RESUMEN
Plerixafor is a CXCR4 antagonist approved in 2008 by the FDA for hematopoietic stem cell collection. Subsequently, plerixafor has shown promise as a potential pathogen-agnostic immunomodulator in a variety of preclinical animal models. Additionally, investigator-led studies demonstrated plerixafor prevents viral and bacterial infections in patients with WHIM syndrome, a rare immunodeficiency with aberrant CXCR4 signaling. Here, we investigated whether plerixafor could be repurposed to treat sepsis or severe wound infections, either alone or as an adjunct therapy. In a Pseudomonas aeruginosa lipopolysaccharide (LPS)-induced zebrafish sepsis model, plerixafor reduced sepsis mortality and morbidity assessed by tail edema. There was a U-shaped response curve with the greatest effect seen at 0.1 µM concentration. We used Acinetobacter baumannii infection in a neutropenic murine thigh infection model. Plerixafor did not show reduced bacterial growth at 24 h in the mouse thigh model, nor did it amplify the effects of a rifampin antibiotic therapy, in varying regimens. While plerixafor did not mitigate or treat bacterial wound infections in mice, it did reduce sepsis mortality in zebra fish. The observed mortality reduction in our LPS model of zebrafish was consistent with prior research demonstrating a mortality benefit in a murine model of sepsis. However, based on our results, plerixafor is unlikely to be successful as an adjunct therapy for wound infections. Further research is needed to better define the scope of plerixafor as a pathogen-agnostic therapy. Future directions may include the use of longer acting CXCR4 antagonists, biased CXCR4 signaling, and optimization of animal models.
Asunto(s)
Bencilaminas , Ciclamas , Modelos Animales de Enfermedad , Compuestos Heterocíclicos , Receptores CXCR4 , Sepsis , Pez Cebra , Animales , Ciclamas/farmacología , Ciclamas/administración & dosificación , Bencilaminas/farmacología , Sepsis/tratamiento farmacológico , Sepsis/microbiología , Compuestos Heterocíclicos/farmacología , Compuestos Heterocíclicos/administración & dosificación , Ratones , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Muslo/microbiología , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/aislamiento & purificación , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Femenino , Lipopolisacáridos , Infección de Heridas/microbiología , Infección de Heridas/tratamiento farmacológico , Antibacterianos/farmacología , Antibacterianos/uso terapéuticoRESUMEN
BACKGROUND: Stem cell therapy is a promising alternative for inflammatory diseases and tissue injury treatment. Exogenous delivery of mesenchymal stem cells is associated with instant blood-mediated inflammatory reactions, mechanical stress during administration, and replicative senescence or change in phenotype during long-term culture in vitro. In this study, we aimed to mobilize endogenous hematopoietic stem cells (HSCs) using AMD-3100 and provide local immune suppression using FK506, an immunosuppressive drug, for the treatment of inflammatory bowel diseases. METHODS: Reactive oxygen species (ROS)-responsive FK506-loaded thioketal microspheres were prepared by emulsification solvent-evaporation method. Thioketal vehicle based FK506 microspheres and AMD3100 were co-administered into male C57BL6/J mice with dextran sulfate sodium (DSS) induced colitis. The effect of FK506-loaded thioketal microspheres in colitis mice were evaluated using disease severity index, myeloperoxidase activity, histology, flow cytometry, and gene expression by qRT-PCR. RESULTS: The delivery of AMD-3100 enhanced mobilization of HSCs from the bone marrow into the inflamed colon of mice. Furthermore, targeted oral delivery of FK506 in an inflamed colon inhibited the immune activation in the colon. In the DSS-induced colitis mouse model, the combination of AMD-3100 and FK506-loaded thioketal microspheres ameliorated the disease, decreased immune cell infiltration and activation, and improved body weight, colon length, and epithelial healing process. CONCLUSION: This study shows that the significant increase in the percentage of mobilized hematopoietic stem cells in the combination therapy of AMD and oral FK506 microspheres may contribute to a synergistic therapeutic effect. Thus, low-dose local delivery of FK506 combined with AMD3100 could be a promising alternative treatment for inflammatory bowel diseases.
Asunto(s)
Bencilaminas , Colitis , Ciclamas , Sulfato de Dextran , Ratones Endogámicos C57BL , Tacrolimus , Animales , Colitis/inducido químicamente , Colitis/terapia , Colitis/tratamiento farmacológico , Colitis/patología , Ratones , Masculino , Ciclamas/farmacología , Ciclamas/uso terapéutico , Tacrolimus/farmacología , Tacrolimus/uso terapéutico , Movilización de Célula Madre Hematopoyética/métodos , Compuestos Heterocíclicos/farmacología , Compuestos Heterocíclicos/uso terapéutico , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Modelos Animales de Enfermedad , Terapia de Inmunosupresión , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Microesferas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
OBJECTIVE: Multiple myeloma (MM) is the leading indication of autologous hematopoietic stem cell transplantation. The aim of this study was to determine the incidence of mobilization failure and characterize the risk factors associated with poor mobilization (PM) of MM patients in novel therapies era. METHODS: We conducted a retrospective study of 211 MM patients who received their first peripheral blood stem cells (PBSC) mobilization at our single center. The following data were collected: age, gender, clinical stage, disease status, complete blood cell count, induction regimen, CD34+ cell count in peripheral blood (PB), and PBSC collections. RESULTS: In addition to conventional drugs, 22 (10.4%) patients received daratumumab containing induction, and 33 (15.6%) patients used plerixafor for poor mobilization (pre-apheresis PB CD34+ cells <20/µL). Failure of collection occurred in 24 (11.4%) patients and was correlated with low white blood cell (WBC), ≥3 cycles of lenalidomide treatment before mobilization, steady-state mobilization and nouse of plerixafor are associated with mobilization failure. Daratumumab-based induction treatment ≥2 courses, albumin >41 g/L before mobilization, and steady-state mobilization were risk factors for PM in subgroups of patients treated with lenalidomide for <3 courses. In addition, Hepatitis B virus infection at baseline, thalassemia and measurable residual disease positivity were recognized as predictive factors for PM in subset of chemo-mobilization patients. CONCLUSION: In addition to some well-recognized risk factors, baseline WBC count and daratumumab exposure ≥2 courses before mobilization were revealed as the predictive factors of mobilization failure, providing consultation for preemptive use of plerixafor.
Asunto(s)
Bencilaminas , Ciclamas , Movilización de Célula Madre Hematopoyética , Mieloma Múltiple , Humanos , Mieloma Múltiple/terapia , Movilización de Célula Madre Hematopoyética/métodos , Femenino , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Anciano , Adulto , Ciclamas/uso terapéutico , Ciclamas/farmacología , Bencilaminas/uso terapéutico , Células Madre de Sangre Periférica/metabolismo , Factores de Riesgo , Anticuerpos Monoclonales/uso terapéutico , Lenalidomida/uso terapéutico , Lenalidomida/administración & dosificación , Compuestos Heterocíclicos/administración & dosificación , Compuestos Heterocíclicos/uso terapéutico , Trasplante de Células Madre de Sangre Periférica/métodos , Trasplante AutólogoAsunto(s)
Bencilaminas , Médula Ósea , Ciclamas , Compuestos Heterocíclicos , Mieloma Múltiple , Receptores CXCR4 , Ciclamas/farmacología , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Bencilaminas/farmacología , Compuestos Heterocíclicos/farmacología , Compuestos Heterocíclicos/uso terapéutico , Médula Ósea/patología , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Microambiente Tumoral/efectos de los fármacosRESUMEN
ABSTRACT: We have reported the direct repair of the sickle cell mutation in vivo in a disease model using vectorized prime editors after hematopoietic stem cell (HSC) mobilization with granulocyte colony-stimulating factor (G-CSF)/AMD3100. The use of G-CSF for HSC mobilization is a hurdle for the clinical translation of this approach. Here, we tested a G-CSF-free mobilization regimen using WU-106, an inhibitor of integrin α4ß1, plus AMD3100 for in vivo HSC prime editing in sickle cell disease (SCD) mice. Mobilization with WU-106 + AMD3100 in SCD mice was rapid and efficient. In contrast to the G-CSF/AMD3100 approach, mobilization of activated granulocytes and elevation of the key proinflammatory cytokine interleukin-6 in the serum were minimal. The combination of WU-106 + AMD3100 mobilization and IV injection of the prime editing vector together with in vivo selection resulted in â¼23% correction of the SCD mutation in the bone marrow and peripheral blood cells of SCD mice. The treated mice demonstrated phenotypic correction, as reflected by normalized blood parameters and spleen size. Editing frequencies were significantly increased (29%) in secondary recipients, indicating the preferential mobilization/transduction of long-term repopulating HSCs. Using this approach, we found <1% undesired insertions/deletions and no detectable off-target editing at the top-scored potential sites. Our study shows that in vivo transduction to treat SCD can now be done within 2 hours involving only simple IV injections with a good safety profile. The same-day mobilization regimen makes in vivo HSC gene therapy more attractive for resource-poor settings, where SCD does the most damage.
Asunto(s)
Anemia de Células Falciformes , Terapia Genética , Factor Estimulante de Colonias de Granulocitos , Movilización de Célula Madre Hematopoyética , Animales , Humanos , Ratones , Anemia de Células Falciformes/terapia , Anemia de Células Falciformes/genética , Bencilaminas , Ciclamas/farmacología , Ciclamas/uso terapéutico , Modelos Animales de Enfermedad , Edición Génica , Terapia Genética/métodos , Factor Estimulante de Colonias de Granulocitos/farmacología , Movilización de Célula Madre Hematopoyética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/metabolismo , Compuestos Heterocíclicos/farmacología , Compuestos Heterocíclicos/uso terapéuticoAsunto(s)
Aprobación de Drogas , Enfermedades de Inmunodeficiencia Primaria , Receptores CXCR4 , United States Food and Drug Administration , Humanos , Receptores CXCR4/antagonistas & inhibidores , Estados Unidos , Enfermedades de Inmunodeficiencia Primaria/tratamiento farmacológico , Enfermedades del Sistema Inmune/tratamiento farmacológico , Ciclamas/uso terapéutico , Ciclamas/farmacología , Verrugas/tratamiento farmacológico , Compuestos Heterocíclicos con 3 Anillos/uso terapéutico , Bencilaminas/uso terapéutico , Bencilaminas/farmacologíaRESUMEN
PURPOSE: This study aimed to evaluate the potential of astragalus polysaccharide (APS) pretreatment in enhancing the homing and anti-peritoneal fibrosis capabilities of bone marrow mesenchymal stromal cells (BMSCs) and to elucidate the underlying mechanisms. METHODS: Forty male Sprague-Dawley rats were allocated into four groups: control, peritoneal dialysis fluid (PDF), PDF + BMSCs, and PDF + APSBMSCs (APS-pre-treated BMSCs). A peritoneal fibrosis model was induced using PDF. Dil-labeled BMSCs were administered intravenously. Post-transplantation, BMSC homing to the peritoneum and pathological alterations were assessed. Stromal cell-derived factor-1 (SDF-1) levels were quantified via enzyme-linked immunosorbent assay (ELISA), while CXCR4 expression in BMSCs was determined using PCR and immunofluorescence. Additionally, a co-culture system involving BMSCs and peritoneal mesothelial cells (PMCs) was established using a Transwell setup to examine the in vitro effects of APS on BMSC migration and therapeutic efficacy, with the CXCR4 inhibitor AMD3100 deployed to dissect the role of the SDF-1/CXCR4 axis and its downstream impacts. RESULTS: In vivo and in vitro experiments confirmed that APS pre-treatment notably facilitated the targeted homing of BMSCs to the peritoneal tissue of PDF-treated rats, thereby amplifying their therapeutic impact. PDF exposure markedly increased SDF-1 levels in peritoneal and serum samples, which encouraged the migration of CXCR4-positive BMSCs. Inhibition of the SDF-1/CXCR4 axis through AMD3100 application diminished BMSC migration, consequently attenuating their therapeutic response to peritoneal mesenchyme-to-mesothelial transition (MMT). Furthermore, APS upregulated CXCR4 expression in BMSCs, intensified the activation of the SDF-1/CXCR4 axis's downstream pathways, and partially reversed the AMD3100-induced effects. CONCLUSION: APS augments the SDF-1/CXCR4 axis's downstream pathway activation by increasing CXCR4 expression in BMSCs. This action bolsters the targeted homing of BMSCs to the peritoneal tissue and amplifies their suppressive influence on MMT, thereby improving peritoneal fibrosis.
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Planta del Astrágalo , Quimiocina CXCL12 , Células Madre Mesenquimatosas , Fibrosis Peritoneal , Polisacáridos , Ratas Sprague-Dawley , Receptores CXCR4 , Animales , Receptores CXCR4/metabolismo , Quimiocina CXCL12/metabolismo , Ratas , Masculino , Fibrosis Peritoneal/tratamiento farmacológico , Fibrosis Peritoneal/metabolismo , Polisacáridos/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Modelos Animales de Enfermedad , Ciclamas/farmacologíaRESUMEN
Most transplant-eligible multiple myeloma (MM) patients undergo autologous peripheral blood stem cell collection (PBSC) using G-CSF with on-demand plerixafor (G ± P). Chemomobilization (CM) can be used as a salvage regimen after G ± P failure or for debulking residual tumor burden ahead of autologous peripheral blood stem cell transplantation (ASCT). Prior studies utilizing cyclophosphamide-based CM have not shown long-term benefits. At our center, intensive CM (ICM) using a PACE- or HyperCVAD-based regimen has been used to mitigate "excessive" residual disease based on plasma cell (PC) burden or MM-related biomarkers. Given the lack of efficacy of non-ICM, we sought to determine the impact of ICM on event-free survival (EFS), defined as death, progressive disease, or unplanned treatment escalation. We performed a retrospective study of newly diagnosed MM patients who collected autologous PBSCs with the intent to proceed immediately to ASCT at our center between 7/2020 and 2/2023. Patients were excluded if they underwent a tandem autologous or sequential autologous-allogeneic transplant, had primary PC leukemia, received non-ICM treatment (i.e., cyclophosphamide and/or etoposide), or had previously failed G ± P mobilization. To appropriately evaluate the impact of ICM among those who potentially could have received it, we utilized a propensity score matching (PSM) approach whereby ICM patients were compared to a cohort of non-CM patients matched on pre-ASCT factors most strongly associated with the receipt of ICM. Of 451 patients identified, 61 (13.5%) received ICM (PACE-based, n = 45; hyper-CVAD-based, n = 16). Post-ICM/pre-ASCT, 11 patients (18%) required admission for neutropenic fever and/or infection. Among 51 evaluable patients, the overall response rate was 31%; however, 46 of 55 evaluable patients (84%) saw a reduction in M-spike and/or involved free light chains. Among those evaluated with longitudinal peripheral blood flow cytometry (n = 8), 5 patients (63%) cleared circulating blood PCs post-ICM. Compared to patients mobilized with non-CM, ICM patients collected a slightly greater median number of CD34+ cells (10.8 versus 10.2 × 106/kg, P = .018). The median follow-up was 30.6 months post-ASCT. In a PSM multivariable analysis, ICM was associated with significantly improved EFS (hazard ratio [HR] 0.30, 95% CI 0.14 to 0.67, P = .003), but not improved OS (HR 0.38, 95% CI 0.10 to 1.44, P = .2). ICM was associated with longer post-ASCT inpatient duration (+4.1 days, 95% CI, 2.4 to 5.8, P < .001), more febrile days (+0.96 days, 95% CI 0.50 to 1.4, P < .001), impaired platelet engraftment (HR 0.23, 95% CI 0.06 to 0.87, P = .031), more bacteremia (OR 3.41, 95% CI 1.20 to 9.31, P = .018), and increased antibiotic usage (cefepime: +2.3 doses, 95% CI 0.39 to 4.1, P = .018; vancomycin: +1.0 doses, 95% CI 0.23 to 1.8, P = .012). ICM was independently associated with improved EFS in a matched analysis involving MM patients with excessive disease burden at pre-ASCT workup. This benefit came at the cost of longer inpatient duration, more febrile days, greater incidence of bacteremia, and increased antibiotic usage in the immediate post-ASCT setting. Our findings suggest that ICM could be considered for a subset of MM patients, but its use must be weighed carefully against additional toxicity.
Asunto(s)
Mieloma Múltiple , Trasplante Autólogo , Humanos , Mieloma Múltiple/terapia , Masculino , Persona de Mediana Edad , Femenino , Estudios Retrospectivos , Anciano , Trasplante de Células Madre de Sangre Periférica/métodos , Ciclamas/uso terapéutico , Ciclamas/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Bencilaminas , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Adulto , Trasplante de Células Madre Hematopoyéticas/métodosRESUMEN
Regenerating gene family member 4 (Reg4) has been implicated in acute pancreatitis, but its precise functions and involved mechanisms have remained unclear. Herein, we sought to investigate the contribution of Reg4 to the pathogenesis of pancreatitis and evaluate its therapeutic effects in experimental pancreatitis. In acute pancreatitis, Reg4 deletion increases inflammatory infiltrates and mitochondrial cell death and decreases autophagy recovery, which are rescued by the administration of recombinant Reg4 (rReg4) protein. In chronic pancreatitis, Reg4 deficiency aggravates inflammation and fibrosis and inhibits compensatory cell proliferation. Moreover, C-X-C motif ligand 12 (CXCL12)/C-X-C motif receptor 4 (CXCR4) axis is sustained and activated in Reg4-deficient pancreas. The detrimental effects of Reg4 deletion are attenuated by the administration of the approved CXCR4 antagonist plerixafor (AMD3100). Mechanistically, Reg4 mediates its function in pancreatitis potentially via binding its receptor exostosin-like glycosyltransferase 3 (Extl3). In conclusion, our findings suggest that Reg4 exerts a therapeutic effect during pancreatitis by limiting inflammation and fibrosis and improving cellular regeneration.
Asunto(s)
Fibrosis , Mitocondrias , Proteínas Asociadas a Pancreatitis , Pancreatitis , Receptores CXCR4 , Animales , Proteínas Asociadas a Pancreatitis/metabolismo , Proteínas Asociadas a Pancreatitis/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Pancreatitis/patología , Pancreatitis/metabolismo , Ratones , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Humanos , Ratones Endogámicos C57BL , Ciclamas/farmacología , Masculino , Ratones Noqueados , Bencilaminas/farmacología , Quimiocina CXCL12/metabolismo , Proliferación Celular , Transducción de Señal , Autofagia , Páncreas/patología , Páncreas/metabolismo , Muerte CelularRESUMEN
BACKGROUND: Increasing indications for cellular therapy collections have stressed our healthcare system, with autologous collections having a longer than desired wait time until apheresis collection. This quality improvement initiative was undertaken to accommodate more patients within existing resources. STUDY DESIGN AND METHODS: Patients with multiple myeloma who underwent autologous peripheral blood stem cell collection from October 2022 to April 2023 were included. Demographic, mobilization, laboratory, and apheresis data were retrospectively collected from the medical record. RESULTS: This cohort included 120 patients (49.2% male), with a median age of 60 years. All received G-CSF and 95% received pre-emptive Plerixafor approximately 18 hours pre-collection. Most (79%) had collection goals of at least 8 × 106/kg CD34 cells, with 63% over 70 years old having this high collection goal (despite 20 years of institutional data showing <1% over 70 years old have a second transplant). With collection efficiencies of 55.9%, 44% of patients achieved their collection goal in a single day apheresis collection. A platelet count <150 × 103/µL on the day of collection was a predictor for poor mobilization; among 27 patients with a low baseline platelet count, 17 did not achieve the collection goal and 2 failed to collect a transplantable dose. CONCLUSIONS: With minor collection goal adjustments, 15% of all collection appointments could have been avoided over this 6-month period. Other strategies to accommodate more patients include mobilization modifications (Plerixafor timing or substituting a longer acting drug), utilizing platelet counts to predict mobilization, and modifying apheresis collection volumes or schedule templates.
Asunto(s)
Bencilaminas , Ciclamas , Factor Estimulante de Colonias de Granulocitos , Movilización de Célula Madre Hematopoyética , Mieloma Múltiple , Trasplante Autólogo , Humanos , Mieloma Múltiple/terapia , Ciclamas/farmacología , Ciclamas/uso terapéutico , Persona de Mediana Edad , Masculino , Femenino , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Movilización de Célula Madre Hematopoyética/métodos , Anciano , Estudios Retrospectivos , Eliminación de Componentes Sanguíneos/métodos , Compuestos Heterocíclicos/administración & dosificación , Compuestos Heterocíclicos/uso terapéutico , Adulto , Trasplante de Células Madre de Sangre Periférica/métodos , Recuento de PlaquetasRESUMEN
BACKGROUND: Despite recent advances in the treatment of multiple myeloma, high-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (ASCT) remains an essential therapeutic keystone. As for the stem cell mobilization procedure, different regimens have been established, usually consisting of a cycle of chemotherapy followed by application of granulocyte-colony stimulating factor (G-CSF), although febrile neutropenia is a common complication. Following national guidelines, our institution decided to primarily use G-CSF only mobilization during the COVID-19 pandemic to minimize the patients' risk of infection and to reduce the burden on the health system. STUDY DESIGN AND METHODS: In this retrospective single-center analysis, the efficacy and safety of G-CSF only mobilization was evaluated and compared to a historic control cohort undergoing chemotherapy-based mobilization by cyclophosphamide and etoposide (CE) plus G-CSF. RESULTS: Although G-CSF only was associated with a higher need for plerixafor administration (p < .0001) and a higher number of apheresis sessions per patient (p = .0002), we were able to collect the target dose of hematopoietic stem cells in the majority of our patients. CE mobilization achieved higher hematopoietic stem cell yields (p = .0015) and shorter apheresis sessions (p < .0001) yet was accompanied by an increased risk of febrile neutropenia (p < .0001). There was no difference in engraftment after ASCT. DISCUSSION: G-CSF only mobilization is a useful option in selected patients with comorbidities and an increased risk of serious infections, especially in the wintertime or in future pandemics.
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Ciclofosfamida , Etopósido , Factor Estimulante de Colonias de Granulocitos , Movilización de Célula Madre Hematopoyética , Mieloma Múltiple , Trasplante Autólogo , Adulto , Femenino , Humanos , Masculino , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Bencilaminas , COVID-19 , Ciclamas/uso terapéutico , Ciclamas/farmacología , Ciclofosfamida/uso terapéutico , Ciclofosfamida/administración & dosificación , Etopósido/uso terapéutico , Etopósido/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Movilización de Célula Madre Hematopoyética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Mieloma Múltiple/terapia , Estudios Retrospectivos , SARS-CoV-2RESUMEN
PURPOSE: Radiopharmaceutical therapies targeting fibroblast activation protein (FAP) have shown promising efficacy against many tumor types. But radiopharmaceuticals alone in most cases are insufficient to completely eradicate tumor cells, which can partially be attributed to the protective interplay between tumor cells and cancer-associated fibroblasts (CAFs). The C-X-C chemokine receptor type 4/C-X-C motif chemokine 12 (CXCR4/CXCL12) interaction plays an important role in orchestrating tumor cells and CAFs. We hereby investigated the feasibility and efficacy of [177Lu]Lu-DOTAGA.(SA.FAPi)2, a FAP-targeting radiopharmaceutical, in combination with AMD3100, a CXCR4 antagonist, in a preclinical murine model of triple-negative breast cancer (TNBC). METHODS: Public database was first interrogated to reveal the correlation between CAFs' scores and the prognosis of TNBC patients, as well as the expression levels of FAP and CXCR4 in normal tissues and tumors. In vitro therapeutic efficacy regarding cell proliferation, migration, and colony formation was assessed in BALB/3T3 fibroblasts and 4T1 murine breast cancer cells. In vivo therapeutic efficacy was longitudinally monitored using serial 18F-FDG, [18F]AlF-NOTA-FAPI-04, and [68Ga]Ga-DOTA-Pentixafor PET/CT scans and validated using tumor sections through immunohistochemical staining of Ki-67, α-SMA, CXCR4, and CXCL12. Intratumoral abundance of myeloid-derived suppressive cells (MDSCs) was analyzed using flow cytometry in accordance with the PET/CT schedules. Treatment toxicity was evaluated by examining major organs including heart, lung, liver, kidney, and spleen. RESULTS: CAFs' scores negatively correlated with the survival of TNBC patients (p < 0.05). The expression of CXCR4 and FAP was both significantly higher in tumors than in normal tissues. The combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 significantly suppressed cell proliferation, migration, and colony formation in cell culture, and exhibited synergistic effects in 4T1 tumor models along with a decreased number of MDSCs. PET/CT imaging revealed lowest tumor accumulation of 18F-FDG and [18F]AlF-NOTA-FAPI-04 on day 13 and day 14 after treatment started, both of which gradually increased at later time points. A similar trend was observed in the IHC staining of Ki-67, α-SMA, and CXCL12. CONCLUSION: The combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 is a feasible treatment against TNBC with minimal toxicity in main organs.
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Quimiocina CXCL12 , Receptores CXCR4 , Neoplasias de la Mama Triple Negativas , Receptores CXCR4/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/diagnóstico por imagen , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/radioterapia , Animales , Ratones , Quimiocina CXCL12/metabolismo , Humanos , Línea Celular Tumoral , Femenino , Ciclamas/farmacología , Ciclamas/uso terapéutico , Lutecio , Bencilaminas/farmacología , Compuestos Heterocíclicos/farmacología , Compuestos Heterocíclicos/química , Radiofármacos/uso terapéutico , Radiofármacos/farmacología , Endopeptidasas , Proliferación Celular/efectos de los fármacos , Gelatinasas/metabolismo , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/metabolismoRESUMEN
In this work, we experimentally investigate the potency of high pressure to drive a protein toward an excited state where an inhibitor targeted for this state can bind. Ras proteins are small GTPases cycling between active GTP-bound and inactive GDP-bound states. Various states of GTP-bound Ras in active conformation coexist in solution, amongst them, state 2 which binds to effectors, and state 1, weakly populated at ambient conditions, which has a low affinity for effectors. Zn2+-cyclen is an allosteric inhibitor of Ras protein, designed to bind specifically to the state 1. In H-Ras(wt).Mg2+.GppNHp crystals soaked with Zn2+-cyclen, no binding could be observed, as expected in the state 2 conformation which is the dominant state at ambient pressure. Interestingly, Zn2+-cyclen binding is observed at 500â MPa pressure, close to the nucleotide, in Ras protein that is driven by pressure to a state 1 conformer. The unknown binding mode of Zn2+-cyclen to H-Ras can thus be fully characterized in atomic details. As a more general conjunction from our study, high pressure x-ray crystallography turns out to be a powerful method to induce transitions allowing drug binding in proteins that are in low-populated conformations at ambient conditions, enabling the design of specific inhibitors.
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Ciclamas , Zinc , Zinc/química , Zinc/metabolismo , Cristalografía por Rayos X , Ciclamas/química , Ciclamas/farmacología , Regulación Alostérica , Presión , Unión Proteica , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Humanos , Sitios de UniónRESUMEN
The laminar flow profiles in microfluidic systems coupled to rapid diffusion at flow streamlines have been widely utilized to create well-controlled chemical gradients in cell cultures for spatially directing cell migration. However, within hydrogel-based closed microfluidic systems of limited depth (≤0.1 mm), the biomechanical cues for the cell culture are dominated by cell interactions with channel surfaces rather than with the hydrogel microenvironment. Also, leaching of poly(dimethylsiloxane) (PDMS) constituents in closed systems and the adsorption of small molecules to PDMS alter chemotactic profiles. To address these limitations, we present the patterning and integration of a PDMS-free open fluidic system, wherein the cell-laden hydrogel directly adjoins longitudinal channels that are designed to create chemotactic gradients across the 3D culture width, while maintaining uniformity across its â¼1 mm depth to enhance cell-biomaterial interactions. This hydrogel-based open fluidic system is assessed for its ability to direct migration of U87 glioma cells using a hybrid hydrogel that includes hyaluronic acid (HA) to mimic the brain tumor microenvironment and gelatin methacrylate (GelMA) to offer the adhesion motifs for promoting cell migration. Chemotactic gradients to induce cell migration across the hydrogel width are assessed using the chemokine CXCL12, and its inhibition by AMD3100 is validated. This open-top hydrogel-based fluidic system to deliver chemoattractant cues over square-centimeter-scale areas and millimeter-scale depths can potentially serve as a robust screening platform to assess emerging glioma models and chemotherapeutic agents to eradicate them.
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Movimiento Celular , Quimiotaxis , Glioma , Hidrogeles , Humanos , Glioma/patología , Glioma/metabolismo , Movimiento Celular/efectos de los fármacos , Hidrogeles/química , Hidrogeles/farmacología , Quimiotaxis/efectos de los fármacos , Línea Celular Tumoral , Técnicas de Cultivo Tridimensional de Células/métodos , Microambiente Tumoral/efectos de los fármacos , Quimiocina CXCL12/farmacología , Quimiocina CXCL12/metabolismo , Ciclamas/farmacología , Ciclamas/química , Técnicas de Cultivo de Célula/métodos , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Gelatina/química , Bencilaminas/farmacología , Bencilaminas/química , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismoRESUMEN
Autologous hematopoietic progenitor cell transplantation (ASCT) has been used for more than five decades to treat malignant and non-malignant diseases. Successful engraftment after high-dose chemotherapy relies on the ability to collect sufficient CD34 + hematopoietic progenitor cells (HPCs), typically from peripheral blood after mobilization. Commonly, either granulocyte colony-stimulating factor (G-CSF) alone as a single agent (i.e. steady-state mobilization) or G-CSF after chemotherapy is administered to collect adequate numbers of HPCs (minimum ≥2 × 106 CD34 + cells/kg for one ASCT; optimal up to 5 × 106 CD34 + cells/kg). However, a significant proportion of patients fail successful HPC mobilization, which is commonly defined as a CD34+ cell count below 10-15/µL after at least 4 days of 10 µg/kg b.w. G-CSF alone, or after chemo-mobilization in combination with 5-10 µg/kg b.w. G-CSF. In these situations plerixafor, a chemokine receptor inhibitor (CXCR4) can be used to enhance HPC collection in patients with multiple myeloma and malignant lymphoma whose cells mobilize poorly. Risk factors for poor mobilization have been evaluated and several strategies (e.g. plerixafor to rescue the mobilization approach or pre-emptive use) have been suggested to optimize mobilization, especially in patients at risk. This manuscript discusses the risk factors of poor CD34+ mobilization and summarizes the current strategies to optimize mobilization and HPC collection.
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Movilización de Célula Madre Hematopoyética , Humanos , Movilización de Célula Madre Hematopoyética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Ciclamas/farmacología , Ciclamas/uso terapéutico , BencilaminasRESUMEN
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is characterized by an immune-suppressive microenvironment, which contributes to tumor progression, metastasis, and immunotherapy resistance. Identification of HCC-intrinsic factors regulating the immunosuppressive microenvironment is urgently needed. Here, we aimed to elucidate the role of SYR-Related High-Mobility Group Box 18 (SOX18) in inducing immunosuppression and to validate novel combination strategies for SOX18-mediated HCC progression and metastasis. METHODS: The role of SOX18 in HCC was investigated in orthotopic allografts and diethylinitrosamine/carbon tetrachloride-induced spontaneous models by using murine cell lines, adeno-associated virus 8, and hepatocyte-specific knockin and knockout mice. The immune cellular composition in the HCC microenvironment was evaluated by flow cytometry and immunofluorescence. RESULTS: SOX18 overexpression promoted the infiltration of tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) while diminishing cytotoxic T cells to facilitate HCC progression and metastasis in cell-derived allografts and chemically induced HCC models. Mechanistically, transforming growth factor-beta 1 (TGF-ß1) upregulated SOX18 expression by activating the Smad2/3 complex. SOX18 transactivated chemokine (C-X-C motif) ligand 12 (CXCL12) and programmed death ligand 1 (PD-L1) to induce the immunosuppressive microenvironment. CXCL12 knockdown significantly attenuated SOX18-induced TAMs and Tregs accumulation and HCC dissemination. Antagonism of chemokine receptor 4 (CXCR4), the cognate receptor of CXCL12, or selective knockout of CXCR4 in TAMs or Tregs likewise abolished SOX18-mediated effects. TGFßR1 inhibitor Vactosertib or CXCR4 inhibitor AMD3100 in combination with anti-PD-L1 dramatically inhibited SOX18-mediated HCC progression and metastasis. CONCLUSIONS: SOX18 promoted the accumulation of immunosuppressive TAMs and Tregs in the microenvironment by transactivating CXCL12 and PD-L1. CXCR4 inhibitor or TGFßR1 inhibitor in synergy with anti-PD-L1 represented a promising combination strategy to suppress HCC progression and metastasis.
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Antígeno B7-H1 , Bencilaminas , Carcinoma Hepatocelular , Quimiocina CXCL12 , Ciclamas , Progresión de la Enfermedad , Neoplasias Hepáticas , Receptores CXCR4 , Factores de Transcripción SOXF , Linfocitos T Reguladores , Factor de Crecimiento Transformador beta1 , Microambiente Tumoral , Macrófagos Asociados a Tumores , Regulación hacia Arriba , Animales , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Factores de Transcripción SOXF/metabolismo , Factores de Transcripción SOXF/genética , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Microambiente Tumoral/inmunología , Humanos , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Factor de Crecimiento Transformador beta1/metabolismo , Ratones , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/genética , Ciclamas/farmacología , Bencilaminas/farmacología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Línea Celular Tumoral , Macrófagos Asociados a Tumores/metabolismo , Macrófagos Asociados a Tumores/inmunología , Ratones Noqueados , Regulación Neoplásica de la Expresión Génica , Transducción de Señal , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Ratones Endogámicos C57BL , Dietilnitrosamina/toxicidad , MasculinoRESUMEN
Adipose-derived stem cells (ASCs) possess therapeutic potential for ischemic brain injury, and the chemokine CXCL12 has been shown to enhance their functional properties. However, the cumulative effects of ASCs when combined with various structures of CXCL12 on ischemic stroke and its underlying molecular mechanisms remain unclear. In this study, we genetically engineered mouse adipose-derived ASCs with CXCL12 variants and transplanted them to the infarct region in a mice transient middle cerebral artery occlusion (tMCAO) model of stroke. We subsequently compared the post-ischemic stroke efficacy of ASC-mCXCL12 with ASC-dCXCL12, ASC-wtCXCL12, and unmodified ASCs. Neurobehavior recovery was assessed using modified neurological severity scores, the hanging wire test, and the elevated body swing test. Changes at the tissue level were evaluated through cresyl violet and immunofluorescent staining, while molecular level alterations were examined via Western blot and real-time PCR. The results of the modified neurological severity score and cresyl violet staining indicated that both ASC-mCXCL12 and ASC-dCXCL12 treatment enhanced neurobehavioral recovery and mitigated brain atrophy at the third and fifth weeks post-tMCAO. Additionally, we observed that ASC-mCXCL12 and ASC-dCXCL12 promoted angiogenesis and neurogenesis, accompanied by an increased expression of bFGF and VEGF in the peri-infarct area of the brain. Notably, in the third week after tMCAO, the ASC-mCXCL12 exhibited superior outcomes compared to ASC-dCXCL12. However, when treated with the CXCR4 antagonist AMD3100, the beneficial effects of ASC-mCXCL12 were reversed. The AMD3100-treated group demonstrated worsened neurological function, aggravated edema volume, and brain atrophy. This outcome is likely attributed to the interaction of monomeric CXCL12 with CXCR4, which regulates the recruitment of bFGF and VEGF. This study introduces an innovative approach to enhance the therapeutic potential of ASCs in treating ischemic stroke by genetically engineering them with the monomeric structure of CXCL12.
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Quimiocina CXCL12 , Accidente Cerebrovascular Isquémico , Células Madre Mesenquimatosas , Trasplante de Células Madre , Animales , Ratones , Bencilaminas/farmacología , Quimiocina CXCL12/genética , Ciclamas/farmacología , Ingeniería Genética , Accidente Cerebrovascular Isquémico/terapia , Células Madre Mesenquimatosas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Oligomeric organization of G protein-coupled receptors is proposed to regulate receptor signaling and function, yet rapid and precise identification of the oligomeric status especially for native receptors on a cell membrane remains an outstanding challenge. By using blinking carbon dots (CDs), we now develop a deep learning (DL)-based blinking fingerprint recognition method, named deep-blinking fingerprint recognition (BFR), which allows automatic classification of CD-labeled receptor organizations on a cell membrane. This DL model integrates convolutional layers, long-short-term memory, and fully connected layers to extract time-dependent blinking features of CDs and is trained to a high accuracy (â¼95%) for identifying receptor organizations. Using deep blinking fingerprint recognition, we found that CXCR4 mainly exists as 87.3% monomers, 12.4% dimers, and <1% higher-order oligomers on a HeLa cell membrane. We further demonstrate that the heterogeneous organizations can be regulated by various stimuli at different degrees. The receptor-binding ligands, agonist SDF-1α and antagonist AMD3100, can induce the dimerization of CXCR4 to 33.1 and 20.3%, respectively. In addition, cytochalasin D, which inhibits actin polymerization, similarly prompts significant dimerization of CXCR4 to 30.9%. The multi-pathway organization regulation will provide an insight for understanding the oligomerization mechanism of CXCR4 as well as for elucidating their physiological functions.
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Carbono , Aprendizaje Profundo , Puntos Cuánticos , Receptores CXCR4 , Bencilaminas/química , Bencilaminas/farmacología , Quimiocina CXCL12/agonistas , Ciclamas/química , Ciclamas/farmacología , Células HeLa , Humanos , Receptores CXCR4/químicaRESUMEN
BACKGROUND: Cross-sensitization of pelvic organs is one theory for why symptoms of gut sickness and interstitial cystitis/bladder pain syndrome overlap. Experimental colitis has been shown to trigger bladder hyperactivity and hyperalgesia in rats. The chemokine receptor CXCR4 plays a key role in bladder function and central sensitization. We aim to study the role of CXCR4 and its inhibitor AMD3100 in colon-bladder cross-organ sensitization. METHODS: The colitis model was established by rectal infusion of trinitrobenzene sulfonic acid. Western blot and immunofluorescence were used to assess the expression and distribution of CXCR4. Intrathecal injection of AMD3100 (a CXCR4 inhibitor) and PD98059 (an ERK inhibitor) were used to inhibit CXCR4 and downstream extracellular signal-regulated kinase (ERK) in the spinal cord and dorsal root ganglion (DRG). Intravesical perfusion of resiniferatoxin was performed to measure the pain behavior counts of rats, and continuous cystometry was performed to evaluate bladder voiding function. RESULTS: Compared to the control group, CXCR4 was expressed more in bladder mucosa and colon mucosa, L6-S1 dorsal root ganglion (DRG), and the corresponding segment of the spinal dorsal horn (SDH) in rats with colitis. Moreover, intrathecal injection of the AMD3100 suppressed bladder overactivity, bladder hyperalgesia, and mastocytosis symptoms caused by colitis. Furthermore, AMD3100 effectively inhibited ERK activation in the spinal cord induced by experimental colitis. Finally, treatment with PD98059 alleviated bladder overactivity and hyperalgesia caused by colitis. CONCLUSION: Increased CXCR4 in the DRG and SDH contributes to colon inflammation-induced bladder overactivity and hyperalgesia partly via the phosphorylation of spinal ERK. Treatment targeting the CXCR4/ERK pathway might provide a potential new approach for the comorbidity between the digestive system and the urinary system.
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Bencilaminas/farmacología , Colitis/tratamiento farmacológico , Colitis/metabolismo , Ciclamas/farmacología , Ganglios Espinales/metabolismo , Receptores CXCR4/efectos de los fármacos , Médula Espinal/metabolismo , Animales , Bencilaminas/administración & dosificación , Colitis/complicaciones , Ciclamas/administración & dosificación , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Flavonoides/administración & dosificación , Flavonoides/farmacología , Hiperalgesia/inducido químicamente , Dimensión del Dolor , Ratas , Ratas Sprague-Dawley , Receptores CXCR4/metabolismo , Transducción de Señal , Enfermedades de la Vejiga Urinaria/tratamiento farmacológico , Enfermedades de la Vejiga Urinaria/etiología , Enfermedades de la Vejiga Urinaria/metabolismo , Micción/efectos de los fármacosRESUMEN
Recent studies have emphasized the role of vascular adventitia inflammation and immune response in hypertension. It has been reported that stromal cell-derived factor-1 (SDF-1) plays various biological functions through its receptors C-X-C motif chemokine receptor 4 (CXCR4) and CXCR7 in tumor growth and tissue repair. However, it is unclear that whether SDF-1/CXCR4/CXCR7 axis is involved in hypertensive vascular remodeling. In the present study, the involvement of SDF-1/CXCR4/CXCR7 axis was evaluated with lentivirus-mediated shRNA of SDF-1 and CXCR7, CXCR4 antagonist AMD3100 and CXCR7 agonist VUF11207 in angiotensin II (AngII)-induced hypertensive mice and in cultured adventitial fibroblasts (AFs). Results showed that AngII infusion markedly increased SDF-1 expressed in vascular adventitia, but not in media and endothelium. Importantly, blockade of SDF-1/CXCR4 axis strikingly potentiated AngII-induced adventitial thickening and fibrosis, as indicated by enhanced collagen I deposition. In contrast, CXCR7 shRNA largely attenuated AngII-induced adventitial thickness and fibrosis, whereas CXCR7 activation with VUF11207 significantly potentiated AngII-induced adventitial thickening and fibrosis. In consistent with these in vivo study, CXCR4 inhibition with AMD3100 and CXCR7 activation with VUF11207 aggravated AngII-induced inflammation, proliferation and migration in cultured AFs. In summary, these results suggested that SDF-1 exerted opposing effects through CXCR4 and CXCR7 in AngII-induced vascular adventitial remodeling.