CCR2 cooperativity promotes hematopoietic stem cell homing to the bone marrow.

Cross-talk between hematopoietic stem and progenitor cells (HSPCs) and bone marrow (BM) cells is critical for homing and sustained engraftment after transplantation. In particular, molecular and physical adaptation of sinusoidal endothelial cells (ECs) promote HSPC BM occupancy; however, signals that govern these events are not well understood. Extracellular vesicles (EVs) are mediators of cell-cell communication crucial in shaping tissue microenvironments. Here, we demonstrate that integrin α4ß7 on murine HSPC EVs targets uptake into ECs. In BM ECs, HSPC EVs induce up-regulation of C-C motif chemokine receptor 2 (CCR2) ligands that synergize with CXCL12-CXCR4 signaling to promote BM homing. In nonirradiated murine models, marrow preconditioning with HSPC EVs or recombinant CCR2 ligands improves homing and early graft occupancy after transplantation. These findings identify a role for HSPC EVs in remodeling ECs, newly define CCR2-dependent graft homing, and inform novel translational conditioning strategies to improve HSPC transplantation.

CXCL12 Gene Polymorphisms and Serum Levels: Associations with Multiple Sclerosis Prevalence and Clinical Parameters in Lithuania.

Our study aimed to investigate the associations between CXCL12 rs1029153, rs1801157, and rs2297630 single-nucleotide polymorphisms (SNPs), CXCL12 protein levels, MS prevalence, and clinical parameters. This study included 250 individuals diagnosed with MS and 250 sex- and age-matched healthy control individuals from Lithuania. The SNPs were genotyped with real-time PCR-based assays. The CXCL12 protein concentration was evaluated in serum using the ELISA method. Of the studied CXCL12 SNPs, we found that the rs1801157 CT genotype in the males was associated with 2.3 times reduced MS odds when compared with the CC genotype according to the overdominant and codominant models (p = 0.011 and p = 0.012, respectively). There was a tendency, which did not reach adjusted statistical significance, for a lower CXCL12 protein concentration in the healthy individuals with the rs1801157 CT genotype (p = 0.028). Sensory symptoms were rarer in the women with the rs1801157 TT genotype (p = 0.004); however, this genotype was also associated with a shorter MS disease duration (p = 0.007). CXCL12 rs1801157 was associated with reduced odds of MS occurrence in the male individuals. In women, rs1801157 was associated with a sensory symptom prevalence.

Recruitment of CXCR4+ type 1 innate lymphoid cells distinguishes sarcoidosis from other skin granulomatous diseases.

Sarcoidosis is a multiorgan granulomatous disease that lacks diagnostic biomarkers and targeted treatments. Using blood and skin from patients with sarcoid and non-sarcoid skin granulomas, we discovered that skin granulomas from different diseases exhibit unique immune cell recruitment and molecular signatures. Sarcoid skin granulomas were specifically enriched for type 1 innate lymphoid cells (ILC1s) and B cells and exhibited molecular programs associated with formation of mature tertiary lymphoid structures (TLSs), including increased CXCL12/CXCR4 signaling. Lung sarcoidosis granulomas also displayed similar immune cell recruitment. Thus, granuloma formation was not a generic molecular response. In addition to tissue-specific effects, patients with sarcoidosis exhibited an 8-fold increase in circulating ILC1s, which correlated with treatment status. Multiple immune cell types induced CXCL12/CXCR4 signaling in sarcoidosis, including Th1 T cells, macrophages, and ILCs. Mechanistically, CXCR4 inhibition reduced sarcoidosis-activated immune cell migration, and targeting CXCR4 or total ILCs attenuated granuloma formation in a noninfectious mouse model. Taken together, our results show that ILC1s are a tissue and circulating biomarker that distinguishes sarcoidosis from other skin granulomatous diseases. Repurposing existing CXCR4 inhibitors may offer a new targeted treatment for this devastating disease.

Heterogeneous expression of the atypical chemokine receptor ACKR3 in glioblastoma patient-derived tissue samples and cell cultures.

Glioblastoma (GBM) is the most aggressive glial tumor of the adult brain, associated with invariably fatal outcome, and a deeper understanding of the underlying malignant mechanisms is necessary to address the current therapeutic failure. We previously demonstrated the role of the CXCL12/CXCR4 axis in GBM cell migration and resistance to ionizing radiation. The atypical chemokine receptor ACKR3, responsible for CXCL12 scavenging, was previously suggested as additional important player in the context of GBM. Following validation of the detection tools, we observed that ACKR3 is expressed within GBM patient tumor tissue, distributed in diverse cell types. In contrast to CXCR4, ACKR3 expression in patient-derived stem-like cells (GSCs) remains however low, while ACKR3 gene expression by tumor cells appears to be modulated by the in-vivo environment. Using overexpression models, we also showed that in vitro ACKR3 had no significant direct effect on cell proliferation or invasion. Altogether, these results suggest that in vitro ACKR3 plays a minor role in malignant GBM cell biology and that its expression is possibly regulated by in-vivo influences. The subtle and multifaceted functions ACKR3 could exert in GBM should therefore only be tackled within a comprehensive tumor microenvironment considering tumoral but also non-tumoral cells.

Allosteric modulation of the CXCR4:CXCL12 axis by targeting receptor nanoclustering via the TMV-TMVI domain.

CXCR4 is a ubiquitously expressed chemokine receptor that regulates leukocyte trafficking and arrest in both homeostatic and pathological states. It also participates in organogenesis, HIV-1 infection, and tumor development. Despite the potential therapeutic benefit of CXCR4 antagonists, only one, plerixafor (AMD3100), which blocks the ligand-binding site, has reached the clinic. Recent advances in imaging and biophysical techniques have provided a richer understanding of the membrane organization and dynamics of this receptor. Activation of CXCR4 by CXCL12 reduces the number of CXCR4 monomers/dimers at the cell membrane and increases the formation of large nanoclusters, which are largely immobile and are required for correct cell orientation to chemoattractant gradients. Mechanistically, CXCR4 activation involves a structural motif defined by residues in TMV and TMVI. Using this structural motif as a template, we performed in silico molecular modeling followed by in vitro screening of a small compound library to identify negative allosteric modulators of CXCR4 that do not affect CXCL12 binding. We identified AGR1.137, a small molecule that abolishes CXCL12-mediated receptor nanoclustering and dynamics and blocks the ability of cells to sense CXCL12 gradients both in vitro and in vivo while preserving ligand binding and receptor internalization.

M2 macrophage-derived exosomes enable osteogenic differentiation and inhibit inflammation in human periodontal ligament stem cells through promotion of CXCL12 expression.

BACKGROUND: Periodontitis is a dental disease characterized by inflammation of periodontal tissues and loss of the periodontal ligaments and alveolar bone. Exosomes are a class of extracellular vesicles that are involved in a variety of diseases by releasing active substances. In this study, we aimed to investigate the effect and mechanism of exosomes from M2 polarized macrophages (M2-exos) on osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs). METHODS: M2-exos were isolated from IL-4-induced RAW264.7 cells (M2 macrophages) and then treated on hPDLSCs. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) staining, alizarin red S (ARS) staining, measurement of osteogenic differentiation-related genes and proteins, and inflammation was evaluated by measuring the levels of inflammatory factors. The mechanism of M2-exo was confirmed through qPCR, western blot, ALP and ARS staining. RESULTS: Results suggested that M2-exo improved osteogenic differentiation and inhibited inflammation in LPS-induced hPDLSCs. CXCL12 expression was elevated in M2 macrophages, but decreased in LPS-induced hPDLSCs. Moreover, the effect of M2-exo on osteogenic differentiation and inflammation in LPS-induced hPDLSCs was reversed by CXCL12 knockdown. CONCLUSION: We demonstrated that M2-exo facilitated osteogenic differentiation and suppressed inflammation in LPS-induced hPDLSCs through promotion of CXCL12 expression. These results suggested the potential of M2-exo in the treatment of periodontitis, which may provide a new theoretical basis for M2-exo treatment of periodontitis.

Type 1 innate lymphoid cells: a biomarker and therapeutic candidate in sarcoidosis.

Sarcoidosis is an inflammatory disease characterized by immune cell-rich granulomas that form in multiple organs. In this issue of the JCI, Sati and colleagues used scRNA-seq and spatial transcriptomics of skin samples from patients with sarcoidosis and non-sarcoidosis granulomatous disease to identify upregulation of a stromal-immune CXCL12/CXCR4 axis and accumulation of type 1 innate lymphoid cells (ILC1s) in sarcoidosis. The accumulation of ILC1s in skin and blood was specific to patients with sarcoidosis and not observed in other granulomatous diseases. The authors used a mouse model of lung granuloma to show that ILCs contribute to granuloma formation and that blockade of CXCR4 reduced the formation of granulomas, providing a proof of concept that sarcoidosis may be treated by CXCR4 blockade to prevent the progression of disease in patients. These results suggest ILC1s could serve as a diagnostic biomarker in sarcoidosis and a potential therapeutic target.

Investigational CXCR4 inhibitors in early phase development for the treatment of hematological malignancies.

INTRODUCTION: CXCR4/CXCL12 axis regulates cell proliferation, survival, and differentiation, as well as the homing and mobilization of hematopoietic stem cells (HSCs) from bone marrow niches to the peripheral blood. Furthermore, CXCR4 and CXCL12 are key mediators of cross-talk between hematological malignancies and their microenvironments. CXCR4 overexpression drives disease progression, boosts tumor cell survival, and promotes chemoresistance, leading to poor prognosis. AREAS COVERED: In light of these discoveries, scientific investigations, and clinical trials have underscored the therapeutic promise found in small-molecule antagonists like plerixafor, peptides/peptidomimetics, such as BKT140, monoclonal antibodies like PF-06747143 and ulocuplumab, as well as microRNAs. Their efficacy is evident in reducing tumor burden, inducing apoptosis and sensitizing malignant cells to conventional chemotherapies. This overview delves into the pathogenic role of the CXC4/CXCL12 axis in hematological neoplasms and examines the clinical application of key CXCR4 antagonists. EXPERT OPINION: The information collectively emphasizes the potential of CXCR4 antagonists as a therapeutic strategy for hematologic malignancies, showcasing advancements in preclinical and clinical studies. As these therapeutic strategies progress through clinical trials, their potential to reshape the prognosis of hematologic malignancies will become increasingly apparent.

Multivalent CXCR4-targeting nanobody formats differently affect affinity, receptor clustering, and antagonism.

The chemokine receptor CXCR4 is involved in the development and migration of stem and immune cells but is also implicated in tumor progression and metastasis for a variety of cancers. Antagonizing ligand (CXCL12)-induced CXCR4 signaling is, therefore, of therapeutic interest. Currently, there are two small-molecule CXCR4 antagonists on the market for the mobilization of hematopoietic stem cells. Other molecules with improved potencies and safety profiles are being developed for different indications, including cancer. Moreover, multiple antagonistic nanobodies targeting CXCR4 displayed similar or better potencies as compared to the CXCR4-targeting molecule AMD3100 (Plerixafor), which was further enhanced through avid binding of bivalent derivatives. In this study, we aimed to compare the affinities of various multivalent nanobody formats which might be differently impacted by avidity. By fusion to a flexible GS-linker, Fc-region of human IgG1, different C4bp/CLR multimerization domains, or via site-directed conjugation to a trivalent linker scaffold, we generated different types of multivalent nanobodies with varying valencies ranging from bivalent to decavalent. Of these, C-terminal fusion, especially to human Fc, was most advantageous with a 2-log-fold and 3-log-fold increased potency in inhibiting CXCL12-mediated Gαi- or ß-arrestin recruitment, respectively. Overall, we describe strategies for generating multivalent and high-potency CXCR4 antagonistic nanobodies able to induce receptor clustering and conclude that fusion to an Fc-tail results in the highest avidity effect irrespective of the hinge linker.

Epigenetic regulation of Nrf2-Mediated angiogenesis in diabetic foot ulcer progression: Role of histone deacetylases.

Nuclear factor E2-related factor 2 (Nrf2), a redox-sensitive transcription factor, regulates proangiogenic mediators, and antioxidant and detoxification enzymes. However, hitherto its regulation in the progression of DFU was poorly examined. The regulation of Nrf2 has been reported to be affected by various factors, including histone deacetylase (HDACs) and DNA methylation. The present study aimed to profile all classes of HDACs and correlate them with Nrf2 and angiogenic markers in the tissue biopsies of different grades of DFU patients (n = 20 in each grade). The gene expression profile of Nrf2 and its downstream targets, angiogenic markers, and all classes of HDACs were assessed using qPCR. Spearman's correlation was performed to analyze the correlation of HDACs with Nrf2 and its downstream targets along with angiogenic markers. We observed a progressive decrease in the gene expression of Nrf2 and angiogenic markers such as VEGF, HIF-1α, and SDF-1α and also an increase in the TSP-2 expression in different grades of DFU. In parallel, a significant downregulation of HDAC2/8 and SIRT1/2/4 has been observed in various grades of DFU subjects. On the other hand, HDAC1/3/4/11 and SIRT3/5/6/7 showed upregulation in different grades of DFU and the maximum increase was observed in Grade 3 patients. A significant negative correlation between Nrf2 and HDAC4, angiogenic markers, and HDAC4 suggested the pivotal role of the HDAC4-regulated Nrf2-mediated angiogenesis among DFU subjects. We have generated a first line of evidence on the epigenetic regulation of Nrf2 and its correlation with angiogenesis in the progression of diabetic foot ulcers.

Cancer-Associated-Fibroblast-Mediated Paracrine and Autocrine SDF-1/CXCR4 Signaling Promotes Stemness and Aggressiveness of Colorectal Cancers.

Colorectal cancer (CRC) is a leading cause of cancer mortality worldwide, and cancer-associated fibroblasts (CAFs) play a major role in the tumor microenvironment (TME), which facilitates the progression of CRC. It is critical to understand how CAFs promote the progression of CRC for the development of novel therapeutic approaches. The purpose of this study was to understand how CAF-derived stromal-derived factor-1 (SDF-1) and its interactions with the corresponding C-X-C motif chemokine receptor 4 (CXCR4) promote CRC progression. Our study focused on their roles in promoting tumor cell migration and invasion and their effects on the characteristics of cancer stem cells (CSCs), which ultimately impact patient outcomes. Here, using in vivo approaches and clinical histological samples, we analyzed the influence of secreted SDF-1 on CRC progression, especially in terms of tumor cell behavior and stemness. We demonstrated that CAF-secreted SDF-1 significantly enhanced CRC cell migration and invasion through paracrine signaling. In addition, the overexpression of SDF-1 in CRC cell lines HT29 and HCT-116 triggered these cells to generate autocrine SDF-1 signaling, which further enhanced their CSC characteristics, including those of migration, invasion, and spheroid formation. An immunohistochemical study showed a close relationship between SDF-1 and CXCR4 expression in CRC tissue, and this significantly affected patient outcomes. The administration of AMD3100, an inhibitor of CXCR4, reversed the entire phenomenon. Our results strongly suggest that targeting this signaling axis in CRC is a feasible approach to attenuating tumor progression, and it may, therefore, serve as an alternative treatment method to improve the prognosis of patients with CRC, especially those with advanced, recurrent, or metastatic CRC following standard therapy.

Cryo-EM structure of monomeric CXCL12-bound CXCR4 in the active state.

CXCR4 binding of its endogenous agonist CXCL12 leads to diverse functions, including bone marrow retention of hematopoietic progenitors and cancer metastasis. However, the structure of the CXCL12-bound CXCR4 remains unresolved despite available structures of CXCR4 in complex with antagonists. Here, we present the cryoelectron microscopy (cryo-EM) structure of the CXCL12-CXCR4-Gi complex at an overall resolution of 2.65 Å. CXCL12 forms a 1:1 stoichiometry complex with CXCR4, following the two-site model. The first 8 amino acids of mature CXCL12 are crucial for CXCR4 activation by forming polar interactions with minor sub-pocket residues in the transmembrane binding pocket. The 3.2-Å distance between V3 of CXCL12 and the "toggle switch" W6.48 marks the deepest insertion among all chemokine-receptor pairs, leading to conformational changes of CXCR4 for G protein activation. These results, combined with functional assays and computational analysis, provide the structural basis for CXCR4 activation by CXCL12.

Protection of stromal cell-derived factor-1 SDF-1/CXCL12 against proteases yields improved skin wound healing.

SDF-1/CXCL12 is a unique chemotactic factor with multiple functions on various types of precursor cells, all carrying the cognate receptor CXCR4. Whereas individual biological functions of SDF-1/CXCL12 have been well documented, practical applications in medicine are insufficiently studied. This is explained by the complex multifunctional biology of SDF-1 with systemic and local effects, critical dependence of SDF-1 activity on aminoterminal proteolytic processing and limited knowledge of applicable modulators of its activity. We here present new insights into modulation of SDF-1 activity in vitro and in vivo by a macromolecular compound, chlorite-oxidized oxyamylose (COAM). COAM prevented the proteolytic inactivation of SDF-1 by two inflammation-associated proteases: matrix metalloproteinase-9/MMP-9 and dipeptidylpeptidase IV/DPPIV/CD26. The inhibition of proteolytic inactivation was functionally measured by receptor-mediated effects, including intracellular calcium mobilization, ERK1/2 phosphorylation, receptor internalization and chemotaxis of CXCR4-positive cells. Protection of SDF-1/CXCL12 against proteolysis was dependent on electrostatic COAM-SDF-1 interactions. By in vivo experiments in mice, we showed that the combination of COAM with SDF-1 delivered through physiological fibrin hydrogel had beneficial effect for the healing of skin wounds. Collectively, we show that COAM protects SDF-1 from proteolytic inactivation, maintaining SDF-1 biological activities. Thus, protection from proteolysis by COAM represents a therapeutic strategy to prolong SDF-1 bioavailability for wound healing applications.

Dipeptidyl Peptidase (DPP)-4 Inhibitors and Pituitary Adenylate Cyclase-Activating Polypeptide, a DPP-4 Substrate, Extend Neurite Outgrowth of Mouse Dorsal Root Ganglia Neurons: A Promising Approach in Diabetic Polyneuropathy Treatment.

Individuals suffering from diabetic polyneuropathy (DPN) experience debilitating symptoms such as pain, paranesthesia, and sensory disturbances, prompting a quest for effective treatments. Dipeptidyl-peptidase (DPP)-4 inhibitors, recognized for their potential in ameliorating DPN, have sparked interest, yet the precise mechanism underlying their neurotrophic impact on the peripheral nerve system (PNS) remains elusive. Our study delves into the neurotrophic effects of DPP-4 inhibitors, including Diprotin A, linagliptin, and sitagliptin, alongside pituitary adenylate cyclase-activating polypeptide (PACAP), Neuropeptide Y (NPY), and Stromal cell-derived factor (SDF)-1a-known DPP-4 substrates with neurotrophic properties. Utilizing primary culture dorsal root ganglia (DRG) neurons, we meticulously evaluated neurite outgrowth in response to these agents. Remarkably, all DPP-4 inhibitors and PACAP demonstrated a significant elongation of neurite length in DRG neurons (PACAP 0.1 µM: 2221 ± 466 µm, control: 1379 ± 420, p < 0.0001), underscoring their potential in nerve regeneration. Conversely, NPY and SDF-1a failed to induce neurite elongation, accentuating the unique neurotrophic properties of DPP-4 inhibition and PACAP. Our findings suggest that the upregulation of PACAP, facilitated by DPP-4 inhibition, plays a pivotal role in promoting neurite elongation within the PNS, presenting a promising avenue for the development of novel DPN therapies with enhanced neurodegenerative capabilities.

Adipose-derived mesenchymal stem cells suppress fibroblast proliferation of hypertrophic scar through CCL5 and CXCL12.

BACKGROUND AND OBJECTIVE: Adipose-derived mesenchymal stem cells (ADSCs) can accelerate wound healing, reduce scar formation, and inhibit hypertrophic scar (HTS). ADSCs can secrete a large amount of CCL5, and CCL5 has been proved to be pro-inflammatory and pro-fibrotic. CXCL12 (SDF-1) is a key chemokine that promotes stem cell migration and survival. Therefore, this study selected normal skin and HTS conditioned medium to simulate different microenvironments, and analyzed the effects of different microenvironments on the expression of CCL5 and CXCL12 in human ADSCs (hADSCs). MATERIALS AND METHODS: hADSCs with silenced expression of CCL5 and CXCL12 were co-cultured with hypertrophic scar fibroblasts to verify the effects of CCL5 and CXCL12 in hADSCs on the proliferation ability of hypertrophic scar fibroblasts. A mouse model of hypertrophic scar was established to further confirm the effect of CCL5 and CXCL12 in hADSCs on hypertrophic scar formation. RESULTS: CCL5 level was found to be significantly high in hADSCs cultured in HTS conditioned medium. CXCL12 in HTS group was prominently lowly expressed compared with the normal group. Inhibition of CCL5 in hADSCs enhanced the effects of untreated hADSCs on proliferation of HTS fibroblasts while CXCL12 knockdown exerted the opposite function. Inhibition of CCL5 in hADSCs increased the percentage of HTS fibroblasts in the G0/G1 phase while down-regulation of CXCL12 decreased those. Meanwhile, the down-regulated levels of fibroblast markers including collagen I, collagen III, and α-SMA induced by CCL5 knockdown were significantly up-regulated by CXCL12 inhibition. hADSCs alleviate the HTS of mice through CCL5 and CXCL12. CONCLUSION: In summary, our results demonstrated that hADSCs efficiently cured HTS by suppressing proliferation of HTS fibroblasts, which may be related to the inhibition of CXCL12 and elevation of CCL5 in hADSCs, suggesting that hADSCs may provide an alternative therapeutic approach for the treatment of HTS.

Association of SDF-1-3' Gene A Variant with Diabetic Retinopathy in the Hungarian Population.

We investigated the association between the SDF-1-3' (c801G > A) variant and the development of diabetic macular edema (DME) or proliferative diabetic retinopathy (PDR) in a Hungarian cohort. SDF-1-3' (c801G > A) was genotyped in 103 patients with diabetic retinopathy and 31 age- and sex-matched non-diabetic controls. Central retinal and choroidal thickness was measured by swept-source optical coherence tomography. The distribution of heterozygous and homozygous SDF-1-3' (c801G > A) genotypes was similar in diabetic and control subjects. The SDF-3'(c801AA) genotype was associated with DME (n = 94 eyes, allele distribution p = 0.006, genotype distribution p = 0.01 OR: 2.48, 95% CL: 1.21-5.08) in both univariable and multivariable modelling, independent of duration and type of diabetes, HbA1C, hypertension and microalbuminuria (p = 0.03). DME occurred earlier in patients carrying the SDF-1 (c801A) allele (Kaplan-Meier analysis, log-rank test p = 0.02). A marginally significant association was found between the presence of the SDF-1 (c801A) allele and the development of PDR (n = 89 eyes, p = 0.06). The SDF-1-3' (c801A) allele also showed a correlation with central retinal (p = 0.006) and choroidal (p = 0.08) thickness. SDF-1-3' (c801G > A) is involved in the development of macular complications in DM independent of critical clinical factors, suggesting that SDF-1 may be a future therapeutic target for high-risk patients, especially those carrying the SDF-1 (c801A) allele.

A chondroitin sulphate hydrogel with sustained release of SDF-1α for extensive cartilage defect repair through induction of cell homing and promotion of chondrogenesis.

Articular cartilage damage represents a prevalent clinical disease in orthopedics, with its regeneration and repair constituting a central focus in ongoing research endeavors. While hydrogel technology has achieved notable progress in the field of cartilage regeneration, addressing the repair of larger cartilage defects remains a significant and formidable challenge. In pursuit of achieving the repair of extensive cartilage defects, this study designed a polydopamine-modified chondroitin sulfate hydrogel loaded with SDF-1α (P-SCMA). This hydrogel, capable of directly providing glycosaminoglycans (GAGs), served as a platform for carrying growth factors and attracting mesenchymal stem cells for the in situ reconstruction of extensive cartilage defects. The results indicate that the P-SCMA hydrogel is capable of not only directly providing GAGs but also sustainably releasing SDF-1α. In the early stages, it promotes cell adhesion and proliferation and induces cell homing, while in the later stages, it further induces chondrogenesis by inhibiting the Wnt/ß-catenin pathway. This bioactive hydrogel, which possesses the functions of providing GAGs, promoting cell proliferation, inducing cell homing and chondrogenesis, is capable of promoting cartilage repair in multiple ways, providing new perspectives for the repair of extensive cartilage defects.

Senescent endothelial cells promote liver metastasis of uveal melanoma in single-cell resolution.

BACKGROUND: Uveal melanoma (UM), the most common adult intraocular tumor, is characterized by high malignancy and poor prognosis in advanced stages. Angiogenesis is critical for UM development, however, not only the role of vascular endothelial dysfunction in UM remains unknown, but also their analysis at the single-cell level has been lacking. A comprehensive analysis is essential to clarify the role of the endothelium in the development of UM. METHODS: By using single-cell RNA transcriptomics data of 11 cases of primary and liver metastasis UM, we analyzed the endothelial cell status. In addition, we analyzed and validated ECs in the in vitro model and collected clinical specimens. Subsequently, we explored the impact of endothelial dysfunction on UM cell migration and explored the mechanisms responsible for the endothelial cell abnormalities and the reasons for their peripheral effects. RESULTS: UM metastasis has a significantly higher percentage of vascular endothelial cells compared to in situ tumors, and endothelial cells in metastasis show significant senescence. Senescent endothelial cells in metastatic tumors showed significant Krüppel-like factor 4 (KLF4) upregulation, overexpression of KLF4 in normal endothelial cells induced senescence, and knockdown of KLF4 in senescent endothelium inhibited senescence, suggesting that KLF4 is a driver gene for endothelial senescence. KLF4-induced endothelial senescence drove tumor cell migration through a senescence-associated secretory phenotype (SASP), of which the most important component of the effector was CXCL12 (C-X-C motif chemokine ligand 12), and participated in the composition of the immunosuppressive microenvironment. CONCLUSION: This study provides an undesirable insight of senescent endothelial cells in promoting UM metastasis.

CXCR4 has a dual role in improving the efficacy of BCMA-redirected CAR-NK cells in multiple myeloma.

Multiple myeloma (MM) is a plasma cell disease with a preferential bone marrow (BM) tropism. Enforced expression of tissue-specific chemokine receptors has been shown to successfully guide adoptively-transferred CAR NK cells towards the malignant milieu in solid cancers, but also to BM-resident AML and MM. For redirection towards BM-associated chemokine CXCL12, we armored BCMA CAR-NK-92 as well as primary NK cells with ectopic expression of either wildtype CXCR4 or a gain-of-function mutant CXCR4R334X. Our data showed that BCMA CAR-NK-92 and -primary NK cells equipped with CXCR4 gained an improved ability to migrate towards CXCL12 in vitro. Beyond its classical role coordinating chemotaxis, CXCR4 has been shown to participate in T cell co-stimulation, which prompted us to examine the functionality of CXCR4-cotransduced BCMA-CAR NK cells. Ectopic CXCR4 expression enhanced the cytotoxic capacity of BCMA CAR-NK cells, as evidenced by the ability to eliminate BCMA-expressing target cell lines and primary MM cells in vitro and through accelerated cytolytic granule release. We show that CXCR4 co-modification prolonged BCMA CAR surface deposition, augmented ZAP-70 recruitment following CAR-engagement, and accelerated distal signal transduction kinetics. BCMA CAR sensitivity towards antigen was enhanced by virtue of an enhanced ZAP-70 recruitment to the immunological synapse, revealing an increased propensity of CARs to become triggered upon CXCR4 overexpression. Unexpectedly, co-stimulation via CXCR4 occurred in the absence of CXCL12 ligand-stimulation. Collectively, our findings imply that co-modification of CAR-NK cells with tissue-relevant chemokine receptors affect adoptive NK cell therapy beyond improved trafficking and retention within tumor sites.

RGS1 and CREB5 are direct and common transcriptional targets of ZNF384-fusion proteins.

BACKGROUND: ZNF384-fusion (Z-fusion) genes were recently identified in B-cell acute lymphoblastic leukemia (B-ALL) and are frequent in Japanese adult patients. The frequency is about 20% in those with Philadelphia chromosome-negative B-ALL. ZNF384 is a transcription factor and Z-fusion proteins have increased transcriptional activity; however, the detailed mechanisms of leukemogenesis of Z-fusion proteins have yet to be clarified. METHODS: We established three transfectants of cell lines expressing different types of Z-fusion proteins, and analyzed their gene expression profile (GEP) by RNA-seq. We also analyzed the GEP of clinical ALL samples using our previous RNA-seq data of 323 Japanese ALL patients. We selected upregulated genes in both Z-fusion gene-expressing transfectants and Z-fusion gene-positive ALL samples, and investigated the binding of Z-fusion proteins to regulatory regions of the candidate genes by ChIP-qPCR. RESULTS: We selected six commonly upregulated genes. After the investigation by ChIP-qPCR, we finally identified CREB5 and RGS1 as direct and common target genes. RGS1 is an inhibitor of CXCL12-CXCR4 signaling that is required for the homing of hematopoietic progenitor cells to the bone marrow microenvironment and development of B cells. Consistent with this, Z-fusion gene transfectants showed impaired migration toward CXCL12. CONCLUSIONS: We identified CREB5 and RGS1 as direct and common transcriptional targets of Z-fusion proteins. The present results provide novel insight into the aberrant transcriptional regulation by Z-fusion proteins.