Automated tumor immunophenotyping predicts clinical benefit from anti-PD-L1 immunotherapy.

Cancer immunotherapy has transformed the clinical approach to patients with malignancies, as profound benefits can be seen in a subset of patients. To identify this subset, biomarker analyses increasingly focus on phenotypic and functional evaluation of the tumor microenvironment to determine if density, spatial distribution, and cellular composition of immune cell infiltrates can provide prognostic and/or predictive information. Attempts have been made to develop standardized methods to evaluate immune infiltrates in the routine assessment of certain tumor types; however, broad adoption of this approach in clinical decision-making is still missing. We developed approaches to categorize solid tumors into 'desert', 'excluded', and 'inflamed' types according to the spatial distribution of CD8+ immune effector cells to determine the prognostic and/or predictive implications of such labels. To overcome the limitations of this subjective approach, we incrementally developed four automated analysis pipelines of increasing granularity and complexity for density and pattern assessment of immune effector cells. We show that categorization based on 'manual' observation is predictive for clinical benefit from anti-programmed death ligand 1 therapy in two large cohorts of patients with non-small cell lung cancer or triple-negative breast cancer. For the automated analysis we demonstrate that a combined approach outperforms individual pipelines and successfully relates spatial features to pathologist-based readouts and the patient's response to therapy. Our findings suggest that tumor immunophenotype generated by automated analysis pipelines should be evaluated further as potential predictive biomarkers for cancer immunotherapy. © 2024 The Pathological Society of Great Britain and Ireland.

Hyperoxia Injury in the Developing Lung Is Mediated by Mesenchymal Expression of Wnt5A.

Rationale: Bronchopulmonary dysplasia (BPD) is a leading complication of preterm birth that affects infants born in the saccular stage of lung development at <32 weeks of gestation. Although the mechanisms driving BPD remain uncertain, exposure to hyperoxia is thought to contribute to disease pathogenesis.Objectives: To determine the effects of hyperoxia on epithelial-mesenchymal interactions and to define the mediators of activated Wnt/ß-catenin signaling after hyperoxia injury.Methods: Three hyperoxia models were used: A three-dimensional organotypic coculture using primary human lung cells, precision-cut lung slices (PCLS), and a murine in vivo hyperoxia model. Comparisons of normoxia- and hyperoxia-exposed samples were made by real-time quantitative PCR, RNA in situ hybridization, quantitative confocal microscopy, and lung morphometry.Measurements and Main Results: Examination of an array of Wnt ligands in the three-dimensional organotypic coculture revealed increased mesenchymal expression of WNT5A. Inhibition of Wnt5A abrogated the BPD transcriptomic phenotype induced by hyperoxia. In the PCLS model, Wnt5A inhibition improved alveolarization following hyperoxia exposure, and treatment with recombinant Wnt5a reproduced features of the BPD phenotype in PCLS cultured in normoxic conditions. Chemical inhibition of NF-κB with BAY11-7082 reduced Wnt5a expression in the PCLS hyperoxia model and in vivo mouse hyperoxia model, with improved alveolarization in the PCLS model.Conclusions: Increased mesenchymal Wnt5A during saccular-stage hyperoxia injury contributes to the impaired alveolarization and septal thickening observed in BPD. Precise targeting of Wnt5A may represent a potential therapeutic strategy for the treatment of BPD.

CD318 is a ligand for CD6.

It has been proposed that CD6, an important regulator of T cells, functions by interacting with its currently identified ligand, CD166, but studies performed during the treatment of autoimmune conditions suggest that the CD6-CD166 interaction might not account for important functions of CD6 in autoimmune diseases. The antigen recognized by mAb 3A11 has been proposed as a new CD6 ligand distinct from CD166, yet the identity of it is hitherto unknown. We have identified this CD6 ligand as CD318, a cell surface protein previously found to be present on various epithelial cells and many tumor cells. We found that, like CD6 knockout (KO) mice, CD318 KO mice are also protected in experimental autoimmune encephalomyelitis. In humans, we found that CD318 is highly expressed in synovial tissues and participates in CD6-dependent adhesion of T cells to synovial fibroblasts. In addition, soluble CD318 is chemoattractive to T cells and levels of soluble CD318 are selectively and significantly elevated in the synovial fluid from patients with rheumatoid arthritis and juvenile inflammatory arthritis. These results establish CD318 as a ligand of CD6 and a potential target for the diagnosis and treatment of autoimmune diseases such as multiple sclerosis and inflammatory arthritis.

Successful Establishment of Primary Type II Alveolar Epithelium with 3D Organotypic Coculture.

Alveolar type II (AT2) epithelial cells are uniquely specialized to produce surfactant in the lung and act as progenitor cells in the process of repair after lung injury. AT2 cell injury has been implicated in several lung diseases, including idiopathic pulmonary fibrosis and bronchopulmonary dysplasia. The inability to maintain primary AT2 cells in culture has been a significant barrier in the investigation of pulmonary biology. We have addressed this knowledge gap by developing a three-dimensional (3D) organotypic coculture using primary human fetal AT2 cells and pulmonary fibroblasts. Grown on top of matrix-embedded fibroblasts, the primary human AT2 cells establish a monolayer and have direct contact with the underlying pulmonary fibroblasts. Unlike conventional two-dimensional (2D) culture, the structural and functional phenotype of the AT2 cells in our 3D organotypic culture was preserved over 7 days of culture, as evidenced by the presence of lamellar bodies and by production of surfactant proteins B and C. Importantly, the AT2 cells in 3D cocultures maintained the ability to replicate, with approximately 60% of AT2 cells staining positive for the proliferation marker Ki67, whereas no such proliferation is evident in 2D cultures of the same primary AT2 cells. This organotypic culture system enables interrogation of AT2 epithelial biology by providing a reductionist in vitro model in which to investigate the response of AT2 epithelial cells and AT2 cell-fibroblast interactions during lung injury and repair.

Endoglin Mediates Vascular Maturation by Promoting Vascular Smooth Muscle Cell Migration and Spreading.

OBJECTIVE: Endoglin, a transforming growth factor-ß superfamily coreceptor, is predominantly expressed in endothelial cells and has essential roles in vascular development. However, whether endoglin is also expressed in vascular smooth muscle cells (VSMCs), especially in vivo, remains controversial. Furthermore, the roles of endoglin in VSMC biology remain largely unknown. Our objective was to examine the expression and determine the function of endoglin in VSMCs during angiogenesis. APPROACH AND RESULTS: Here, we determine that endoglin is robustly expressed in VSMCs. Using CRISPR/CAS9 knockout and short hairpin RNA knockdown in the VSMC/endothelial coculture model system, we determine that endoglin in VSMCs, but not in endothelial cells, promotes VSMCs recruitment by the endothelial cells both in vitro and in vivo. Using an unbiased bioinformatics analysis of RNA sequencing data and further study, we determine that, mechanistically, endoglin mediates VSMC recruitment by promoting VSMC migration and spreading on endothelial cells via increasing integrin/FAK pathway signaling, whereas endoglin has minimal effects on VSMC adhesion to endothelial cells. In addition, we further determine that loss of endoglin in VSMCs inhibits VSMC recruitment in vivo. CONCLUSIONS: These studies demonstrate that endoglin has an important role in VSMC recruitment and blood vessel maturation during angiogenesis and also provide novel insights into how discordant endoglin function in endothelial and VSMCs may regulate vascular maturation and angiogenesis.

Larger core size has superior technical and analytical accuracy in bladder tissue microarray.

The construction of tissue microarrays (TMAs) with cores from a large number of paraffin-embedded tissues (donors) into a single paraffin block (recipient) is an effective method of analyzing samples from many patient specimens simultaneously. For the TMA to be successful, the cores within it must capture the correct histologic areas from the donor blocks (technical accuracy) and maintain concordance with the tissue of origin (analytical accuracy). This can be particularly challenging for tissues with small histological features such as small islands of carcinoma in situ (CIS), thin layers of normal urothelial lining of the bladder, or cancers that exhibit intratumor heterogeneity. In an effort to create a comprehensive TMA of a bladder cancer patient cohort that accurately represents the tumor heterogeneity and captures the small features of normal and CIS, we determined how core size (0.6 vs 1.0 mm) impacted the technical and analytical accuracy of the TMA. The larger 1.0 mm core exhibited better technical accuracy for all tissue types at 80.9% (normal), 94.2% (tumor), and 71.4% (CIS) compared with 58.6%, 85.9%, and 63.8% for 0.6 mm cores. Although the 1.0 mm core provided better tissue capture, increasing the number of replicates from two to three allowed with the 0.6 mm core compensated for this reduced technical accuracy. However, quantitative image analysis of proliferation using both Ki67+ immunofluorescence counts and manual mitotic counts demonstrated that the 1.0 mm core size also exhibited significantly greater analytical accuracy (P=0.004 and 0.035, respectively, r2=0.979 and 0.669, respectively). Ultimately, our findings demonstrate that capturing two or more 1.0 mm cores for TMA construction provides superior technical and analytical accuracy over the smaller 0.6 mm cores, especially for tissues harboring small histological features or substantial heterogeneity.

Stimulation of host bone marrow stromal cells by sympathetic nerves promotes breast cancer bone metastasis in mice.

Bone and lung metastases are responsible for the majority of deaths in patients with breast cancer. Following treatment of the primary cancer, emotional and psychosocial factors within this population precipitate time to recurrence and death, however the underlying mechanism(s) remain unclear. Using a mouse model of bone metastasis, we provide experimental evidence that activation of the sympathetic nervous system, which is one of many pathophysiological consequences of severe stress and depression, promotes MDA-231 breast cancer cell colonization of bone via a neurohormonal effect on the host bone marrow stroma. We demonstrate that induction of RANKL expression in bone marrow osteoblasts, following ß2AR stimulation, increases the migration of metastatic MDA-231 cells in vitro, independently of SDF1-CXCR4 signaling. We also show that the stimulatory effect of endogenous (chronic stress) or pharmacologic sympathetic activation on breast cancer bone metastasis in vivo can be blocked with the ß-blocker propranolol, and by knockdown of RANK expression in MDA-231 cells. These findings indicate that RANKL promotes breast cancer cell metastasis to bone via its pro-migratory effect on breast cancer cells, independently of its effect on bone turnover. The emerging clinical implication, supported by recent epidemiological studies, is that ßAR-blockers and drugs interfering with RANKL signaling, such as Denosumab, could increase patient survival if used as adjuvant therapy to inhibit both the early colonization of bone by metastatic breast cancer cells and the initiation of the "vicious cycle" of bone destruction induced by these cells.

The chick embryo as an expanding experimental model for cancer and cardiovascular research.

A long and productive history in biomedical research defines the chick as a model for human biology. Fundamental discoveries, including the description of directional circulation propelled by the heart and the link between oncogenes and the formation of cancer, indicate its utility in cardiac biology and cancer. Despite the more recent arrival of several vertebrate and invertebrate animal models during the last century, the chick embryo remains a commonly used model for vertebrate biology and provides a tractable biological template. With new molecular and genetic tools applied to the avian genome, the chick embryo is accelerating the discovery of normal development and elusive disease processes. Moreover, progress in imaging and chick culture technologies is advancing real-time visualization of dynamic biological events, such as tissue morphogenesis, angiogenesis, and cancer metastasis. A rich background of information, coupled with new technologies and relative ease of maintenance, suggest an expanding utility for the chick embryo in cardiac biology and cancer research.

Transforming growth factor beta receptor type III is a tumor promoter in mesenchymal-stem like triple negative breast cancer.

INTRODUCTION: There is a major need to better understand the molecular basis of triple negative breast cancer (TNBC) in order to develop effective therapeutic strategies. Using gene expression data from 587 TNBC patients we previously identified six subtypes of the disease, among which a mesenchymal-stem like (MSL) subtype. The MSL subtype has significantly higher expression of the transforming growth factor beta (TGF-ß) pathway-associated genes relative to other subtypes, including the TGF-ß receptor type III (TßRIII). We hypothesize that TßRIII is tumor promoter in mesenchymal-stem like TNBC cells. METHODS: Representative MSL cell lines SUM159, MDA-MB-231 and MDA-MB-157 were used to study the roles of TßRIII in the MSL subtype. We stably expressed short hairpin RNAs specific to TßRIII (TßRIII-KD). These cells were then used for xenograft tumor studies in vivo; and migration, invasion, proliferation and three dimensional culture studies in vitro. Furthermore, we utilized human gene expression datasets to examine TßRIII expression patterns across all TNBC subtypes. RESULTS: TßRIII was the most differentially expressed TGF-ß signaling gene in the MSL subtype. Silencing TßRIII expression in MSL cell lines significantly decreased cell motility and invasion. In addition, when TßRIII-KD cells were grown in a three dimensional (3D) culture system or nude mice, there was a loss of invasive protrusions and a significant decrease in xenograft tumor growth, respectively. In pursuit of the mechanistic underpinnings for the observed TßRIII-dependent phenotypes, we discovered that integrin-α2 was expressed at higher level in MSL cells after TßRIII-KD. Stable knockdown of integrin-α2 in TßRIII-KD MSL cells rescued the ability of the MSL cells to migrate and invade at the same level as MSL control cells. CONCLUSIONS: We have found that TßRIII is required for migration and invasion in vitro and xenograft growth in vivo. We also show that TßRIII-KD elevates expression of integrin-α2, which is required for the reduced migration and invasion, as determined by siRNA knockdown studies of both TßRIII and integrin-α2. Overall, our results indicate a potential mechanism in which TßRIII modulates integrin-α2 expression to effect MSL cell migration, invasion, and tumorigenicity.

The DARC side of metastasis: shining a light on KAI1-mediated metastasis suppression in the vascular tunnel.

Tumor cell metastasis to distant organs is an inefficient process that is limited in part by recently identified metastasis suppressors. Interactions between tumor cells and the surrounding stroma are thought to control much of cancer progression. In the August issue of Nature Medicine, demonstrate that specific cell surface interactions between the metastasis suppressor KAI1 on tumor cells and the decoy cytokine receptor DARC on adjacent vascular cells triggers senescence in the tumor cells and suppresses metastasis. These new observations demonstrate how metastasis suppressors can relay the restraint imposed by the stroma onto disseminating tumor cells.

Multiparametric Single-Vesicle Flow Cytometry Resolves Extracellular Vesicle Heterogeneity and Reveals Selective Regulation of Biogenesis and Cargo Distribution.

Mammalian cells release a heterogeneous array of extracellular vesicles (EVs) that contribute to intercellular communication by means of the cargo that they carry. To resolve EV heterogeneity and determine if cargo is partitioned into select EV populations, we developed a method named "EV Fingerprinting" that discerns distinct vesicle populations using dimensional reduction of multiparametric data collected by quantitative single-EV flow cytometry. EV populations were found to be discernible by a combination of membrane order and EV size, both of which were obtained through multiparametric analysis of fluorescent features from the lipophilic dye Di-8-ANEPPS incorporated into the lipid bilayer. Molecular perturbation of EV secretion and biogenesis through respective ablation of the small GTPase Rab27a and overexpression of the EV-associated tetraspanin CD63 revealed distinct and selective alterations in EV populations, as well as cargo distribution. While Rab27a disproportionately affects all small EV populations with high membrane order, the overexpression of CD63 selectively increased the production of one small EV population of intermediate membrane order. Multiplexing experiments subsequently revealed that EV cargos have a distinct, nonrandom distribution with CD63 and CD81 selectively partitioning into smaller vs larger EVs, respectively. These studies not only present a method to probe EV biogenesis but also reveal how the selective partitioning of cargo contributes to EV heterogeneity.

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Viral nanoparticles as tools for intravital vascular imaging.

A significant impediment to the widespread use of noninvasive in vivo vascular imaging techniques is the current lack of suitable intravital imaging probes. We describe here a new strategy to use viral nanoparticles as a platform for the multivalent display of fluorescent dyes to image tissues deep inside living organisms. The bioavailable cowpea mosaic virus (CPMV) can be fluorescently labeled to high densities with no measurable quenching, resulting in exceptionally bright particles with in vivo dispersion properties that allow high-resolution intravital imaging of vascular endothelium for periods of at least 72 h. We show that CPMV nanoparticles can be used to visualize the vasculature and blood flow in living mouse and chick embryos to a depth of up to 500 microm. Furthermore, we show that the intravital visualization of human fibrosarcoma-mediated tumor angiogenesis using fluorescent CPMV provides a means to identify arterial and venous vessels and to monitor the neovascularization of the tumor microenvironment.

Lack of transforming growth factor-ß signaling promotes collective cancer cell invasion through tumor-stromal crosstalk.

INTRODUCTION: Transforming growth factor beta (TGF-ß) has a dual role during tumor progression, initially as a suppressor and then as a promoter. Epithelial TGF-ß signaling regulates fibroblast recruitment and activation. Concurrently, TGF-ß signaling in stromal fibroblasts suppresses tumorigenesis in adjacent epithelia, while its ablation potentiates tumor formation. Much is known about the contribution of TGF-ß signaling to tumorigenesis, yet the role of TGF-ß in epithelial-stromal migration during tumor progression is poorly understood. We hypothesize that TGF-ß is a critical regulator of tumor-stromal interactions that promote mammary tumor cell migration and invasion. METHODS: Fluorescently labeled murine mammary carcinoma cells, isolated from either MMTV-PyVmT transforming growth factor-beta receptor II knockout (TßRII KO) or TßRIIfl/fl control mice, were combined with mammary fibroblasts and xenografted onto the chicken embryo chorioallantoic membrane. These combinatorial xenografts were used as a model to study epithelial-stromal crosstalk. Intravital imaging of migration was monitored ex ovo, and metastasis was investigated in ovo. Epithelial RNA from in ovo tumors was isolated by laser capture microdissection and analyzed to identify gene expression changes in response to TGF-ß signaling loss. RESULTS: Intravital microscopy of xenografts revealed that mammary fibroblasts promoted two migratory phenotypes dependent on epithelial TGF-ß signaling: single cell/strand migration or collective migration. At epithelial-stromal boundaries, single cell/strand migration of TßRIIfl/fl carcinoma cells was characterized by expression of α-smooth muscle actin and vimentin, while collective migration of TßRII KO carcinoma cells was identified by E-cadherin+/p120+/ß-catenin+ clusters. TßRII KO tumors also exhibited a twofold greater metastasis than TßRIIfl/fl tumors, attributed to enhanced extravasation ability. In TßRII KO tumor epithelium compared with TßRIIfl/fl epithelium, Igfbp4 and Tspan13 expression was upregulated while Col1α2, Bmp7, Gng11, Vcan, Tmeff1, and Dsc2 expression was downregulated. Immunoblotting and quantitative PCR analyses on cultured cells validated these targets and correlated Tmeff1 expression with disease progression of TGF-ß-insensitive mammary cancer. CONCLUSION: Fibroblast-stimulated carcinoma cells utilize TGF-ß signaling to drive single cell/strand migration but migrate collectively in the absence of TGF-ß signaling. These migration patterns involve the signaling regulation of several epithelial-to-mesenchymal transition pathways. Our findings concerning TGF-ß signaling in epithelial-stromal interactions are important in identifying migratory mechanisms that can be targeted as recourse for breast cancer treatment.

CD98hc (SLC3A2) participates in fibronectin matrix assembly by mediating integrin signaling.

Integrin-dependent assembly of the fibronectin (Fn) matrix plays a central role in vertebrate development. We identify CD98hc, a membrane protein, as an important component of the matrix assembly machinery both in vitro and in vivo. CD98hc was not required for biosynthesis of cellular Fn or the maintenance of the repertoire or affinity of cellular Fn binding integrins, which are important contributors to Fn assembly. Instead, CD98hc was involved in the cell's ability to exert force on the matrix and did so by dint of its capacity to interact with integrins to support downstream signals that lead to activation of RhoA small GTPase. Thus, we identify CD98hc as a membrane protein that enables matrix assembly and establish that it functions by interacting with integrins to support RhoA-driven contractility. CD98hc expression can vary widely; our data show that these variations in CD98hc expression can control the capacity of cells to assemble an Fn matrix, a process important in development, wound healing, and tumorigenesis.

A versatile valve-enabled microfluidic cell co-culture platform and demonstration of its applications to neurobiology and cancer biology.

A versatile microfluidic platform allowing co-culture of multiple cell populations in close proximity with separate control of their microenvironments would be extremely valuable for many biological applications. Here, we report a simple and compact microfluidic platform that has these desirable features and allows for real-time, live-cell imaging of cell-cell interactions. Using a pneumatically/hydraulically controlled poly(dimethylsiloxane) (PDMS) valve barrier, distinct cell types can be cultured in side-by-side microfluidic chambers with their optimum culture media and treated separately without affecting the other cell population. The platform is capable of both two-dimensional and three-dimensional cell co-culture and through variations of the valve barrier design, the platform allows for cell-cell interactions through either direct cell contact or soluble factors alone. The platform has been used to perform dynamic imaging of synapse formation in hippocampal neurons by separate transfection of two groups of neurons with fluorescent pre- and post-synaptic protein markers. In addition, cross-migration of 4T1 tumor cells and endothelial cells has been studied under normoxic and hypoxic conditions, which revealed different migration patterns, suggesting the importance of the microenvironments in cell-cell interactions and biological activities.

Integrated Cells and Collagen Fibers Spatial Image Analysis.

Modern technologies designed for tissue structure visualization like brightfield microscopy, fluorescent microscopy, mass cytometry imaging (MCI) and mass spectrometry imaging (MSI) provide large amounts of quantitative and spatial information about cells and tissue structures like vessels, bronchioles etc. Many published reports have demonstrated that the structural features of cells and extracellular matrix (ECM) and their interactions strongly predict disease development and progression. Computational image analysis methods in combination with spatial analysis and machine learning can reveal novel structural patterns in normal and diseased tissue. Here, we have developed a Python package designed for integrated analysis of cells and ECM in a spatially dependent manner. The package performs segmentation, labeling and feature analysis of ECM fibers, combines this information with pre-generated single-cell based datasets and realizes cell-cell and cell-fiber spatial analysis. To demonstrate performance and compatibility of our computational tool, we integrated it with a pipeline designed for cell segmentation, classification, and feature analysis in the KNIME analytical platform. For validation, we used a set of mouse mammary gland tumors and human lung adenocarcinoma tissue samples stained for multiple cellular markers and collagen as the main ECM protein. The developed package provides sufficient performance and precision to be used as a novel method to investigate cell-ECM relationships in the tissue, as well as detect structural patterns correlated with specific disease outcomes.

HLA-DR cancer cells expression correlates with T cell infiltration and is enriched in lung adenocarcinoma with indolent behavior.

Lung adenocarcinoma (ADC) is a heterogeneous group of tumors associated with different survival rates, even when detected at an early stage. Here, we aim to investigate whether CyTOF identifies cellular and molecular predictors of tumor behavior. We developed and validated a CyTOF panel of 34 antibodies in four ADC cell lines and PBMC. We tested our panel in a set of 10 ADCs, classified into long- (LPS) (n = 4) and short-predicted survival (SPS) (n = 6) based on radiomics features. We identified cellular subpopulations of epithelial cancer cells (ECC) and their microenvironment and validated our results by multiplex immunofluorescence (mIF) applied to a tissue microarray (TMA) of LPS and SPS ADCs. The antibody panel captured the phenotypical differences in ADC cell lines and PBMC. LPS ADCs had a higher proportion of immune cells. ECC clusters (ECCc) were identified and uncovered two ADC groups. ECCc with high HLA-DR expression were correlated with CD4+ and CD8+ T cells, with LPS samples being enriched for those clusters. We confirmed a positive correlation between HLA-DR expression on ECC and T cell number by mIF staining on TMA slides. Spatial analysis demonstrated shorter distances from T cells to the nearest ECC in LPS. Our results demonstrate a distinctive cellular profile of ECC and their microenvironment in ADC. We showed that HLA-DR expression in ECC is correlated with T cell infiltration, and that a set of ADCs with high abundance of HLA-DR+ ECCc and T cells is enriched in LPS samples. This suggests new insights into the role of antigen presenting tumor cells in tumorigenesis.

Depletion of Embryonic Macrophages Leads to a Reduction in Angiogenesis in the Ex Ovo Chick Chorioallantoic Membrane Assay.

Macrophages play an important but poorly understood role in angiogenesis. To investigate their role in vessel formation, relevant in vivo models are crucial. Although the chick chorioallantoic membrane (CAM) model has been frequently used as an angiogenesis assay, limited data are available on the involvement of chicken macrophages in this process. Here, we describe a method to deplete macrophages in the ex ovo chick CAM assay by injection of clodronate liposomes and show that this depletion directly affects vascularisation of collagen onplants. Chicken embryos were injected intravenously with either clodronate or phosphate-buffered saline (PBS) liposomes, followed by placement of collagen type I plugs on the CAM to quantify angiogenic ingrowth. Clodronate liposome injection led to a significant 3.4-fold reduction of macrophages compared with control embryos as measured by immunohistochemistry and flow cytometry. Furthermore, analysis of vessel ingrowth into the collagen plugs revealed a significantly lower angiogenic response in macrophage-depleted embryos compared with control embryos, indicating that chicken embryonic macrophages play an essential function in the development of blood vessels. These results demonstrate that the chick CAM assay provides a promising model to investigate the role of macrophages in angiogenesis.

Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles.

Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, frequently called palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyse the palmitoyl-proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers.