Disease Microenvironment Research Articles

High endothelial venules in the pleura: MECA-79 expression in mesothelioma, pleural metastasis and pleuritis

IntroductionHigh endothelial venules (HEVs) are vessels specialized in the extravasation of lymphocytes from the blood to the tissue implicated in the immune microenvironment of several tumors. Their presence has been never studied in the pleural tissue. Material and methodsWe retrospectively studied 149 surgical pleural biopsies by immunohistochemistry for MECA-79 expression, a marker specifically recognizing HEVs. The tissues included 44 (44 %) inflammatory and 105 (56 %) neoplastic diseases. The latter corresponded to 34 (22.8 %) mesotheliomas and 71 (47.7 %) metastases from lung (n=50) or breast (n=21) primaries. ResultsHEVs were present in 102 (68 %) of all pleural specimens with a mean number of foci containing HEVs of 13.33 (±20.64). Neoplastic pleural pathologies harbored HEVs in 73.3 % of the cases compared to the non-neoplastic pathologies which harbored HEVs in 56.8 % of the cases (p=0.048). Their presence did not differ between pulmonary or mammary metastasis (p=0.7). ConclusionWe show for the first time that HEVs are present in the pleural cavity probably participating in the immune microenvironment of inflammatory and neoplastic pleural disease.

Targeting Lactate: An Emerging Strategy for Macrophage Regulation in Chronic Inflammation and Cancer.

Lactate accumulation and macrophage infiltration are pivotal features of both chronic inflammation and cancer. Lactate, once regarded merely as an aftereffect of glucose metabolism, is now gaining recognition for its burgeoning spectrum of biological roles and immunomodulatory significance. Recent studies have evidenced that macrophages display divergent immunophenotypes in different diseases, which play a pivotal role in disease management by modulating macrophage polarization within the disease microenvironment. The specific polarization patterns of macrophages in a high-lactate environment and their contribution to the progression of chronic inflammation and cancer remain contentious. This review presents current evidence on the crosstalk of lactate and macrophage in chronic inflammation and cancer. Additionally, we provide an in-depth exploration of the pivotal yet enigmatic mechanisms through which lactate orchestrates disease pathogenesis, thereby offering novel perspectives to the development of targeted therapeutic interventions for chronic inflammation and cancer.

Dissecting the microenvironment of gestational trophoblastic disease through deconvolution of bulk RNA-seq data

Dissecting the microenvironment of gestational trophoblastic disease through deconvolution of bulk RNA-seq data

Promotes M1-polarization and diabetic wound healing using Prussian blue nanozymes

Long-term inflammation and impaired angiogenesis are the main reasons for the difficulty of diabetic wound healing. What to do to effectively promote vascular endothelial cell response and immune cell reprogramming is the key to diabetic skin healing. However, contemporary therapies cannot simultaneously coordinate the promotion of vascular endothelial cells and macrophage polarization, which leads to an increased rate of disability in patients with chronic diabetes. Therefore, we developed a method of repair composed of self-assembling Prussian blue nanoenzymes, which achieved synergistic support for the immune microenvironment, and also contributed to macrophage polarization in the tissue regeneration cycle, and enhanced vascular endothelial cell activity. The template hydrothermal synthesis PB-Zr nanoplatform was prepared and locally applied to wounds to accelerate wound healing through the synergistic effect of reactive oxygen species (ROS). PB-Zr significantly normalized the wound microenvironment, thereby inhibiting ROS production and inflammatory response, which may be because it inhibited the M1 polarization of macrophages in a rat model of wound. PB-Zr treatment significantly promoted the activity of vascular endothelial cells, which better promoted the growth and regeneration of other tissues in the body. The results confirmed the disease microenvironment of PB-Zr-mediated wound therapy and indicated its application in other inflammation-related diseases.

Engineered Extracellular Vesicle-Based Nanoformulations That Coordinate Neuroinflammation and Immune Homeostasis, Enhancing Parkinson's Disease Therapy.

Although conventional intervention to microglia can mitigate neuroinflammation in the short term, immune disorders by peripheral inflammatory cells can infiltrate continuously, resulting in an overactivated immune microenvironment of Parkinson's disease (PD). Here, we design engineered extracellular vesicle-based nanoformulations (EVNs) to address multiple factors for the management of PD. Specifically, EVN is developed by coating CCR2-enriched mesenchymal stem cell-derived extracellular vesicles (MSCCCR2 EVs) onto a dihydrotanshinone I-loaded nanocarrier (MSeN-DT). The MSCCCR2 EVs (the shell of EVN) can actively show homing to specific chemokines CCL2 in the substantia nigra, which enables them to block the infiltration of peripheral inflammatory cells. Interestingly, MSeN-DT (the core of EVN) can promote the Nrf2-GPX4 pathway for the suppression of the source of inflammation by inhibiting ferroptosis in microglia. In the PD model mice, a satisfactory therapeutic effect is achieved, with inhibition of peripheral inflammatory cell infiltration, precise regulation of inflammatory microglia in the substantia nigra, as well as promotion of behavioral improvement and repairing damaged neurons. In this way, the combinatorial code of alleviation of inflammation and modulation of immune homeostasis can reshape the immune microenvironment in PD, which bridges internal anti-inflammatory and external immunity. This finding reveals a comprehensive therapeutic paradigm for PD that breaks the vicious cycle of immune overactivation.

TET3-overexpressing macrophages promote endometriosis.

Endometriosis is a debilitating, chronic inflammatory disease affecting ~10% of reproductive age women worldwide with no cure. While macrophages have been intrinsically linked to the pathophysiology of endometriosis, targeting them therapeutically has been extremely challenging due to their high heterogeneity and because these disease-associated macrophages (DAMs) can be either pathogenic or protective. Here, we reported identification of pathogenic macrophages characterized by TET3 overexpression in human endometriosis lesions. We showed that factors from the disease microenvironment upregulated TET3 expression transforming macrophages into pathogenic DAMs. TET3 overexpression stimulated pro-inflammatory cytokine production via a feedback mechanism involving inhibition of let-7 miRNA expression. Remarkably, these cells relied on TET3 overexpression for survival, hence vulnerable to TET3 knockdown. We demonstrated that Bobcat339, a synthetic cytosine derivative, triggered TET3 degradation both in human and mouse macrophages. This degradation was dependent on a VHL E3 ubiquitin ligase whose expression was also upregulated in TET3-overexpressing macrophages. Furthermore, depleting TET3-overexpressing macrophages either through myeloid-specific Tet3 ablation or using Bobcat339 strongly inhibited endometriosis progression in mice. Our results defined TET3-overexpressing macrophages as key pathogenic contributors to and attractive therapeutic targets for endometriosis. Our findings may also be applicable to other chronic inflammatory diseases where DAMs have important roles.

Imbalance of B-Cell Subpopulations in the Microenvironment of Sarcoidosis or Lung Cancer.

Although the role of T lymphocytes in sarcoidosis (SA) and lung cancer (LC) is quite well reported, the occurrence of B cells in disease microenvironments may suggest their potential role as natural modifiers of the immune response. The aim of this study was to investigate the B-cell profile and lymphocyte-related hematological parameters between patients with SA, LC and healthy controls (HCs). The cells were assessed by flow cytometry and a hematological analyzer in peripheral blood (PB) and material from lymph nodes (LNs) obtained by the EBUS/TBNA method. We showed that in SA patients, there were higher percentages of naïve B and CD21low B cells and a lower percentage of class-switched memory B cells than LC patients in LNs. We observed a higher median proportion of non-switched memory and transitional B cells in the PB of SA patients than in LC patients. We noticed the lowest median proportion of class-switched memory B cells in the PB from SA patients. LC patients had a higher percentage of RE-LYMP and AS-LYMP than SA patients. Our study presented a different profile of B-cell subpopulations in SA and LC patients, distinguishing dominant subpopulations, and showed the relocation from distant compartments of the circulation to the disease microenvironment, thus emphasizing their role.

Carrier-free cryptotanshinone-peptide conjugates self-assembled nanoparticles: An efficient and low-risk strategy for acne vulgaris

Acne vulgaris ranks as the second most prevalent dermatological condition worldwide, and there are still insufficient safe and reliable drugs to treat it. Cryptotanshinone (CTS), a bioactive compound derived from traditional Chinese medicine Salvia miltiorrhiza, has shown promise for treating acne vulgaris due to its broad-spectrum antimicrobial and significant anti-inflammatory properties. Nevertheless, its local application is hindered by its low solubility and poor skin permeability. To overcome these challenges, a carrier-free pure drug self-assembled nanosystem is employed, which can specifically modify drug molecules based on the disease type and microenvironment, offering a potential for more effective treatment. We designed and synthesized three distinct structures of cationic CTS-peptide conjugates, creating self-assembled nanoparticles. This study has explored their self-assembly behavior, skin permeation, cellular uptake, and both in vitro and in vivo anti-acne effects. Molecular dynamics simulations revealed these nanoparticles form through intermolecular hydrogen bonding and π-π stacking interactions. Notably, self-assembled nanoparticles demonstrated enhanced bioavailability with higher skin permeation and cellular uptake rates. Furthermore, the nanoparticles exhibited superior anti-acne effects compared to the parent drug, attributed to heightened antimicrobial activity and significant downregulation of the MAPK/NF-κB pathway, leading to reduced expression of pro-inflammatory factors including TNF-α, IL-1β and IL-8. In summary, the carrier-free self-assembled nanoparticles based on CTS-peptide conjugate effectively address the issue of poor skin bioavailability, offering a promising new approach for acne treatment.

Oral mitochondrial transplantation using nanomotors to treat ischaemic heart disease.

Mitochondrial transplantation is an important therapeutic strategy for restoring energy supply in patients with ischaemic heart disease (IHD); however, it is limited by the invasiveness of the transplantation method and loss of mitochondrial activity. Here we report successful mitochondrial transplantation by oral administration for IHD therapy. A nitric-oxide-releasing nanomotor is modified on the mitochondria surface to obtain nanomotorized mitochondria with chemotactic targeting ability towards damaged heart tissue due to nanomotor action. The nanomotorized mitochondria are packaged in enteric capsules to protect them from gastric acid erosion. After oral delivery the mitochondria are released in the intestine, where they are quickly absorbed by intestinal cells and secreted into the bloodstream, allowing delivery to the damaged heart tissue. The regulation of disease microenvironment by the nanomotorized mitochondria can not only achieve rapid uptake and high retention of mitochondria by damaged cardiomyocytes but also maintains high activity of the transplanted mitochondria. Furthermore, results from animal models of IHD indicate that the accumulated nanomotorized mitochondria in the damaged heart tissue can regulate cardiac metabolism at the transcriptional level, thus preventing IHD progression. This strategy has the potential to change the therapeutic strategy used to treat IHD.

PathFinder: a novel graph transformer model to infer multi-cell intra- and inter-cellular signaling pathways and communications.

Recently, large-scale scRNA-seq datasets have been generated to understand the complex signaling mechanisms within the microenvironment of Alzheimer's Disease (AD), which are critical for identifying novel therapeutic targets and precision medicine. However, the background signaling networks are highly complex and interactive. It remains challenging to infer the core intra- and inter-multi-cell signaling communication networks using scRNA-seq data. In this study, we introduced a novel graph transformer model, PathFinder, to infer multi-cell intra- and inter-cellular signaling pathways and communications among multi-cell types. Compared with existing models, the novel and unique design of PathFinder is based on the divide-and-conquer strategy. This model divides complex signaling networks into signaling paths, which are then scored and ranked using a novel graph transformer architecture to infer intra- and inter-cell signaling communications. We evaluated the performance of PathFinder using two scRNA-seq data cohorts. The first cohort is an APOE4 genotype-specific AD, and the second is a human cirrhosis cohort. The evaluation confirms the promising potential of using PathFinder as a general signaling network inference model.

Uncovering the Cellular Microenvironment in Chronic Rhinosinusitis via Single-Cell RNA Sequencing: Application and Future Directions.

Chronic rhinosinusitis (CRS) is a heterogenic disease characterized by persistent mucosal inflammation of the upper airway. Researches of CRS have progressed from phenotype-based to endotype-based, looking more deeply into molecular biomarkers, signaling pathways, and immune microenvironment. Single-cell RNA sequencing is an effective tool in analyzing composition, function, and interaction of cells in disease microenvironment at transcriptome level, showing great advantage in analyzing potential biomarkers, pathogenesis, and heterogeneity of chronic airway inflammation in an unbiased manner. In this article, we will review the latest advances in scRNA-seq studies of CRS to provide new perspectives for the diagnosis and treatment of this heterogeneous disease.

P148 CCN3-derived peptides as anti-fibrotic therapies for systemic sclerosis

Abstract Background/Aims Effective therapy for skin fibrosis in scleroderma (systemic sclerosis: SSc) remains an unmet clinical challenge. Persistently activated M2 macrophages are believed to stimulate myofibroblasts in the disease microenvironment creating a pro-fibrotic niche in the skin. Expression of the anti-fibrotic matricellular protein CCN3 is reduced in animal models of fibrosis. We have identified a small peptide based on an amino acid sequence in CCN3, BLR-200, which mimics the anti-fibrotic activity of CCN3, and propose that BLR-200 could be used to treat patients in whom lack of CCN3 activity is permissive for fibrosis. However, whether there is lack of CCN3 activity in SSc and whether BLR-200 has effective anti-fibrotic activity in SSc are unknown. Methods We used ELISAs (R&D Systems) to detect CCN3 and the M2 macrophage marker CD206 in the serum of scleroderma patients with early stage diffuse subset disease (within two years of disease onset), those with late-stage diffuse subset disease (over five years duration) as well as healthy controls (all n = 20). The bleomycin-induced mouse model of skin scleroderma, in which bleomycin is injected subcutaneously every day for 21 days, was used to assess the antifibrotic ability of BLR200 in vivo. Results In scleroderma serum, CCN3 was significantly reduced, relative to matched healthy controls and patients with inactive disease, in early onset scleroderma patients with active disease that show elevated CD206 levels (p < 0.01). In the mouse model of human SSc, compared to injection with a scrambled control peptide and relative to control mice treated with phosphate buffered saline, injection with BLR-200 prevented bleomycin-induced changes in collagen deposition, myofibroblast differentiation and skin thickness as measured by Trichrome stain, indirect immunofluorescence analysis with an anti-a-smooth muscle actin antibody, and morphometric analysis, respectively (N = 8 mice per group, p < 0.05). Similarly, RNA sequencing and real-time polymerase chain reaction analysis revealed that BLR-200 significantly impaired the ability of bleomycin to induce expression of fibrogenic genes such as: integrin alpha 11, CCN2, CCN1, Smad3, wnt4, YAP1 and tenascin-C (all N = 5, p < 0.05). Spatial transcriptomics analysis revealed that BLR-200 impaired bleomycin-induced alterations in skin cell populations, including the activation and expansion of epithelial and reticular fibroblast niches and the induction of Wnt, hippo, focal adhesion and actin cytoskeleton gene expression cluster, all of which are known to be activators of fibrosis and myofibroblast activation and persistence. Conclusion CCN3 expression is reduced in serum of scleroderma patients with active skin disease that show M2 macrophage activation as visualized by elevated CD206 levels. Since BLR-200 has antifibrotic activity in the bleomycin model of skin scleroderma, our data are consistent with the hypothesis that BLR-200 could be used to block fibrosis progression in early onset diffuse scleroderma patients who possess both low CCN3 and high CD206 in serum. Disclosure J. Nguyen: None. K. Zestranjyan: None. S. Xu: None. B.L. Riser: Corporate appointments; CEO of BLR Bio. R.J. Stratton: None. A. Leask: None.

Pyxinol Fatty Acid Ester Derivatives J16 against AKI by Selectively Promoting M1 Transition to M2c Macrophages.

Acute kidney injury (AKI) is a common, multicause clinical condition that, if ignored, often progresses to chronic kidney disease (CKD) and end-stage kidney disease, with a mortality rate of 40-50%. However, there is a lack of universal treatment for AKI. Inflammation is the basic pathological change of early kidney injury, and inflammation can exacerbate AKI. Macrophages are the primary immune cells involved in the inflammatory microenvironment of kidney disease. Therefore, regulating the function of macrophages is a crucial breakthrough for the AKI intervention. Our team chemically modified pyxinol, an ocotillol-type ginsenoside, to prepare PJ16 with higher solubility and bioavailability. In vitro, using a model of macrophages stimulated by LPS, it was found that PJ16 could regulate macrophage function, including inhibiting the secretion of inflammatory factors, promoting phagocytosis, inhibiting M1 macrophages, and promoting M1 transition to the M2c macrophage. Further investigation revealed that PJ16 may shield renal tubular epithelial cells (HK-2) damaged by LPS in vitro. Based on this, PJ16 was validated in the animal model of unilateral ureteral obstruction, which showed that it improves renal function and inhibits renal tissue fibrosis by decreasing inflammatory responses, reducing macrophage inflammatory infiltration, and preferentially upregulating M2c macrophages. In conclusion, our study is the first to show that PJ16 resists AKI and fibrosis by mechanistically regulating macrophage function by modulating the phenotypic transition from M1 to M2 macrophages, mainly M2c macrophages.

Abstract 5330: Intestine-specific homeobox edits macrophage polarization in the disease microenvironment by activating inflammasome activity

Abstract Inflammasome activity has a pivotal role in the innate immune system, highlighting its importance in the development of various inflammatory diseases, including cancers. However, the specific impact of inflammasome-produced cytokines from tumor cells on disease progression remains uncertain. In this study, we show that the expression levels of inflammasome-related genes (NLRP1 and NLRP3), combined with levels of the intestine-specific homeobox (ISX), significantly correlate with factors like lesion count, disease stage, and lymph vascular invasion. Tumor cells enhance the secretion of key cytokines such as IL-1β and IL-18, exacerbating disease progression via the ISX-TWIST1 complex. In terms of mechanism, transcriptomic analysis and molecular evidence reveal that ISX interacts with the basic helix-loop-helix (bHLH) domain of TWIST1 through its homeobox domain. This interaction forms a transcriptional complex that activates inflammasomes by directly binding to the conserved sequence "-GGDWYR-" in promoter regions of several NF-κB and inflammasome-related genes (NFKBs, RELs, NLRP1, NLRP3, NEK7, AIM2, and ASC). Elevated levels of IL-18, induced by active inflammasomes, lead to the shift of M0 macrophages into the M2-like phenotype within the hepatic microenvironment. In mice with liver-specific ISX mutations, inflammasome-related genes and activities were eliminated, stopping disease progression in an acute hepatic disease model. These findings underscore the critical role of the ISX-TWIST1 complex in liver disease and tumor progression on both mechanistic and clinical levels. They also offer new insights for potential preventive and therapeutic targets in liver disease. Citation Format: Li-Ting Wang, Shih-Hsien Hsu. Intestine-specific homeobox edits macrophage polarization in the disease microenvironment by activating inflammasome activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5330.

Abstract 1048: Loss of MTAP expression is strongly linked to homozygous 9p21 deletion, unfavorable tumor phenotype and non-inflamed microenvironment in urothelial bladder cancer

Abstract 9p21 deletions are common in urothelial carcinoma and mostly include S-methyl-5′-thioadenosine phosphorylase (MTAP) a critical enzyme for DNA and RNA synthesis. Homozygous MTAP deletions result in a complete loss of expression which can be visualized by immunohistochemistry (IHC). Evidence is emerging that MTAP deficiency results in a critical vulnerability of cancer cells towards drugs targeting multiple different pathways that depend on a functional MTAP gene and causes resistance to immune checkpoint inhibitors. To determine the prevalence and clinical significance of MTAP deficiency and its relationship with the tumor microenvironment in urothelial bladder carcinomas, more than 2,500 tumors were analyzed by fluorescence in-situ hybridization (FISH) and IHC in a tissue microarray format, and the data were compared with existing data on intratumoral lymphocyte subsets and PD-L1 expression. The cohort included 1,826 patients that underwent radical cystectomy for muscle-invasive disease (pT2-4). 9p21 deletion was homozygous in 364 (21.3%) and heterozygous in 334 (19.5%) of 1,711 analyzable tumors. The rate of 9p21 deleted tumors increased from pTa G2 low (9.2% homozygous, 25.8% heterozygous) to pTa G2 high (32.6%, 20.9%; p<0.0001) but was slightly lower in pTa G3 (16.7% each). In pT2-pT4 carcinomas, 9p21 deletions were homozygous in 23.3% and heterozygous in 17.9%. Within muscle-invasive cancers, 9p21 deletions were tied to high pT (p=0.0014) and poor overall survival (p=0.0461). MTAP expression loss was strongly linked to homozygous 9p21 deletions. Complete absence of MTAP staining was seen in 96.1% of homozygous deleted cancers while only 4 of 249 MTAP negative cancers had retained at least one 9p21 copy (p<0.0001). Accordingly, absence of MTAP staining was also linked to advanced pT status and poor overall survival (p<0.05 each). Tumors with heterozygous 9p21 deletion did often show preserved but low-level expression of MTAP. 9p21 deletions were associated with low numbers of PD-L1 positive tumor cells (p<0.0001), CD8 (p=0.01) and CD4 positive lymphocytes (p=0.009), M2-macrophages (p<0.0001), and dendritic cells (p=0.0007). A similar tendency was observed for MTAP expression loss, but the level of significance was only reached for PD-L1 positive tumor cells (p<0.0001), M2 macrophages (p=0.001) and dendritic cells (p=0.012). Complete MTAP expression loss is common in urinary bladder cancer and strongly tied to homozygous 9p21 loss, aggressive disease, and non-inflamed microenvironment. Drugs targeting MTAP-deficiency may be highly useful in bladder cancer. MTAP IHC is an excellent surrogate for the detection of MTAP deficiency in this tumor entity. Citation Format: Natalia Gorbokon, Niklas Wößner, Viktoria Ahlburg, Simon Schallenberg, Henning Plage, Sebastian Hofbauer, Kira Furlano, Sarah Weinberger, Paul G. Bruch, Florian Roßner, Sefer Elezkurtaj, Maximilian Lennartz, Niclas C. Blessin, Andreas H. Marx, Henrik Samtleben, Margit Fisch, Michael Rink, Marcin Slojewski, Krystian Kaczmarek, Thorsten Ecke, Steffen Hallmann, Stefan Koch, Nico Adamini, Sarah Minner, Ronald Simon, Guido Sauter, Henrik Zecha, David Horst, Tobias Klatte, Thorsten Schlomm, Martina Kluth. Loss of MTAP expression is strongly linked to homozygous 9p21 deletion, unfavorable tumor phenotype and non-inflamed microenvironment in urothelial bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1048.

Comparative analysis of single-cell transcriptome reveals heterogeneity and commonality in the immune microenvironment of colorectal cancer and inflammatory bowel disease.

During aging, chronic inflammation can promote tumor development and metastasis. Patients with chronic inflammatory bowel diseases (IBD) are at an increased risk of developing colorectal cancer (CRC). However, the molecular mechanism underlying is still unclear. We conducted a large-scale single-cell sequencing analysis comprising 432,314 single cells from 92 CRC and 24 IBD patients. The analysis focused on the heterogeneity and commonality of CRC and IBD with respect to immune cell landscape, cellular communication, aging and inflammatory response, and Meta programs. The CRC and IBD had significantly different propensities in terms of cell proportions, differential genes and their functions, and cellular communication. The progression of CRC was mainly associated with epithelial cells, fibroblasts, and monocyte-macrophages, which displayed pronounced metabolic functions. In particular, monocyte-macrophages were enriched for the aging and inflammation-associated NF-κB pathway. And IBD was enriched in immune-related functions with B cells and T cells. Cellular communication analysis in CRC samples displayed an increase in MIF signaling from epithelial cells to T cells, and an increase in the efferent signal of senescence-associated SPP1 signaling from monocyte-macrophages. Notably, we also found some commonalities between CRC and IBD. The efferent and afferent signals showed that the pro-inflammatory cytokine played an important role. And the activity of aging and inflammatory response with AUCell analysis also showed a high degree of commonality. Furthermore, using the Meta programs (MPs) with the NMF algorithm, we found that the CRC non-malignant cells shared a substantial proportion of the MP genes with CRC malignant cells (68% overlap) and IBD epithelial cells (52% overlap), respectively. And it was extensively involved in functions of cell cycle and immune response, revealing its dual properties of inflammation and cancer. In addition, CRC malignant and non-malignant cells were enriched for the senescence-related cell cycle G2M phase transition and the p53 signaling pathway. Our study highlights the characteristics of aging, inflammation and tumor in CRC and IBD at the single-cell level, and the dual property of inflammation-cancer in CRC non-malignant cells may provide a more up-to-date understanding of disease transformation.

Advances and challenges in immunoPET methodology.

Immuno-positron emission tomography (immunoPET) enables imaging of specific targets that play a role in targeted therapy and immunotherapy, such as antigens on cell membranes, targets in the disease microenvironment, or immune cells. The most common immunoPET applications use a monoclonal antibody labeled with a relatively long-lived positron emitter such as 89Zr (T 1/2 = 78.4 h), but smaller antibody-based constructs labeled with various other positron emitting radionuclides are also being investigated. This molecular imaging technique can thus guide the development of new drugs and may have a pivotal role in selecting patients for a particular therapy. In early phase immunoPET trials, multiple imaging time points are used to examine the time-dependent biodistribution and to determine the optimal imaging time point, which may be several days after tracer injection due to the slow kinetics of larger molecules. Once this has been established, usually only one static scan is performed and semi-quantitative values are reported. However, total PET uptake of a tracer is the sum of specific and nonspecific uptake. In addition, uptake may be affected by other factors such as perfusion, pre-/co-administration of the unlabeled molecule, and the treatment schedule. This article reviews imaging methodologies used in immunoPET studies and is divided into two parts. The first part summarizes the vast majority of clinical immunoPET studies applying semi-quantitative methodologies. The second part focuses on a handful of studies applying pharmacokinetic models and includes preclinical and simulation studies. Finally, the potential and challenges of immunoPET quantification methodologies are discussed within the context of the recent technological advancements provided by long axial field of view PET/CT scanners.

Programming temporal stiffness cues within extracellular matrix hydrogels for modelling cancer niches

Extracellular matrix (ECM) stiffening is a common occurrence during the progression of many diseases, such as breast cancer. To accurately mimic the pathophysiological context of disease within 3D in vitro models, there is high demand for smart biomaterials which replicate the dynamic and temporal mechanical cues of diseased states. This study describes a preclinical disease model, using breast cancer as an example, which replicates the dynamic plasticity of the tumour microenvironment by incorporating temporal (3-week progression) biomechanical cues within a tissue-specific hydrogel microenvironment. The composite hydrogel formulation, integrating adipose-derived decellularised ECM (AdECM) and silk fibroin, was initially crosslinked using a visible light-mediated system, and then progressively stiffened through spontaneous secondary structure interactions inherent between the polymer chains (∼10–15 kPa increase, with a final stiffness of 25 kPa). When encapsulated and cultured in vitro, MCF-7 breast cancer cells initially formed numerous, large spheroids (>1000 μm2 in area), however, with progressive temporal stiffening, cells demonstrated growth arrest and underwent phenotypic changes resulting in intratumoral heterogeneity. Unlike widely-investigated static mechanical models, this stiffening hydrogel allowed for progressive phenotypic changes to be observed, and fostered the development of mature organoid-like spheroids, which mimicked both the organisation and acinar-structures of mature breast epithelium. The spheroids contained a central population of cells which expressed aggressive cellular programs, evidenced by increased fibronectin expression and reduction of E-cadherin. The phenotypic heterogeneity observed using this model is more reflective of physiological tumours, demonstrating the importance of establishing temporal cues within preclinical models in future work. Overall, the developed model demonstrated a novel strategy to uncouple ECM biomechanical properties from the cellular complexities of the disease microenvironment and offers the potential for wide applicability in other 3D in vitro disease models through addition of tissue-specific dECM materials.

ZNF787 and HDAC1 Mediate Blood-Brain Barrier Permeability in an In Vitro Model of Alzheimer's Disease Microenvironment.

The permeability of the blood-brain barrier (BBB) is increased in Alzheimer's disease (AD). This plays a key role in the instigation and maintenance of chronic inflammation during AD. Experiments using AD models showed that the increased permeability of the BBB was mainly caused by the decreased expression of tight junction-related proteins occludin and claudin-5. In this study, we found that ZNF787 and HDAC1 were upregulated in β-amyloid (Aβ)1-42-incubated endothelial cells, resulting in increased BBB permeability. Conversely, the silencing of ZNF787 and HDAC1 by RNAi led to reduced BBB permeability. The silencing of ZNF787 and HDAC1 enhanced the expression of occludin and claudin-5. Mechanistically, ZNF787 binds to promoter regions for occludin and claudin-5 and functions as a transcriptional regulator. Furthermore, we demonstrate that ZNF787 interacts with HDAC1, and this resulted in the downregulation of the expression of genes encoding tight junction-related proteins to increase in BBB permeability. Taken together, our study identifies critical roles for the interaction between ZNF787 and HDAC1 in regulating BBB permeability and the pathogenesis of AD.

P128 Salt-Inducible Kinase 2 is a promising therapeutic target in patients with ulcerative colitis

Abstract Background Salt-Inducible Kinases (SIKs) are a family of 3 serine/threonine kinases which represent promising therapeutic targets in a number of inflammatory diseases including ulcerative colitis (UC). Their catalytic activity is highly phosphoregulated in response to changes in cellular homeostasis. Inactivation of SIKs results in proinflammatory cytokine downregulation and the concomitant upregulation of anti-inflammatory IL-10. Of the three isozymes, SIK2 is the most active in myeloid cells and is thought to be the key driver of IL-10 production. Selective pharmacological inhibition of SIKs has thus far proved challenging: a number of SIK inhibitors have previously been developed and whilst they have demonstrated biological activity in UC, dosing has been limited due to potential side effects underlining the need for improved selectivity. We hypothesized that more selective inhibition of SIK2 represents a promising approach in ulcerative colitis. Methods We sought to examine the effects of inhibiting SIK2 with a potent small molecule in both in vitro cell cultures and in ex vivo biopsies from UC patients. CD14+ monocytes were isolated from donor PBMCs and differentiated into hMDMs (monocyte-derived macrophages) and moDCs (monocyte-derived DCs). Cultures were treated with compound followed by LPS stimulation for 4 hours (hMDMs) or maturation for 48 hours (moDCs). To recapitulate the disease microenvironment ex vivo, a colonic explant culture model was used: colonic biopsies were collected from patients with active (Mayo 1 /2) and inactive UC and cultured in the presence of compound or control for 24-48h. Supernatants were assayed for cytokine and chemokine release. Cells or biopsies were lysed for qPCR. Surface markers were assessed by flow cytometry Results Using more SIK2-selective small molecules, we show that SIK inhibition modulates inflammatory and immunometabolic pathways in human myeloid cells. Furthermore, SIK2 inhibition drives a tolerogenic phenotype in dendritic cells, as demonstrated by suppression of proinflammatory cytokines and chemokines such as TNFα and the CC/CXC chemokine family as well as downregulation of activation markers such as CD80 and 83; functionally these modulated DCs can suppress Th1 CD4 T cell IFNγ production. Modulation of cytokine and chemokine production by SIK2 inhibition was also observed in colonic explant cultures from UC patients with a reduction in levels of TNFα and the CC chemokine family. Conclusion In both myeloid cellular cultures and UC biopsies, SIK2 inhibition modulates an inflammatory environment into a pro-resolution state supporting the therapeutic potential of SIK2 inhibition in UC.