CXCR4 Axis Research Articles

Lipid-Associated Macrophages Are Induced by Cancer-Associated Fibroblasts and Mediate Immune Suppression in Breast Cancer.

This work identifies a novel lipid-associated macrophage subpopulation with immune suppressive functions, offering new leads for therapeutic interventions in triple-negative breast cancer.

Abstract 1489: MicroRNA-1 targets CXCR4/FOXM1/RRM2 axis regulating small cell lung cancer growth and metastasis

Abstract Small cell lung cancer (SCLC) is a high-grade neuroendocrine metastatic lung cancer subtype having a universal relapse and poor prognosis. Limited or extensive-stage SCLC patients have limited therapeutic options due to a lack of potential drug targets. In the quest for novel therapeutic molecules, we performed micro-RNA sequencing from the serum samples and also analyzed bulk RNA-sequencing data from tumors of SCLC patients. We found a consistent downregulation of miR-1 in SCLC. These results were recapitulated in SCLC cell lines and tumor tissues compared to their matched normal. To assess the therapeutic potential of miR-1, we overexpressed miR-1 in SCLC cell lines that translated into decreased cell growth and oncogenic signaling. Mechanistic studies revealed that CXCR4 is a direct target for miR-1 in SCLC. Intracardiac injection of SCLC cell lines in the mouse models showed that overexpression of miR-1 decreases the distant organ metastasis, whereas miR-1 sponging potentiates aggressiveness and metastasis. Furthermore, we identified FOXM1-RRM2 as a unique downstream target of the miR-1/CXCR4 axis involved in the growth and metastasis of SCLC. Our results showed that FOXM1 directly binds to the promoter site of RRM2 and regulates its activity. Overexpression of miR-1 decreases the expression and activity of FOXM1-RRM2 through CXCR4 that reduces cell growth and metastasis. Taken together, our results suggest that miR-1 decreases SCLC metastasis by targeting the CXCR4/FOXM1-RRM2 axis and has a high potential for the development of novel SCLC therapies. Citation Format: Parvez Khan, Jawed A. Siddiqui, Shailendra Kumar Maurya, Tamara Mirzapoiazova, Ranjana Kanchan, Ramakanth Chirravuri Venkata, Pranita Atri, Wemin Tang, NaveenKumar Perumal, Prakash Kshirsagar, Mahek Fatima, Md Arafat Khan, Sanjib Chaudhary, Asad Ur Rehman, Imayavaramban Lakshmanan, Sidharth Mahapatra, Prakash Kulkarni, Apar Kishor Ganti, Maneesh Jain, David Oupicky, Ravi Salgia, Surinder Kumar Batra, Mohd Wasim Nasser. MicroRNA-1 targets CXCR4/FOXM1/RRM2 axis regulating small cell lung cancer growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1489.

METTL3 Inhibits Antitumor Immunity by Targeting m6A-BHLHE41-CXCL1/CXCR2 Axis to Promote Colorectal Cancer

N6-Methyladenosine (m6A) is the most prevalent RNA modification and recognized as an important epitranscriptomic mechanism in colorectal cancer (CRC). We aimed to exploit whether and how tumor-intrinsic m6A modification driven by methyltransferase like 3 (METTL3) can dictate the immune landscape of CRC. Mettl3 knockout mice, CD34+ humanized mice, and different syngeneic mice models were used. Immune cell composition and cytokine level were analyzed by flow cytometry and Cytokine 23-Plex immunoassay, respectively. M6A sequencing and RNA sequencing were performed to identify downstream targets and pathways of METTL3. Human CRC specimens (n= 176) were used to evaluate correlation between METTL3 expression and myeloid-derived suppressor cell (MDSC) infiltration. We demonstrated that silencing of METTL3 in CRC cells reduced MDSC accumulation to sustain activation and proliferation of CD4+ and CD8+ T cells, and eventually suppressed CRC in ApcMin/+Mettl3+/- mice, CD34+ humanized mice, and syngeneic mice models. Mechanistically, METTL3 activated the m6A-BHLHE41-CXCL1 axis by analysis of m6A sequencing, RNA sequencing, and cytokine arrays. METTL3 promoted BHLHE41 expression in an m6A-dependent manner, which subsequently induced CXCL1 transcription to enhance MDSC migration invitro. However, the effect was negligible on BHLHE41 depletion, CXCL1 protein or CXCR2 inhibitor SB265610 administration, inferring that METTL3 promotes MDSC migration via BHLHE41-CXCL1/CXCR2. Consistently, depletion of MDSCs by anti-Gr1 antibody or SB265610 blocked the tumor-promoting effect of METTL3 invivo. Importantly, targeting METTL3 by METTL3-single guide RNA or specific inhibitor potentiated the effect of anti-programmed cell death protein 1 (anti-PD1) treatment. Our study identifies METTL3 as a potential therapeutic target for CRC immunotherapy whose inhibition reverses immune suppression through the m6A-BHLHE41-CXCL1 axis. METTL3 inhibition plus anti-PD1 treatment shows promising antitumor efficacy against CRC.

STOPTRAFFIC-1: A phase I/II trial of SX-682 in combination with nivolumab for refractory RAS-mutated microsatellite stable (MSS) metastatic colorectal cancer (mCRC).

TPS3638 Background: Refractory RAS mutated MSS mCRC represents a critical unmet need with minimal response to immune checkpoint blockade (ICB). Preclinical CRC models reveal that KRAS mutations activate the CXCR2 axis promoting an immunosuppressive tumor microenvironment (TME). This occurs via KRAS repression of interferon regulatory factor 2 (IRF2) resulting in upregulation of CXCL3 chemokines, which bind CXCR2 and recruit myeloid-derived suppressor cells (MDSC; Liao et al, Cancer Cell 2019; Grover et al, Cancer Discovery, 2022). SX-682 is a novel oral small-molecule inhibitor of the CXCR1/2 chemokine receptors involved in MDSC-recruitment to the TME. This proof-of-concept study investigates whether de novo immunotherapy resistance in MSS RAS mutated mCRC can be overcome by treatment with a small molecule CXCR1/2 antagonist in combination with anti-PD-1 therapy. Methods: STOPTRAFFIC-1 (NCT04599140, enrolling) is a phase I/II, open-label, dose-escalation/dose expansion study of SX-682 in combination with nivolumab to evaluate safety and clinical activity for patients (pts) with refractory RAS ( KRAS and NRAS) mutated MSS mCRC. Key eligibility criteria: patients (pts) with MSS mCRC with measurable disease, progression or intolerance to at least 2 prior lines of standard therapy, and ECOG 0 or 1. Pts will receive SX-682 (5 dose levels: 25 mg, 50 mg, 100mg, 200mg, or 400 mg by mouth twice daily) administered in an 8-week cycle with intravenous nivolumab (480 mg) on days 1 and 29 of each cycle. Adverse events are assessed according to CTCAE v5.0. Response assessments (per RECIST) occur every 8 weeks. In dose escalation, pts enter a 3-week monotherapy safety run-in of SX-682 followed by 3-week combination with nivolumab for a six-week dose-limiting toxicity (DLT) period. Cohorts 1-4 have been completed without DLT. Enrollment to cohort 5 began in January 2022. Following determination of maximum tolerated dose (MTD), dose expansion design is a Simon's optimal two-stage design. Efficacy will be assessed in the first 15 pts with a requirement of at least 2 responses in order to enroll 14 additional pts (N=29) in the second stage. Pre- and on-treatment tissue biopsies will be collected in the expansion phase. The primary objectives are to determine the safety profile of SX-682 alone and in combination with nivolumab, including the MTD, recommended phase 2 dose, and the DLT. The secondary objectives include overall response rate, progression-free survival, overall survival, and pharmacokinetic profiles of SX-682. Translational analyses include correlations of clinical outcomes with genomic and immune biomarkers from paired tissue and plasma samples. Clinical trial information: NCT04599140.

Magnetic Nano-Sized SDF-1 Particles Show Promise for Application in Stem Cell-Based Repair of Damaged Tissues.

Stem cell-based therapy is a promising option for repair of injured tissue. Stem cells have homing characteristics and can be mobilized to the injury sites following activation, under the regulation of the SDF-1/CXCR4 axis. However, a sufficient level of stem cell aggregation and retention is essential for ensuring favorable repair outcomes. Problems related to stem cell delivery/recruitment efficiency and retention in the injury site are among the main challenges faced during in vivo studies on stem cell therapy. In this study, we designed an SDF-1(alpha) magnetic nanoparticle delivery system for stem cell recruitment. We expressed and purified a biotin-labeled SDF-1(alpha) protein and immobilized it on streptavidin-modified magnetic nanoparticles (MNP) through the streptavidin–biotin linkage, with an efficiency of approximately 14%. The physicochemical properties of the SDF-MNP in glycerol buffer were similar to those of the streptavidin-modified MNP. Further evidence suggested that SDF-MNP barely show cytotoxicity even at a concentration of 125 µg/ml MNP and have a promising chemotaxis effect on mesenchymal stem cells in vitro and in vivo. Our study provides a strategy for the assembly of magnetic nanoparticle carrier systems for protein factors, as well as preliminary evidence for the application of SDF-MNP in stem cell-based therapy for the regeneration of injured bone tissue.

Effects of local vs systemic administration of CXCR4 inhibitor AMD3100 on orthodontic tooth movement in rats

Chemokines play pivotal roles in orthodontic tooth movement (OTM) through osteoclast-mediated bone resorption, but the underlying mechanism remains unclear. We aimed to elucidate the effects of serial local vs systemic administration of the chemokine receptor CXCR4 antagonist AMD3100 on OTM. The maxillary first molar (M1) of rats was moved mesially using a 10 g of force nickel-titanium coil spring. The injections were performed every other day with phosphate-buffered saline as a control, whereas local and systemic animals were injected with AMD3100 at the buccal palatal mucosa adjacent to M1 and subcutaneously, respectively. OTM distance and alveolar bone were examined by microcomputed tomography and histologic analysis. Osteoclast numbers were quantified using TRAP staining. Cathepsin K and stromal cell-derived factor-1 (SDF-1) were evaluated using immunohistochemistry. Reverse transcriptase polymerase chain reaction for cathepsin K, Runx2, SDF-1, CXCR4, RANKL, and OPG were also examined. OTM and osteoclast numbers were significantly decreased in the local and systemic groups compared with the control group, whereas there was no significant difference among the experimental groups. Local administration inhibited molar but not incisor movement. Trabecular thickness and trabecular spacing of the alveolar bone significantly increased, and trabecular number significantly decreased in the systemic group compared with the control group, whereas local injection also affected bone quality in the same tendency as a systemic injection. AMD3100 significantly downregulated the mRNA expression levels of cathepsin K, Runx2, SDF-1, RANKL, and RANKL/OPG ratio in both experimental groups. Local administration of AMD3100 can control initial OTM and diminish bone resorption processes during OTM via inhibition of the SDF-1/CXCR4 axis, similar to the systemic administration.

Important role of the SDF-1/CXCR4 axis in the homing of systemically transplanted human amnion-derived mesenchymal stem cells (hAD-MSCs) to ovaries in rats with chemotherapy-induced premature ovarian insufficiency (POI)

BackgroundChemotherapy can induce premature ovarian insufficiency (POI). POI causes multiple sequelae and is currently incurable. As shown in our previous studies, systemically transplanted human amnion-derived mesenchymal stem cells (hAD-MSCs) home to ovaries with chemotherapy-induced POI and subsequently reduce ovarian injury and improve ovarian function in rats with POI. However, the cellular mechanisms that direct the migration and homing of hAD-MSCs to ovaries with chemotherapy-induced POI are incompletely understood. This study investigated the role of the SDF-1/CXCR4 axis in the migration and homing of systemically transplanted hAD-MSCs to ovaries with chemotherapy-induced POI and its relevant downstream signalling pathways.MethodsCXCR4 expression in hAD-MSCs was assessed using Western blotting and immunofluorescence staining. hAD-MSC migration was tested using Transwell migration assays. SDF-1 levels were detected using ELISA. Seventy-two female SD rats were randomly divided into the control, POI, hAD-MSCs and hAD-MSCs + AMD3100 groups. Cyclophosphamide was used to establish rat POI models. For inhibitor treatment, hAD-MSCs were pretreated with AMD3100 before transplantation. PKH26-labeled hAD-MSCs were injected into the tail vein of POI rats 24 h after chemotherapy. After hAD-MSC transplantation, the homing of hAD-MSCs to ovaries and ovarian function and pathological changes were examined. We further investigated the molecular mechanisms by detecting the PI3K/Akt and ERK1/2 signalling pathways.ResultshAD-MSCs expressed CXCR4. SDF-1 induced hAD-MSC migration in vitro. SDF-1 levels in ovaries and serum were significantly increased in rats with chemotherapy-induced POI, and ovaries with POI induced the homing of hAD-MSCs expressing CXCR4. Blocking the SDF-1/CXCR4 axis with AMD3100 significantly reduced the number of hAD-MSCs homing to ovaries with POI and further reduced their efficacy in POI treatment. The binding of SDF-1 to CXCR4 activated the PI3K/Akt signalling pathway, and LY294002 significantly inhibited hAD-MSC migration induced by SDF-1 in vitro. Moreover, inhibition of the PI3K/Akt signalling pathway significantly reduced the number of systemically transplanted hAD-MSCs homing to chemotherapy-induced ovaries in rats with POI.ConclusionsSDF-1/CXCR4 axis partially mediates the migration and homing of systemically transplanted hAD-MSCs to the ovaries of rats with chemotherapy-induced POI, and the PI3K/Akt signalling pathway might be involved in the migration and homing of hAD-MSCs mediated by the SDF-1/CXCR4 axis.

Role of BPIFB4 in Immune System and Cardiovascular Disease: the Lesson from Centenarians

Recent discoveries have shed light on the participation of the immune system in the physio pathology of the cardiovascular system underpinning the importance of keeping the balance of the first to preserve the latter. Aging, along with other risk factors, can challenge such balance triggering the onset of cardiovascular diseases. Among several mediators ensuring the proper cross-talk between the two systems, bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) has been shown to have a pivotal role, also by sustaining important signals such as eNOS and PKC-alpha. In addition, the Longevity-associated variant (LAV), which is an haplotype allele in BPIFB4characterized by 4 missense polymorphisms, enriched in homozygosity in Long Living Individuals (LLIs), has been shown to be efficient, if administered systemically through gene therapy, in improving many aspects of cardiovascular diseases (CVDs). This occurs mainly through a fine immune system remodeling across: 1) a M2 macrophage polarizing effect, 2) a favorable redistribution of the circulating monocyte cell subsets and 3) the reduction of T-cell activation. Furthermore, LAV-BPIFB4 treatment induced a desirable recovery of the inflammatory balance by mitigating the pro-inflammatory factor levels and enhancing the anti-inflammatory boost through a mechanism that is partially dependent on SDF-1/CXCR4 axis. Importantly, the remarkable effects of LAV-BPIFB4 treatment, which translates in increased BPIFB4 circulating levels, mirror what occurs in long-living individuals (LLIs) in whom the high circulating levels of BPIFB4 are protective from age-related and CVDs and emphasize the reason why LLIs are considered a model of successful aging. Here, we review the mechanisms by which LAV-BPIFB4 exerts its immunomodulatory activity in improving the cardiovascular-immune system dialogue that might strengthen its role as a key mediator in CVDs.

Abstract P5-06-08: Triple-negative breast cancer CAFs induce a metastatic phenotype in MCF-7 cells via the SDF-1/CXCR4 axis

Abstract Background: Metastatic disease is the foremost cause of breast cancer (BC) related mortality in women. One of the crucial challenges in reducing metastasis-related mortality is in identifying and understanding why certain BCs metastasize and recur. Although cancer cell-intrinsic factors are a key determinant in BC metastasis, host-intrinsic factors like the cells of the tumor microenvironment (TME), such as cancer-associated fibroblasts (CAFs) are what might be driving certain BCs to metastasize and recur. We recently showed that CAFs from the primary (TME) enter into circulation as cCAFs, form heterotypic clusters with tumor cells, and arrive at metastatic sites. This interaction between CAFs and tumor cells is crucial in furthering the metastatic cascade. However, the functional heterogeneity of the different CAF phenotypes and how that impacts BC metastasis is unknown. Stromal derived factor-1 (SDF-1/CXCL12) is an important chemokine that is known to be involved in promoting tumor cell invasion and metastasis. In this study, we examine the functional differences between CAF isolated from different molecular subtypes of BC. Furthermore, we elucidate the role of CAF secreted SDF-1 as being one of the mechanisms by which a subset of CAF cells permanently reprograms poorly-metastatic MCF-7 cells to augment EMT-driven genes and become metastatic in vivo. Methods: We used primary CAF cell lines derived from BCs of different molecular subtypes and examined their global gene expression profiles using RNAseq. We used NSG mice xenografted with MCF-7 BC cells and primary CAF cells to evaluate the contribution of two different CAF phenotypes in promoting BC progression and metastasis. We developed dissociated tumor cell lines from CAF co-injected MCF-7 xenografts to determine the molecular changes that CAFs impart to MCF-7 cells in vivo. Using limiting dilution tumor xenograft assays and gene expression profiles we examined the molecular pathways, BC stemness markers, and tumor-initiating capacity of the dissociated tumor cell lines. By long-term SDF-1 treatment of MCF-7 cells in vitro and using these cells in various RNASeq, in vitro, and in vivo assays we determined the role of SDF-1 in altering the phenotype of MCF-7 cells. Results: We found that CAFs from different molecular subtypes have a differential effect on tumor initiation, progression, and metastasis in xenograft assays. We also found that a subset of CAFs has the ability to make the poorly-metastatic MCF-7 cells metastatic in vivo. We also found that this subset of CAF cells has a higher expression of SDF-1 and the tumors formed with the co-injection of MCF-7 cells with this subset of CAF cells are enriched in cancer stem cell-like metastasis initiating cells. We also found that upon serial transplant, the tumors formed by CAF-reprogramed MCF-7 cells have a gene expression profile that shows enrichment for EMT-driven genes and these tumors are robustly metastatic. Furthermore, we found that the SDF-1/CXCR4 axis is one of the critical mechanisms by which this subset of CAF cells permanently reprograms the phenotype of the MCF-7 cells. Conclusions: CAFs are a highly heterogeneous population of cells in the breast TME. This study examines the contribution of the functional heterogeneity of the CAF cells in BC metastasis. We have found that a unique subset of CAF cells has the ability to reprogram a poorly metastatic BC cell to not only become metastatic but induce a permanent EMT-driven phenotypic shift in the BC cells. SDF-1 may be one of the mechanisms that drive this CAF-induced change and understanding the CAF functional landscape and its role in BC metastasis might be crucial to developing new therapeutic modalities to abrogate BC metastasis. Citation Format: Utsav Sharma, Jun Sun, Kelsie Medina-Saenz, Susan Bare, Philip Miller, Manuel Picon-Ruiz, Joyce Slingerland, Dorraya El-Ashry, Marc Lippman. Triple-negative breast cancer CAFs induce a metastatic phenotype in MCF-7 cells via the SDF-1/CXCR4 axis [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-08.

Engagement of the CXCL12-CXCR4 Axis in the Interaction of Endothelial Progenitor Cell and Smooth Muscle Cell to Promote Phenotype Control and Guard Vascular Homeostasis.

Endothelial progenitor cells (EPCs) are involved in vascular repair and modulate properties of smooth muscle cells (SMCs) relevant for their contribution to neointima formation following injury. Considering the relevant role of the CXCL12–CXCR4 axis in vascular homeostasis and the potential of EPCs and SMCs to release CXCL12 and express CXCR4, we analyzed the engagement of the CXCL12–CXCR4 axis in various modes of EPC–SMC interaction relevant for injury- and lipid-induced atherosclerosis. We now demonstrate that the expression and release of CXCL12 is synergistically increased in a CXCR4-dependent mechanism following EPC–SMC interaction during co-cultivation or in response to recombinant CXCL12, thus establishing an amplifying feedback loop Additionally, mechanical injury of SMCs induces increased release of CXCL12, resulting in enhanced CXCR4-dependent recruitment of EPCs to SMCs. The CXCL12–CXCR4 axis is crucially engaged in the EPC-triggered augmentation of SMC migration and the attenuation of SMC apoptosis but not in the EPC-mediated increase in SMC proliferation. Compared to EPCs alone, the alliance of EPC–SMC is superior in promoting the CXCR4-dependent proliferation and migration of endothelial cells. When direct cell–cell contact is established, EPCs protect the contractile phenotype of SMCs via CXCL12–CXCR4 and reverse cholesterol-induced transdifferentiation toward a synthetic, macrophage-like phenotype. In conclusion we show that the interaction of EPCs and SMCs unleashes a CXCL12–CXCR4-based autoregulatory feedback loop promoting regenerative processes and mediating SMC phenotype control to potentially guard vascular homeostasis.

CircN4BP2L2 promotes colorectal cancer growth and metastasis through regulation of the miR-340-5p/CXCR4 axis

AbstractColorectal cancer (CRC) is the third leading cause of cancer-related death worldwide. Dysregulation of circular RNAs (circRNAs) appears to be a critical factor in CRC progression. However, mechanistic studies delineating the role of circRNAs in CRC remain limited. In this study, qRT-PCR and western blot assays were used to measure the expression of genes and proteins. Migration, invasion, proliferation, and apoptosis were examined by wound-healing, transwell, CCK-8, colony formation, and flow cytometry assays, respectively. Molecular interactions were validated by a dual-luciferase report system. A xenograft animal model was established to examine in vivo tumor growth and lung metastasis. Our data indicated that circN4BP2L2 expression was increased in CRC tissues and cell lines. Notably, inhibition of circN4BP2L2 effectively inhibited proliferation, migration, and invasion of LoVo cells, and inhibited tumor growth and metastasis in vivo, whereas the forced expression of circN4BP2L2 facilitated the proliferation, migration, and invasion of HT-29 cells. Mechanistic studies revealed that circN4BP2L2 acted as a molecular sponge of miR-340-5p to competitively promote CXCR4 expression. Furthermore, inhibition of miR-340-5p reversed the anti-cancer effects of circN4BP2L2 or CXCR4 silencing. Our data indicated an oncogenic role of circN4BP2L2 in CRC via regulation of the miR-340-5p/CXCR4 axis, which may be a promising biomarker and target for CRC treatment.

Effects of Low-Intensity Pulsed Ultrasound on the Migration and Homing of Human Amnion–Derived Mesenchymal Stem Cells to Ovaries in Rats With Premature Ovarian Insufficiency

Premature ovarian insufficiency (POI) can cause multiple sequelae and is currently incurable. Mesenchymal stem cell (MSC) transplantation might provide an effective treatment method for POI. However, the clinical application of systemic MSC transplantation is limited by the low efficiency of cell homing to target tissue in vivo, including systemic MSC transplantation for POI treatment. Thus, exploration of methods to promote MSC homing is necessary. This study was to investigate the effects of low-intensity pulsed ultrasound (LIPUS) on the migration and homing of transplanted human amnion–derived MSCs (hAD-MSCs) to ovaries in rats with chemotherapy-induced POI. For LIPUS treatment, hAD-MSCs were exposed to LIPUS or sham irradiation. Chemokine receptor expressions in hAD-MSCs were detected by polymerase chain reaction (PCR), Western blot, and immunofluorescence assays. hAD-MSC migration was detected by wound healing and transwell migration assays. Cyclophosphamide-induced POI rat models were established to evaluate the effects of LIPUS on the homing of systemically transplanted hAD-MSCs to chemotherapy-induced POI ovaries in vivo. We found that hAD-MSCs expressed chemokine receptors. The LIPUS promoted the expression of chemokine receptors, especially CXCR4, in hAD-MSCs. SDF-1 induced hAD-MSC migration. The LIPUS promoted hAD-MSC migration induced by SDF-1 through SDF-1/CXCR4 axis. SDF-1 levels significantly increased in ovaries induced by chemotherapy in POI rats. Pretreating hAD-MSCs with LIPUS increased the number of hAD-MSCs homing to ovaries in rats with chemotherapy-induced POI to some extent. However, the difference was not significant. Both hAD-MSC and LIPUS-pretreated hAD-MSC transplantation reduced ovarian injuries and improved ovarian function in rats with chemotherapy-induced POI. CXCR4 antagonist significantly reduced the number of hAD-MSCs- and LIPUS-pretreated hAD-MSCs homing to POI ovaries, and further reduced their efficacy in POI treatment. According to these findings, pretreating MSCs with LIPUS before transplantation might provide a novel, convenient, and safe technique to explore for improving the homing of systemically transplanted MSCs to target tissue.

A new approach based on CXCR4-targeted combination liposomes for the treatment of liver fibrosis.

Liver fibrosis results from excessive extracellular matrix accumulation due to injury and leads to cirrhosis, cancer, and death. Herein, we propose a chemokine receptor 4 (CXCR4)-targeted combination (CTC) liposomal therapy to treat carbon tetrachloride (CCl4)-induced liver fibrosis in a mouse model. This study aims to combine small molecules such as pirfenidone and AMD3100 in a single nanoplatform to investigate their synergistic antifibrotic effects in a setting of CCl4-induced liver fibrosis. CTC liposomes (CTC lipo) were prepared using the thin-film hydration method. CTC lipo exhibited a spherical shape, and the particle size was recorded at the nanoscale which confirms its appropriateness for in vitro and in vivo applications. CTC lipo had good storage and serum stability. The entrapped drugs in CTC lipo showed reduced toxicity at higher concentrations. CTC lipo displayed CXCR4 mediated cell uptake and were internalized by caveolae-mediated endocytosis. CTC lipo showed CXCR4 targeting and stromal cell-derived factor 1α (SDF1-α)/CXCR4 axis blocking activity. CTC lipo reduced the elevated serum aspartate aminotransferase (AST), alanine transaminase (ALT), and hydroxyproline (HYP) levels. The histological studies showed improved liver architecture and reduced collagen deposition after treatment. Transforming growth factor β (TGFβ), alpha-smooth muscle actin (α-SMA), and collagen I were elevated by CCl4 in comparison with the Sham. Upon CTC liposomal treatment, the quantitative score for the elevated fibrotic proteins such as TGFβ, α-SMA, and collagen I was normalized. CTC lipo displayed significant downregulation of the upregulated TGFβ, α-SMA, collagen I, and P-p38 expressions at the molecular level. The CXCR4 targeted liposomes showed prolonged biodistribution at 24 h. Our findings indicated that CTC lipo might be an alternative antifibrotic therapy that may offer new access to research and development. In a nutshell, the present study suggests that systemic administration of CTC lipo has efficient antifibrotic potential and deserves to be investigated for further clinical applications.

Bone marrow mesenchymal stem cells promote the progression of prostate cancer through the SDF-1/CXCR4 axis in vivo and vitro.

The aim of this study was to investigate the involvement of the SDF-1/CXCR4 axis in the process of BMMSC homing in prostate cancer (PCa) in vivo and in vitro. After verification of BMMSCs, we fixed the concentration gradient of SDF-1 for BMMSC cultivation to analyze CXCR4 expression by qRT-PCR and flow cytometric analysis. Furthermore, we developed a non-contact co-culture system and explored the participation of the SDF-1/CXCR4 axis in PCa using qRT-PCR, flow cytometry, and ELISA. In addition, A green fluorescent protein (GFP)-transplanted methylnitrosourea (MNU)-induced PCa mouse model was established to investigate the CXCR4 expression in vivo. The CXCR4 expression was up-regulated with the increase in SDF-1 concentrations, and elevated SDF-1 had a significant promoting effect on cell proliferation and migration in BMMSCs. Moreover, the CXCR4 expression of BMMSCs was significantly increased in the non-contact co-culture model with vascular endothelial cells (VECs), and analysis of this model also showed that the proliferation and migration of BMMSCs were promoted in the presence of VECs. The ELISA assay showed that the SDF-1 levels in the co-culture model at 48h were significantly increased. Twenty of the GFP-transplanted mice were divided into a PCa group and a control group, and four GFP-transplanted mice were observed to have prostate tumorigenesis. It also showed that CXCR4 was obviously increased in the prostate tissue of PCa mice. Our findings suggest thatBMMSCs could home and promote the proliferation and migration of PCa through the SDF-1/CXCR4 axis in vivo and in vitro.

Schwann cell-derived EVs facilitate dental pulp regeneration through endogenous stem cell recruitment via SDF-1/CXCR4 axis

The dental pulp is critical for physiological vitality of the tooth, and dental pulp regeneration has great potential for rebuilding live pulp tissue after pulp disease. Schwann cells (SCs) play a critical role in the support, maintenance, and regeneration of nerve fibers in dental pulp. Extracellular vesicles (EVs), which possess cell homing and tissue repair potential, derived from SCs (SC-EVs), can regulate dental mesenchymal stem cells (MSCs) proliferation, multipotency, and self-renewal. However, the role of SC-EVs in dental pulp tissue regeneration remains unclear. To address this question, we treated dental pulp stem cells (DPSCs) and bone marrow stem cells (BMSCs) with SC-EVs, and the results showed an obvious increase in the proliferation, migration, and osteogenic differentiation of both cell types. SC-EVs also promoted neurite outgrowth and neuron migration of rat dorsal root ganglia, as well as vessel formation in vitro. In an in vivo model of subcutaneous, SC-EVs enhanced the recruitment of endogenous vascular endothelioid-like cells and MSCs, and promoted the formation of a pulpo-dentinal complex-like structure. Finally, mass spectrometry analyses and western blot revealed that stromal cell-derived factor 1 (SDF-1, also known as CXCL12) plays a dominant role in SC-EVs. Together, these data suggest that SC-EVs successfully recruit endogenous stem cells to promote dental pulp regeneration. Our results provide a cell-free strategy for pulp regeneration that avoids the risks associated with stem cell transplantation. Statement of SignificanceDental pulp is vulnerable to infections resulting from dental care, trauma, and multiple restorations, with such infections resulting in pulpitis and pulp necrosis. The current endodontic treatment of irreversible pulp disease cannot restore the function of dental pulp and tissue engineering strategies using cell-based approaches are limited by several disadvantages, including immune rejection and limited cell sources. In this study, we found that schwann cells-derived EVs facilitated dental pulp regeneration through endogenous stem cells recruitment via SDF-1/CXCR4 axis without exogenous cell transplantation. We believe that our study makes a significant contribution to describe a cell-free strategy to promote dental pulp regeneration.

Effects of SDF-1/CXCR7 on the Migration, Invasion and Epithelial-Mesenchymal Transition of Gastric Cancer Cells.

We found that SDF-1/CXCR7 axis played an important role in the growth and proliferation of gastric cancer in the previous studies. The objectives of this study were to explore the effects of SDF-1/CXCR7 on the metastatic ability of gastric cancer cells and the possible mechanisms. CXCR7 expression in SGC-7901 gastric cancer cells was stably knocked down via lentiviral vectors. The cell migration and invasion abilities were detected by transwell migration and invasion assays. The expressions of matrix metalloproteinase 2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), epithelial-mesenchymal transition (EMT) markers and Akt phosphorylation were detected with real-time PCR and/or western blot. We found that SDF-1 markedly enhanced the migration and invasion abilities of SGC-7901 gastric cancer cells; CXCR7 knockdown inhibited these effects. SDF-1/CXCR7 increased the expressions of MMP-2, MMP-9 and VEGF. SDF-1/CXCR7 also downregulated E-cadherin expression but upregulated N-cadherin, vimentin and Snail expressions, suggesting that SDF-1/CXCR7 could promote the development of EMT in gastric cancer cells. Furthermore, SDF-1/CXCR7 could promote Akt phosphorylation. Our results indicated that SDF-1/CXCR7 enhanced the migration, invasion and EMT of gastric cancer cells and thus CXCR7 supression may be a strategy for inhibiting gastric cancer metastasis.

Inflammatory Modulation of Polyethylene Glycol-AuNP for Regulation of the Neural Differentiation Capacity of Mesenchymal Stem Cells.

A nanocomposite composed of polyethylene glycol (PEG) incorporated with various concentrations (~17.4, ~43.5, ~174 ppm) of gold nanoparticles (Au) was created to investigate its biocompatibility and biological performance in vitro and in vivo. First, surface topography and chemical composition was determined through UV-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), scanning electron microscopy (SEM), free radical scavenging ability, and water contact angle measurement. Additionally, the diameters of the PEG-Au nanocomposites were also evaluated through dynamic light scattering (DLS) assay. According to the results, PEG containing 43.5 ppm of Au demonstrated superior biocompatibility and biological properties for mesenchymal stem cells (MSCs), as well as superior osteogenic differentiation, adipocyte differentiation, and, particularly, neuronal differentiation. Indeed, PEG-Au 43.5 ppm induced better cell adhesion, proliferation and migration in MSCs. The higher expression of the SDF-1α/CXCR4 axis may be associated with MMPs activation and may have also promoted the differentiation capacity of MSCs. Moreover, it also prevented MSCs from apoptosis and inhibited macrophage and platelet activation, as well as reactive oxygen species (ROS) generation. Furthermore, the anti-inflammatory, biocompatibility, and endothelialization capacity of PEG-Au was measured in a rat model. After implanting the nanocomposites into rats subcutaneously for 4 weeks, PEG-Au 43.5 ppm was able to enhance the anti-immune response through inhibiting CD86 expression (M1 polarization), while also reducing leukocyte infiltration (CD45). Moreover, PEG-Au 43.5 ppm facilitated CD31 expression and anti-fibrosis ability. Above all, the PEG-Au nanocomposite was evidenced to strengthen the differentiation of MSCs into various cells, including fat, vessel, and bone tissue and, particularly, nerve cells. This research has elucidated that PEG combined with the appropriate amount of Au nanoparticles could become a potential biomaterial able to cooperate with MSCs for tissue regeneration engineering.

Experimental Study on the Effect of Allogeneic Endothelial Progenitor Cells on Wound Healing in Diabetic Mice.

Endothelial progenitor cells (EPCs) are involved in the neovascularization in traumatic and ischemic sites, but EPCs are “detained” in bone marrow under diabetic conditions, which results in reduction of the number of EPCs and their biological activity in peripheral blood. Based on our previous study to mobilize autologous bone marrow EPCs by administering AMD3100+G-CSF to realize the optimal effect, our present study is aimed at exploring the effects of transplanting EPCs locally in a wound model of diabetic mice. First, we prepared and identified EPCs, and the biological functions and molecular characteristics were compared between EPCs from DB/+ and DB/DB mice. Then, we performed full-thickness skin resection in DB/DB mice and tested the effect of local transplantation of EPCs on skin wound healing. The wound healing process was recorded using digital photographs. The animals were sacrificed on postoperative days 7, 14, and 17 for histological and molecular analysis. Our results showed that DB/+ EPCs were biologically more active than those of DB/DB EPCs. When compared with the control group, local transplantation of EPCs accelerated wound healing in DB/DB mice by promoting wound granulation tissue formation, angiogenesis, and collagen fiber deposition, but there was no significant difference in wound healing between DB/+ EPCs and DB/DB EPCs transplanted into the wound. Furthermore, local transplantation of EPCs promoted the expression of SDF-1, CXCR4, and VEGF. We speculated that EPC transplantation may promote wound healing through the SDF-1/CXCR4 axis. This point is worth exploring further. Present data are of considerable significance because they raise the possibility of promoting wound healing by isolating autologous EPCs from the patient, which provides a new approach for the clinical treatment of diabetic wounds in the future.

Mesenchymal Stem Cells With Cancer-Associated Fibroblast-Like Phenotype Stimulate SDF-1/CXCR4 Axis to Enhance the Growth and Invasion of B-Cell Acute Lymphoblastic Leukemia Cells Through Cell-to-Cell Communication.

Background: Bone marrow mesenchymal stem cells (BM-MSCs) are the stromal cells in the leukemia microenvironment, and can obtain cancer-associated fibroblast (CAF)-like phenotype under certain conditions to further promote leukemia progression. However, the mechanism of MSCs with CAF-like phenotype interacting with leukemia cells in B-cell acute lymphoblastic leukemia (B-ALL) and promoting the progression of B-ALL remains unclear.Methods: Mesenchymal stem cells with CAF-like phenotype were obtained by treating MSCs with recombinant human transforming growth factor-β (rhTGF-β), hereafter referred to as TGF-β conditioned MSCs. In vivo mouse model experiments, in vitro transwell chamber experiments, three-dimensional (3D) cell culture models, lentiviral transfection and other experimental methods were used to investigate the possible mechanism of the interaction between TGF-β conditioned MSCs and leukemia cells in promoting the growth, migration and invasion of B-ALL cells.Results: Compared with untreated MSCs, TGF-β conditioned MSCs significantly promoted the growth and proliferation of leukemia cells in mice, and increased the expression of CXCR4 in tumor tissues. In vitro cell experiments, TGF-β conditioned MSCs obviously promoted the migration and invasion of Nalm-6/RS4;11 cells, which were effectively blocked by the CXCR4 inhibitor AMD3100, thereby inhibiting the secretion of MMP-9 in TGF-β conditioned MSCs and inhibiting the activation of the PI3K/AKT signaling pathway in leukemia cells. Further, findings were made that the interaction between TGF-β conditioned MSCs and leukemia cells were mediated by the interaction between the integrin receptor α5β1 on the surface of leukemia cells and the increased expression of fibronectin on TGF-β conditioned MSCs. AMD3100 could weaken such effect by reducing the expression of integrin α5β1 on leukemia cells. Further regulation of integrin β1 could effectively interfere with the interaction between TGF-β conditioned MSCs and leukemia cells.Conclusion: Mesenchymal stem cells with CAF-like phenotype could be a key factor in promoting the growth and invasion of B-ALL cells, and the SDF-1/CXCR4 axis might be a significant factor in mediating the communication of MSCs with CAF-like phenotype and leukemia cells. To prevent the progression of B-ALL cells, blocking the SDF-1/CXCR4 axis with AMD3100 or targeting integrin β1 might be a potential therapeutic strategy.

SDF-1/CXCR4 axis promotes osteogenic differentiation of BMSCs through the JAK2/STAT3 pathway.

This study aimed to investigate the effects of stromal cell-derived factor-1 (SDF-1) and activation of its receptor, chemokine receptor 4 (CXCR4), on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), and the key signaling mechanisms involved in these effects. BMSCs were treated with 100 μg/L SDF-1 and cultured in osteogenic medium for 7 days. RT-qPCR and western blotting were used to detect the protein and mRNA levels of Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN). Alizarin-red staining was used to detect the mineralization-inducing ability of the cells. After BMSCs were treated with SDF-1, the levels of JAK2 mRNA, STAT3 mRNA, and protein phosphorylation increased, the number of mineralized nodules of BMSCs increased, and the osteogenic-differentiation ability was enhanced. In addition, after BMSCs were treated with an inhibitor of JAK2 phosphorylation, the levels of JAK2, STAT3, Runx2, and OCN decreased significantly, the number of mineralized nodules of BMSCs also decreased, and the osteogenic-differentiation ability decreased. The inhibition of CXCR4-treated BMSCs further confirmed that SDF-1/CXCR4 activated JAK2/STAT3 to regulate the osteogenic differentiation of BMSCs. SDF-1/CXCR4 promoted the osteogenic differentiation of BMSCs through JAK2/STAT3 activation.