence (EV+) or absence (EV-), as well asPD-L1 positivity or negativity in EVs, to compare PFS & OS with hazard ratios (HRs) at 60 months by censored univariate and multivariate analyses. Results: CAMLs were identified in 93% (n=97/104) of all samples, with EV budding (EV+) identified in 62% (n=60/97) of CAMLs. At 60 month follow-up, it was determined that EV+ CAMLs was associated with significantly worse PFS (HR=1.67 95%CI=0.4-1.0, p=0.0410) and OS (HR=1.88, 95%CI=0.3-0.9, p=0.0108). Further, in patients not treated with IMT, EV+ CAMLs was associated with significantly worse PFS (HR=2.51, 95%CI=0.2-0.8, p=0.0251) and OS (HR=2.32, 95%CI=0.2-0.9, 0.0407). In contrast, in patients treated with IMT, EV+ CAMLs had no correlation to PFS (1.18, 95%CI=0.5-1.6, p=0.7050) or OS (HR=1.53, 95%CI=0.3-1.3, p=0.2858), suggesting a clinical benefit in treating EV+ patients with PD-L1 IMT. Conclusions: EV budding on phagocytic stromal cells found in the blood of mNSCLC patients appears to predict for poorer PFS and OS, which is reduced with additional of PD-L1 immunotherapies. Larger validation studies are ongoing in both mNSCLC and stage III NSCLC patients, along with analysis of PD-L1 EV staining.

Effects of concomitant chronic obstructive pulmonary disease on immune microenvironment in non-small cell lung cancer patients.

e20522 Background: Chronic obstructive pulmonary disease (COPD) as a progressive deterioration of lung function, has caused significant morbidity and mortality, especially in developing countries. Persistent lung inflammation and potential lung stroma remodeling in COPD may be important causes of lung cancer. Previous studies have shown that non-small cell lung cancer (NSCLC) patients with COPD have a worse prognosis than those without, but the reasons have not been determined. Due to the increasing application of ICIs in the NSCLC, the characteristics of the immune microenvironment in the NSCLC patients concomitant COPD are worth exploring. Methods: Immune microenvironment and the next generation sequencing (NGS) data of twenty NSCLC patients from Sun Yat-sen Memorial Hospital were analyzed. Formalin-fixed paraffin-embedded biopsy tissue before first-line treatment to prepare tumor tissue slides. Samples were stained using an Opal automation mIF Detection Kit (Akoya) for immune cells. The immune cell infiltration were reported as percentages and density from each individual cell subpopulation in the tumor or stromal area. The landscape of gene mutantions was obtained by NGS detection of 733-gene panel, sequencing depth was 35000x. Results: There were 8 (40%) patients concomitant COPD, and the median age was 65.5-year-old. Analysis the immune cell infiltration in all patients, including T cells, B cells, macrophages and NK cells through 13 markers, showed no difference between COPD and non-COPD groups. However, comparing the differences between tumor and stromal immune microenvironment, it was found that there was a significant difference in the percentages of M1 macrophages between tumor and stromal in COPD group (P = 0.031), but no difference in non-COPD group (P = 0.47). Further analysis of the difference in cell density between tumor and stroma showed that the stromal M1 macrophages in the COPD group was significantly higher than that in the non-COPD group (P = 0.017), as well as PD-1+CD8+ T cells (P = 0.05). Additionally, a preliminary analysis of the gene mutation characteristics had done. In COPD patients with somatic mutations, the most frequently mutated gene was EGFR (60%), followed by TP53 (50%) and GLI2 (50%), which were similar as non-COPD patients. The most common variant of COPD patients are the missense mutation of EGFR L858R, but it was EGFR 19del in non-COPD. Conclusions: The results showed that the COPD was correlated with the infiltration status of M1 macrophages and PD-1+CD8+ T cell, and poor immune cell infiltration in tumor of COPD patients may be the potential influencing factors for the worse prognosis. The characteristics of immune microenvironment suggest that promoting the entry of immune cells from stroma into tumor may be a potential mechanism for the benefit of immunotherapy in NSCLC patients with COPD.

Artificial intelligence (AI) –powered spatial analysis of macrophages in tumor microenvironment and its association with interferon-gamma (IFNG) signature and immune phenotype (IP) in pan-cancer dataset.

2621 Background: Macrophages are known to play a crucial role in cancer progression, but few studies have reported macrophage spatial distribution and its association with genomic signatures and tumor-infiltrating lymphocytes (TIL). Here, we applied an AI-powered Whole Slide Image (WSI) analyzer in The Cancer Genome Atlas (TCGA) pan-cancer dataset to show correlation between specific localization of macrophage and anti-tumor activity, which is reflected by IFNG signature and IP based on spatial TIL density. Methods: For spatial macrophage analysis in TCGA pan-cancer dataset, we used Lunit SCOPE IO, an AI-powered H&E WSI analyzer, which identifies and quantifies various classes of cells such as tumor cell, macrophage, and TIL within cancer or stroma area. IPs were defined as follows: inflamed IP (IIP) as high intratumoral TIL (iTIL) and stromal TIL (sTIL); immune-excluded IP (IEP) as low iTIL and high sTIL; immune-desert IP (IDP) as low TIL overall. The immune cell profiling and quantification of the IFNG signature score were analyzed by using CIBERSORT and RSEM. Results: In TCGA dataset (N = 7,402), median macrophage density in total TME area (tMP) was 19.9 /mm2 (interquartile range [IQR] 9.0-44.9), and it was highly enriched in lung adenocarcinoma (median 65.1, IQR 34.3-110.0), lung squamous cell carcinoma (61, 32.4-101.3), and head and neck squamous cell carcinoma (39.4, 19.6-77.3). Based on IP classification, tMP was highly enriched in IIP (37.8, 18.7-73.8) compared to IEP (25.5, 13.8-48.6) and IDP (8.9, 4.4-17.1). Interestingly, spatial analysis showed that intratumoral macrophage density (iMP) in IIP was highly enriched compared to the others (mean fold change 2.57) and the difference of stromal macrophage density (sMP) among IPs was relatively smaller (mean fold change x1.85). Classified into four groups based on median values of iMP and sMP, high-iMP/high-sMP group had the highest proportion of IIP (47.3% vs 18.7%, p < 0.001), whereas low-iMP/high-sMP group had the highest one of IEP (54.9% vs 32.3%, p < 0.001) compared to the others, respectively. Moreover, the high-iMP/high-sMP group had the highest level of IFNG signature score among 4 groups (x2.13 times higher, p < 0.001). Notably, both iMP and sMP were significantly correlated with CD68 expression (rho 0.292 and 0.287) but CSF1R expression was more strongly correlated with iMP (rho 0.259) compared to sMP (rho 0.078). Conclusions: IIP, IFNG signature, and CSF1R expression are more strongly correlated with intratumoral macrophage density versus stromal macrophage density, suggesting a relationship between macrophage location and function in the TME warranting further investigation.

Metabolic profile of mesenchymal stromal cells and macrophages in the presence of polyethylene particles in a 3D model

BackgroundContinuous cross talk between MSCs and macrophages is integral to acute and chronic inflammation resulting from contaminated polyethylene particles (cPE); however, the effect of this inflammatory microenvironment on mitochondrial metabolism has not been fully elucidated. We hypothesized that (a) exposure to cPE leads to impaired mitochondrial metabolism and glycolytic reprogramming and (b) macrophages play a key role in this pathway.MethodsWe cultured MSCs with/without uncommitted M0 macrophages, with/without cPE in 3-dimensional gelatin methacrylate (3D GelMA) constructs/scaffolds. We evaluated mitochondrial function (membrane potential and reactive oxygen species—ROS production), metabolic pathways for adenosine triphosphate (ATP) production (glycolysis or oxidative phosphorylation) and response to stress mechanisms. We also studied macrophage polarization toward the pro-inflammatory M1 or the anti-inflammatory M2 phenotype and the osteogenic differentiation of MSCs.ResultsExposure to cPE impaired mitochondrial metabolism of MSCs; addition of M0 macrophages restored healthy mitochondrial function. Macrophages exposed to cPE-induced glycolytic reprogramming, but also initiated a response to this stress to restore mitochondrial biogenesis and homeostatic oxidative phosphorylation. Uncommitted M0 macrophages in coculture with MSC polarized to both M1 and M2 phenotypes. Osteogenesis was comparable among groups after 21 days.ConclusionThis work confirmed that cPE exposure triggers impaired mitochondrial metabolism and glycolytic reprogramming in a 3D coculture model of MSCs and macrophages and demonstrated that macrophages cocultured with MSCs undergo metabolic changes to maintain energy production and restore homeostatic metabolism.

LDL receptor related protein 1 is an adverse prognostic biomarker that correlates with stromal remodeling and macrophages infiltration in bladder cancer.

Bladder cancer (BLCA) is a highly heterogeneous disease influenced by the tumor microenvironment, which may affect patients' response to immune checkpoint blockade therapy. Therefore, identifying molecular markers and therapeutic targets to improve treatment is essential. In this study, we aimed to investigate the prognostic significance of LRP1 in BLCA. We analyzed TCGA and IMvigor210 cohorts to investigate the relationship of LRP1 with BLCA prognosis. We utilized gene mutation analysis and enrichment to identify LRP1-associated mutated genes and biological processes. Deconvolution algorithms and single-cell analysis were used to understand the tumor-infiltrated cells and biological pathways associated with LRP1 expression. Immunohistochemistry was conducted to validate the bioinformatics analysis. Our study revealed that LRP1 was an independent risk factor for overall survival in BLCA patients and was associated with clinicopathological features and FGFR3 mutation frequency. Enrichment analysis demonstrated that LRP1 was involved in extracellular matrix remodeling and tumor metabolic processes. Furthermore, the ssGSEA algorithm revealed that LRP1 was positively correlated with the activities of tumor-associated pathways. Our study also found that high LRP1 expression impaired patients' responsiveness to ICB therapy in BLCA, which was predicted by TIDE prediction and validated by IMvigor210 cohort. Immunohistochemistry confirmed the expression of LRP1 in Cancer-Associated Fibroblasts (CAFs) and macrophages in the tumor microenvironment of BLCA. Our study suggests that LRP1 may be a potential prognostic biomarker and therapeutic target in BLCA. Further research on LRP1 may improve BLCA precision medicine and enhance the efficacy of immune checkpoint blockade therapy.

Abstract 3310: Extracellular vesicles budding from stromal macrophages in the blood of metastatic non-small cell lung cancer patients correlates with poor survival

Abstract Purpose: Cancer extracellular vesicles (EVs) are involved in cellular communication, tumor growth, progression, and metastasis in cancer. The origins of EVs, their formation, and potential clinical use as biomarkers are not well understood. Recently, budding of extracellular structures on Cancer Associated Macrophage-Like Cells [CAMLs] a specific subtype of phagocytic circulating stromal cells has been observed in metastatic non-small cell lung carcinoma (mNSCLC) patients. In this prospective analysis of n=40 mNSCLC samples, we enumerated EV budding on CAMLs to determine if their formation had an effect on clinical outcomes. These preliminary results suggest that EV budding from a specific subtype of circulating tumor associated macrophage prognosticates for worse clinical outcome which may serve as the mechanism for cancer EV formation and spread throughout the body. Patients and Methods: We initiated a single blind prospective pilot study to evaluate extracellular budding on the CAMLs of mNSCLC patients from blood samples obtained prior to therapy to determine their prevalence and clinical utility. Anonymized blood was procured and filtered to isolate CAMLs and stained for cytokeratin, CD45, CD31 and PD-L1. EV budding was observed as small (≤1 µm) bulbous protrusions from the cell periphery. EVs were quantified and compared against patient progression free survival (PFS) and overall survival (OS) with hazard ratios (HRs) at 24 months by censored univariate analysis. The imaged EVs were also characterized by their PD-L1 biomarker expression. Results: CAMLs were identified in 88% (n=35/40) of all samples, with EV budding identified in 60% (n=21/35) of CAMLs. These EVs appeared with tumor positive proteins (i.e. CD31, CD45, PD-L1 and cytokeratin). With a minimum of 24 months of follow-up, it was determined that the presence of EV budding in patients’ CAMLs was associated with significantly worse PFS (HR=4.00, 95%CI=1.4-12, p=0.0251) and borderline significant OS (HR=3.57, 95%CI=1.1-12, p=0.0747). Conclusions: EV budding found on phagocytic stromal cells found in the blood appear with tumor positive biomarkers and predict poor survival. These findings suggest that CAMLs are an origin cell for some cancer EVs. Larger validation studies and cross comparison of PD-L1 on EVs as it related to immunotherapy response is ongoing. Citation Format: Jillian A. Moran, Steven H. Lin, Martin J. Edelman, Pablo Lopez, Jianzhong He, Yawei Qiao, Ting Xu, Zhongxing Liao, Kirby P. Gardner, Cha-Mei Tang, Daniel L. Adams. Extracellular vesicles budding from stromal macrophages in the blood of metastatic non-small cell lung cancer patients correlates with poor survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3310.

Stromal and therapy-induced macrophage proliferation promotes PDAC progression and susceptibility to innate immunotherapy.

Tumor-associated macrophages (TAMs) are abundant in pancreatic ductal adenocarcinomas (PDACs). While TAMs are known to proliferate in cancer tissues, the impact of this on macrophage phenotype and disease progression is poorly understood. We showed that in PDAC, proliferation of TAMs could be driven by colony stimulating factor-1 (CSF1) produced by cancer-associated fibroblasts. CSF1 induced high levels of p21 in macrophages, which regulated both TAM proliferation and phenotype. TAMs in human and mouse PDACs with high levels of p21 had more inflammatory and immunosuppressive phenotypes. p21 expression in TAMs was induced by both stromal interaction and/or chemotherapy treatment. Finally, by modeling p21 expression levels in TAMs, we found that p21-driven macrophage immunosuppression in vivo drove tumor progression. Serendipitously, the same p21-driven pathways that drive tumor progression also drove response to CD40 agonist. These data suggest that stromal or therapy-induced regulation of cell cycle machinery can regulate both macrophage-mediated immune suppression and susceptibility to innate immunotherapy.

METTL14 alleviates the development of osteoporosis in ovariectomized mice by upregulating m6A level of SIRT1 mRNA.

The purpose of this study was to investigate whether METTL14 participated in ovariectomized (OVX)-induced osteoporosis (OP) in mice by regulating the m6A level of SIRT1 mRNA. OVX was performed on mice to induce OP, and mouse bone marrow stromal cells (BMSCs) and bone marrow mononuclear macrophages (BMMs) were isolated to induce osteoblast differentiation and osteoclast differentiation, respectively. The morphology of bone trabeculae was evaluated under a micro-CT scanner. The changes in pathology of bone tissues were observed through staining using hematoxylin-eosin. The number of osteoclasts was measured by tartrate-resistant acid phosphatase staining, and the content of serum calcium, PINP, and CTX-I was tested by enzyme-linked immunosorbent assay, accompanied by the measurement of the expression of SIRT1, METTL14, osteogenic marker genes, and osteoclast marker genes. The m6A modification level of SIRT1 and the binding between METTL14 and SIRT1 were verified. In OVX mice, SIRT1 and METTL14 were downregulated. Overexpression of SIRT1 or METTL14 increased the expression of osteogenic marker genes but decreased the expression of osteoclast marker genes. Additionally, METTL14 overexpression increased m6A level of SIRT1 mRNA. Furthermore, overexpression of METTL14 promoted osteoblast differentiation and suppressed osteoclast differentiation, which were reversed by knockdown of SIRT1. METTL14 promoted osteoblast differentiation and repressed osteoclast differentiation by m6A-dependent upregulation of SIRT1 mRNA, thereby alleviating OP development.

Laser-induced microinjury of the corneal basal epithelium and imaging of resident macrophage responses in a live, whole-eye preparation.

The corneal epithelium is continuously subjected to external stimuli that results in varying degrees of cellular damage. The use of live-cell imaging approaches has facilitated understanding of the cellular and molecular mechanisms underlying the corneal epithelial wound healing process. Here, we describe a live, ex vivo, whole-eye approach using laser scanning confocal microscopy to simultaneously induce and visualize short-term cellular responses following microdamage to the corneal epithelium. Live-cell imaging of corneal cell layers was enabled using the lipophilic fluorescent dyes, SGC5 or FM4-64, which, when injected into the anterior chamber of enucleated eyes, readily penetrated and labelled cell membranes. Necrotic microdamage to a defined region (30 μm x 30 μm) through the central plane of the corneal basal epithelium was induced by continuously scanning for at least one minute using high laser power and was dependent on the presence of lipophilic fluorescent dye. This whole-mount live-cell imaging and microdamage approach was used to examine the behavior of Cx3cr1:GFP-expressing resident corneal stromal macrophages (RCSMs). In undamaged corneas, RCSMs remained stationary, but exhibited a constant extension and retraction of short (~5 μm) semicircular, pseudopodia-like processes reminiscent of what has previously been reported in corneal dendritic cells. Within minutes of microdamage, nearby anterior RCSMs became highly polarized and extended projections towards the damaged region. The extension of the processes plateaued after about 30 minutes and remained stable over the course of 2-3 hours of imaging. Retrospective immunolabeling showed that these responding RCSMs were MHC class II+. This study adds to existing knowledge of immune cell behavior in response to corneal damage and introduces a simple corneal epithelial microdamage and wound healing paradigm.