RESUMEN
Due to immunosuppressive properties and confirmed tropism towards cancer cells mesenchymal stromal cells (MSC) have been used in many trials. In our study we used these cells as carriers of IL-12 in the treatment of mice with primary and metastatic B16-F10 melanomas. IL-12 has confirmed anti-cancer activity, induces a strong immune response against cancer cells and acts as an anti-angiogenic agent. A major limitation of the use of IL-12 in therapy is its systemic toxicity. The aim of the work was to develop a system in which cytokine may be administered intravenously without toxic side effects. In this study MSC were used as carriers of the IL-12. We confirmed antitumor effectiveness of the cells secreting IL-12 (MSC/IL-12) in primary and metastatic murine melanoma models. We observed inhibition of tumor growth and a significant reduction in the number of metastases in mice after MSC/IL-12 administration. MSC/IL-12 decreased vascular density and increased the number of anticancer M1 macrophages and CD8+ cytotoxic T lymphocytes in tumors of treated mice. To summarize, we showed that MSC are an effective, safe carrier of IL-12 cytokine. Administered systemically they exert therapeutic properties of IL-12 cytokine without toxicity. Therapeutic effect may be a result of pleiotropic (proinflammatory and anti-angiogenic) properties of IL-12 released by modified MSC.
Asunto(s)
Interleucina-12/metabolismo , Melanoma/terapia , Trasplante de Células Madre Mesenquimatosas/métodos , Animales , Línea Celular Tumoral , Células Cultivadas , Interleucina-12/genética , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Linfocitos T Citotóxicos/inmunología , Microambiente Tumoral/inmunología , Macrófagos Asociados a Tumores/inmunologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
BACKGROUND: Adipose tissue-derived mesenchymal stromal cells (ASCs) have been shown to exhibit some promising properties of their use in regenerative medicine as advanced therapy medicinal products (ATMP). However, different sources of their origin, methods of isolation, and expansion procedures cause the laboratory and clinical results difficult to compare. METHODS: ASCs were isolated from lipoaspirates and cultured in three different medium formulations: αMEM and DMEM as a basal medium supplemented with 10% of human platelet lysate (hPL) and DMEM supplemented with 20% fetal bovine serum (FBS) and bFGF as a gold standard medium. Subsequently, the impact of culture media on ASCs growth kinetics, their morphology and immunophenotype, ability to differentiate, clonogenic potential, and secretion profile was evaluated. RESULTS: All cultured ASCs lines showed similar morphology and similar clonogenic potential and have the ability to differentiate into three lines: adipocytes, osteoblasts, and chondroblasts. The immunophenotype of all cultured ASCs was consistent with the guidelines of the International Society for Cell Therapy (ISCT) allowing to define cells as mesenchymal stromal cell (MSC) (≥ 95% CD105, CD73, CD90 and ≤ 2% CD45, CD34, CD14, CD19, HLA-DR). The immunophenotype stabilized after the second passage and did not differ between ASCs cultured in different conditions. The exception was the ASCs grown in the presence of FBS and bFGF, which expressed CD146 antigens. The secretion profile of ASCs cultured in different media was similar. The main secreted cytokine was IL-6, and its level was donor-specific. However, we observed a strong influence of the medium formulation on ASCs growth kinetics. The proliferation rate of ASCs in medium supplemented with hPL was the highest. CONCLUSIONS: Culture media that do not contain animal-derived antigens (xeno-free) can be used to culture cells defined as MSC. Xeno-free medium is a safe alternative for the production of clinical-grade MSC as an advanced therapy medicinal product. Additionally, in such culture conditions, MSC can be easily expanded in accordance with the Good Manufacturing Process (GMP) requirements to a desired amount of cells for clinical applications.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Medios de Cultivo/farmacología , Adipogénesis , Tejido Adiposo/citología , Adulto , Antígeno CD146/metabolismo , Proliferación Celular , Células Cultivadas , Condrogénesis , Medios de Cultivo/química , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Inmunofenotipificación , Interleucina-6/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , OsteogénesisRESUMEN
OBJECTIVES: Application of non-invasive imaging methods plays an important role in the assessment of cellular therapy effects in peripheral artery disease. The purpose of this work was to evaluate the kinetics of MRI-derived parameters characterizing ischaemic hindlimb muscle after administration of human mesenchymal stromal cells derived from adipose tissue (hADSC) in mice. MATERIALS AND METHODS: MRI experiments were performed on a 9.4T Bruker system. The measurement protocol included transverse relaxation time mapping and diffusion tensor imaging. The monitoring period encompassed 14 days after femoral artery ligation and subsequent cell administration. The effect of hADSC transplantation was compared with the effect of normal human dermal fibroblasts (NHDFs) and phosphate-buffered saline injection. RESULTS: The most significant differences between the hADSC group and the remaining ones were observed around day 3 after ischaemia induction (increased transverse relaxation time in the hADSC group in comparison with the control group) and around day 7 (increased transverse relaxation time and decreased third eigenvalue of the diffusion tensor in the hADSC group in comparison with the control and NHDF groups) at the site of hADSC injection. Histologically, it was associated with increased macrophage infiltration at days 3-7 and with the presence of small regenerating fibres in the ischaemic tissue at day 7. CONCLUSIONS: Our results underscore the important role of macrophages in mediating the therapeutic effects of hADSCs and confirm the huge potential of magnetic resonance imaging in monitoring of cellular therapy effects.
Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular/fisiología , Isquemia/patología , Células Madre Mesenquimatosas/citología , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Imagen de Difusión Tensora/métodos , Fibroblastos/patología , Humanos , Masculino , Ratones Transgénicos , Neovascularización Fisiológica/fisiología , Regeneración/efectos de los fármacos , Regeneración/fisiologíaRESUMEN
BACKGROUND: Adipose-derived mesenchymal stromal cells (ADSCs) are multipotent stromal cells. The cells secrete a number of cytokines and growth factors and show immunoregulatory and proangiogenic properties. Their properties may be used to repair damaged tissues. The aim of our work is to explain the muscle damage repair mechanism with the utilization of the human adipose-derived mesenchymal stromal cells (hADSCs). METHODS: For the hADSCs isolation, we used the subcutaneous adipose tissue collected during the surgery. The murine hind limb ischemia was used as a model. The unilateral femoral artery ligation was performed on 10-12-week-old male C57BL/6NCrl and NOD SCID mice. The mice received PBS- (controls) or 1 × 106 hADSCs. One, 3, 7, 14 and 21 days after the surgery, we collected the gastrocnemius muscles for the immunohistochemical analysis. The results were analyzed with relevant tests using the Statistica software. RESULTS: The retention time of hADSCs in the limb lasted about 14 days. In the mice receiving hADSCs, the improvement in the functionality of the damaged limb occurred faster than in the control mice. More new blood vessels were formed in the limbs of the mice receiving hADSCs than in limbs of the control mice. hADSCs also increased the infiltration of the macrophages with the M2 phenotype (7-AAD-/CD45+/F4/80+/CD206+) into the ischemic limbs. hADSCs introduced into the limb of mice secreted interleukin-6. This cytokine stimulates the emergence of the proangiogenic M2 macrophages, involved, among others, in the repair of a damaged tissue. Both macrophage depletion and IL-6 blockage suppressed the therapeutic effect of hADSCs. In the mice treated with hADSCs and liposomes with clodronate (macrophages depletion), the number of capillaries formed was lower than in the mice treated with hADSCs alone. Administration of hADSCs to the mice that received siltuximab (human IL-6 blocker) did not cause an influx of the M2 macrophages, and the number of capillaries formed was at the level of the control group, as in contrast to the mice that received only the hADSCs. CONCLUSIONS: The proposed mechanism for the repair of the damaged muscle using hADSCs is based on the activity of IL-6. In our opinion, the cytokine, secreted by the hADSCs, stimulates the M2 macrophages responsible for repairing damaged muscle and forming new blood vessels.
Asunto(s)
Tejido Adiposo/metabolismo , Interleucina-6/biosíntesis , Macrófagos/metabolismo , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Músculo Esquelético , Tejido Adiposo/patología , Animales , Xenoinjertos , Humanos , Macrófagos/patología , Masculino , Células Madre Mesenquimatosas/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Músculo Esquelético/lesiones , Músculo Esquelético/metabolismo , Músculo Esquelético/patologíaRESUMEN
Vascular disrupting agents as DMXAA inhibit tumor growth only for a short period of time followed by rapid tumor regrowth. Among others, hypoxia and presence of transcription factor HIF-1α are responsible for tumors regrowth. The aim of our study was to investigate the inhibition of murine melanoma growth by combining two agents: anti-vascular - DMXAA and the HIF-1α inhibitor - digoxin and explaining the mechanism of action of this combination. After DMXAA treatment tumor size was reduced only for a limited time. After 7 days regrowth of tumors was observed and number of vessels was increased especially in tumor's peripheral areas. DMXAA also induced an influx of immune cells: macrophages, CD8+ cytotoxic lymphocytes, NK cells, CD4+ lymphocytes. Administration of digoxin alone inhibited the growth of tumors. Administration of both agents in the proper sequence significantly inhibited the regrowth of tumors better than either agents alone. Combination therapy reduced number of newly formed vessels. In tumors of mice treated with combination therapy, the number of macrophages M1, CD8+ cytotoxic lymphocytes, NK cells and to a lesser extent CD4+ cells was increased. The combination of anti-vascular agents with HIF-1α inhibitors appears to be an effective therapeutic option.
Asunto(s)
Antineoplásicos/farmacología , Digoxina/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Melanoma Experimental/patología , Melanoma/patología , Neovascularización Patológica/tratamiento farmacológico , Xantonas/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Melanoma/irrigación sanguínea , Melanoma/inmunología , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/tratamiento farmacológico , Ratones , Xantonas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor-associated macrophages (TAMs) play a significant role in at least two key processes underlying neoplastic progression: angiogenesis and immune surveillance. TAMs phenotypic changes play important role in tumor vessel abnormalization/ normalization. M2-like TAMs stimulate immunosuppression and formation of defective tumor blood vessels leading to tumor progression. In contrast M1-like TAMs trigger immune response and normalize irregular tumor vascular network which should sensitize cancer cells to chemo- and radiotherapy and lead to tumor growth regression. Here, we demonstrated that combination of endoglin-based DNA vaccine with interleukin 12 repolarizes TAMs from tumor growth-promoting M2-like phenotype to tumor growth-inhibiting M1-like phenotype. Combined therapy enhances tumor infiltration by CD4+, CD8+ lymphocytes and NK cells. Depletion of TAMs as well as CD8+ lymphocytes and NK cells, but not CD4+ lymphocytes, reduces the effect of combined therapy. Furthermore, combined therapy improves tumor vessel maturation, perfusion and reduces hypoxia. It caused that suboptimal doses of doxorubicin reduced the growth of tumors in mice treated with combined therapy. To summarize, combination of antiangiogenic drug and immunostimulatory agent repolarizes TAMs phenotype from M2-like (pro-tumor) into M1-like (anti-tumor) which affects the structure of tumor blood vessels, improves the effect of chemotherapy and leads to tumor growth regression.
Asunto(s)
Interleucina-12/administración & dosificación , Macrófagos/fisiología , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/inmunología , Neovascularización Patológica/patología , Microambiente Tumoral/inmunología , Inhibidores de la Angiogénesis/administración & dosificación , Animales , Antibióticos Antineoplásicos/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Proliferación Celular/efectos de los fármacos , Doxorrubicina/farmacología , Femenino , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Macrófagos/efectos de los fármacos , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/inmunología , Células Tumorales Cultivadas , Vacunas de ADN/administración & dosificaciónRESUMEN
AIMS: The aim of the present study was to isolate mesenchymal stromal cells (MSC) with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI). The study aimed also to investigate the feasibility of xenogeneic MSCs implantation. METHODS AND RESULTS: MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO). Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb). MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed. CONCLUSION: In two murine models (hindlimb ischemia and MI) we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart.
Asunto(s)
Antígenos CD34/metabolismo , Endoglina/metabolismo , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Infarto del Miocardio/terapia , Animales , Modelos Animales de Enfermedad , Fibrosis/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocardio/patologíaRESUMEN
Tumor progression depends on tumor milieu, which influences neovasculature formation and immunosuppression. Combining immunotherapy with antiangiogenic/antivascular therapy might be an effective therapeutic approach. The aim of our study was to elaborate an anticancer therapeutic strategy based on the induction of immune response which leads to polarization of tumor milieu. To achieve this, we developed a tumor cell-based vaccine. CAMEL peptide was used as a B16-F10 cell death-inducing agent. The lysates were used as a vaccine to immunize mice bearing B16-F10 melanoma tumors. To further improve the therapeutic effect of the vaccine, we combined it with interleukin (IL)-12 gene therapy. IL-12, a cytokine with antiangiogenic properties, activates nonspecific and specific immune responses. We observed that combined therapy is significantly more effective (as compared with monotherapies) in inhibiting tumor growth. Furthermore, the tested combination polarizes the tumor microenvironment, which results in a switch from a proangiogenic/immunosuppressive to an antiangiogenic/immunostimulatory one. The switch manifests itself as a decreased number of tumor blood vessels, increased levels of tumor-infiltrating CD4(+), CD8(+) and NK cells, as well as lower level of suppressor lymphocytes (Treg). Our results suggest that polarizing tumor milieu by such combined therapy does inhibit tumor growth and seems to be a promising therapeutic strategy.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Vacunas contra el Cáncer , Inmunoterapia Adoptiva/métodos , Interleucina-12/administración & dosificación , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Melanoma Experimental/terapia , Neoplasias Cutáneas/terapia , Animales , Péptidos Catiónicos Antimicrobianos/inmunología , Péptidos Catiónicos Antimicrobianos/metabolismo , Proliferación Celular/efectos de los fármacos , Terapia Combinada , Femenino , Terapia Genética , Humanos , Inmunización , Interleucina-12/genética , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Neoplasias Cutáneas/inmunología , Microambiente Tumoral/efectos de los fármacosRESUMEN
Mesenchymal stromal cells (MSCs) have been among the most intensively studied cells in recent years. Lack of specific unique markers for these cells makes it difficult to distinguish MSCs from other types of cells, such as fibroblasts or pericytes. MSCs are a mixture of morphologically different cells with expression of various cellular markers, with varying degrees of differentiation, as well as varying proliferation capacities. The majority of phenotypic features of these cells have been identified through cell culture. One of their basic features is the capacity to differentiate into three cell lines: osteoblasts, adipocytes and chondroblasts. Under in vivo conditions, MSCs form an important functional element of the hematopoietic stem cell niche. Residing within the blood vessel wall, MSCs assist in its formation and functioning. MSCs release antiapoptotic and proangiogenic factors, as well as agents that stimulate cell proliferation and also immunostimulating factors. In this study, we focused in particular on therapeutic strategies employing MSCs to improve the performance of the infarcted heart as well as on their involvement in the repair of hard-to-heal wounds. Thanks to the released anti-inflammatory agents, MSCs can inhibit inflammatory reactions. Owing to cytokines and growth factors they can also stimulate regeneration of damaged tissues and organs. The therapeutic effect that follows MSCs administration is linked to their paracrine activity.
Asunto(s)
Células Madre Mesenquimatosas/fisiología , Nicho de Células Madre/fisiología , Adipocitos/fisiología , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Humanos , Osteoblastos/fisiología , Comunicación Paracrina/fisiología , RegeneraciónRESUMEN
According to literature data, self-renewing, multipotent, and clonogenic cardiac c-Kit(+) progenitor cells occur within human myocardium. The aim of this study was to isolate and characterize c-Kit(+) progenitor cells from explanted human hearts. Experimental material was obtained from 19 adult and 7 pediatric patients. Successful isolation and culture was achieved for 95 samples (84.1%) derived from five different regions of the heart: right and left ventricles, atrium, intraventricular septum, and apex. The average percentage of c-Kit(+) cells, as assessed by FACS, ranged between 0.7 and 0.9%. In contrast to published data we do not observed statistically significant differences in the number of c-Kit(+) cells between disease-specific groups, parts of the heart or sexes. Nevertheless, c-Kit(+) cells were present in significant numbers (11-24%) in samples derived from three explanted pediatric hearts. c-Kit(+) cells were also positive for CD105 and a majority of them was positive for CD31 and CD34 (83.7 ± 8.6 and 75.7 ± 11.4%, respectively). Immunohistochemical analysis of the heart tissue revealed that most cells possessing the c-Kit antigen were also positive for tryptase, a specific mast cell marker. However, flow cytometry analysis has shown cultured c-Kit(+) cells to be negative for hematopoietic marker CD45 and mast cell marker CD33. Isolated c-Kit(+) cells display mesenchymal stem cell features and are thought to differentiate into endothelial cells.
Asunto(s)
Células Madre Mesenquimatosas/citología , Miocardio/citología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Adolescente , Adulto , Factores de Edad , Anciano , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular , Niño , Preescolar , Femenino , Citometría de Flujo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Triptasas/metabolismo , Adulto JovenRESUMEN
Myocardial infarction is underoxygenation-driven limited necrosis of heart tissues which results in elimination of ca. 0.5 to 1 billion spontaneously contracting cardiomyocytes (CM). Since the ability of human heart to regenerate is limited, efforts have been undertaken to increase the number of cardiomyocytes in post-infarction myocardium. Theoretically, such proposals might involve transplantation of 1) skeletal myoblasts and cardiomyocytes, or 2) progenitor/stem cells, theoretically capable of differentiating into cardiomyocytes, or 3) pluripotent cells such as embryonal stem cells (ESC) and induced pluripotent stem cells (iPSC) differentiating into cardiomyocytes. The efforts to increase CM could also involve 4) in situ reprogramming of fibroblasts into active cardiomyocyte-like cells, or 5) stimulating in situ proliferation of cardiomyocytes using pharmacological agents. Only three proposals merit closer scrutiny (2, 4 and 5). However, preclinical and clinical data have demonstrated weak ability of progenitor cells to differentiate (proposal 2). Nevertheless, transplanted cell-induced paracrine effects accompanying such therapy do improve functioning of the damaged heart muscle. The proposals that would permit the number of CM to be increased include in situ reprogramming of fibroblasts into active cardiomyocytes (proposal 4), as well as in situ stimulation of quiescent cardiomyocytes' proliferation (proposal 5). It appears that an optimized therapeutic solution (increasing left ventricular ejection fraction and decreasing the post-infarct scar) might combine agents stimulating paracrine effects and reprogramming of fibroblasts.
Asunto(s)
Cardiomioplastia/métodos , Células Madre Embrionarias/trasplante , Células Madre Pluripotentes Inducidas/trasplante , Mioblastos/trasplante , Infarto del Miocardio/cirugía , Miocitos Cardíacos/trasplante , Células Madre Embrionarias/citología , Fibroblastos/citología , Humanos , Miocitos Cardíacos/citologíaRESUMEN
D-K6L9 peptide is bound by phosphatidylserine and induces necrosis in cancer cells. In our therapeutic experience, this peptide, when administered directly into B16-F10 murine melanoma tumors, inhibited their growth. Cessation of therapy results, however, in tumor relapse. We aimed at developing a combined therapy involving D-K6L9 and additional factors that would yield complete elimination of tumor cells in experimental animals. To this purpose, we employed glycyrrhizin, an inhibitor of HMGB1 protein, BP1 peptide and interleukin (IL)-12. Glycyrrhizin or BP1, when combined with D-K6L9, inhibits growth of primary tumors only during the period of their administration. A long-term tumor growth inhibitory effect was obtained only in combining D-K6L9 with IL-12. At 2 months following therapy cessation, 60 % of animals were alive. Prolonged survival was noted in mice bearing B16-F10 tumors as well as in mice bearing C26 colon carcinoma tumors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma/terapia , Neoplasias del Colon/terapia , Melanoma Experimental/terapia , Fragmentos de Péptidos/administración & dosificación , Neoplasias Cutáneas/terapia , Animales , Antiinflamatorios/administración & dosificación , Carcinoma/inmunología , Procesos de Crecimiento Celular/efectos de los fármacos , Neoplasias del Colon/inmunología , Femenino , Ácido Glicirrínico/administración & dosificación , Humanos , Interleucina-12/administración & dosificación , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Péptidos/administración & dosificación , Péptidos/química , Recurrencia , Neoplasias Cutáneas/inmunología , Factores de Tiempo , Carga Tumoral/efectos de los fármacosRESUMEN
Development and neoplastic progression strongly rely on tumor microenvironment cells. Various kinds of cells that form such tumor milieu play substantial roles in angiogenesis and immunosuppression. Attempts to inhibit tumor vascularization alter tumor milieu and enhance immune response against the tumor. Anticancer therapeutic strategy bringing together antiangiogenic and immunostimulating agents has emerged as a promising approach. We here investigated whether therapy directed against preexisting vessels, combined with an immunomodulatory factor would be equally effective in arresting tumor growth. To this goal, we investigated the effectiveness of ABRaA-vascular endothelial growth factor isoform 121 (VEGF121), an antivascular drug constructed by us. It is a fusion protein composed of VEGF121, and abrin A chain (translation-inhibiting toxin). We used it in combination with interleukin (IL-12) gene therapy and tried to inhibit B16-F10 melanoma tumor growth. ABRaA-VEGF121 is a chimeric recombinant protein capable of destroying tumor vasculature and triggering necrosis in the vicinity of damaged vessels. IL-12 cytokine, in turn, activates both specific and non-specific immune responses. Our results demonstrate that combination of ABRaA-VEGF121 antivascular agent with immunostimulatory cytokine IL-12 indeed inhibits tumor growth more effectively than either agent alone, leading to complete cure of ca. 20 % mice. Post-therapeutic analysis of tumors excised from mice treated with combination therapy showed decreased numbers of blood microvessels in the tumor microenvironment, lowered numbers of regulatory T lymphocytes, as well as showed higher levels of CD4(+) and CD8(+) as compared to control mice. It seems that bringing together antivascular strategy and the action of immunostimulating agents indeed inhibits growth of tumors.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Interleucina-12/metabolismo , Melanoma Experimental/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Proteínas Recombinantes de Fusión/administración & dosificación , Linfocitos T Reguladores/efectos de los fármacos , Abrina/genética , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Procesos de Crecimiento Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Terapia Genética , Humanos , Interleucina-12/genética , Melanoma Experimental/irrigación sanguínea , Ratones , Ratones Endogámicos C57BL , Microvasos/efectos de los fármacos , Microvasos/patología , Proteínas Recombinantes de Fusión/genética , Linfocitos T Reguladores/inmunología , Microambiente Tumoral , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
A potential anti-melanoma prodrug containing a phenolic activator, a hydrazine linker, and a nitrogen mustard effector - (N-{4-[bis-(2-chloroethyl)amino]benzoyl}-N'-(4-hydroxybenzyl)hydrazine) has been synthesized in seven steps. Spectrophotometric measurements of its oxidation by tyrosinase showed a rapid increase of absorbance at 337 nm. HPLC analysis demonstrated that two major products were formed. However, during the reaction one of the products was converted into the other. The stable product with a maximum of absorption at 337 nm was isolated and identified as 5,6-dihydroxy-1H-indazol-1-yl 4-[bis-(2-chloroethyl)amino]benzoate. It was formed by a cyclization of the enzymatically generated o-quinone. This reaction was unexpected, since the acylated hydrazine nitrogen atom should not be sufficiently nucleophilic to attack the o-quinone ring. This cyclization prevented the effector release from the enzyme-activated prodrug. As a result, the prodrug showed only limited specificity for B16-F10 murine melanoma cells compared to reference cell lines. When applied in solid tumors in mice it showed slightly higher activity than the parent mustard drug (4-[bis-(2-chloroethyl)amino]benzoic cid), but significantly lower activity than melphalan, a commercial mustard drug with a structure resembling tyrosine, occasionally used in the treatment of melanoma.
Asunto(s)
Hidrazinas/química , Hidrazinas/farmacología , Mecloretamina/química , Mecloretamina/farmacología , Melanoma Experimental/tratamiento farmacológico , Profármacos/química , Profármacos/farmacología , Animales , Línea Celular Tumoral , Ciclización , Hidrazinas/síntesis química , Hidrazinas/metabolismo , Mecloretamina/síntesis química , Mecloretamina/metabolismo , Ratones , Monofenol Monooxigenasa/metabolismo , Profármacos/síntesis química , Profármacos/metabolismoRESUMEN
Blood vascular supply significantly affects progression of tumor growth. Inhibition of endothelial cell proliferation by antiangiogenic drugs should lead to growth arrest of both primary tumors and metastases. During the course of lengthy therapy, endothelial cells may, however, become refractory to the action of antiangiogenic agents. Novel approaches to anticancer treatment should explore the issue of drug resistance shown by endothelial cells. One possible therapeutic solution might be tumor immunotherapy directed against antigens expressed on the surface of endothelial cells which co-form tumor blood vasculature. Such therapy is supposed to break immune tolerance to own antigens and to eliminate tumor blood vessel endothelial cells by activating cytotoxic T lymphocytes. This kind of response can be obtained against endoglin (CD105). Endoglin is overexpressed in proliferating endothelial cells which line tumor blood vessels. Presence of endoglin in solid tumor blood vessels has prognostic value in cancer treatment. CD105 is also expressed by certain cancer cells (prostate, melanoma and Ewing sarcoma). It appears that therapeutic strategies directed against endoglin allow several mechanisms of resistance to antiangiogenic drugs to be omitted. The therapeutic approach that we propose, i.e. a tumor blood vessel-destroying strategy combined with immunotherapy, may become an effective therapeutic tool.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antígenos CD/efectos de los fármacos , Antígenos CD/inmunología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/inmunología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/inmunología , Animales , Endoglina , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Inmunoterapia , Neoplasias/irrigación sanguínea , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Glioblastoma multiforme is the most common and a particularly aggressive form of glial primary brain tumors. This malignancy accounts for ca. 70% of all diagnosed cases. Unfortunately, average survival of glioma patients does not exceed one year from diagnosis. Specific vascularization pattern (presence of numerous microvessels and glomerular vessels) and exceptional invasiveness are characteristic features of glioblastoma tumors. Both of these features reflect complex underlying processes forming two vicious circles. Common to both of these circles is the state of tumor underoxygenation. Hypoxia that occurs in the vicinity of abnormal tumor blood vessels stimulates formation of novel microvessels and invasiveness of tumor cells. In their essence, both of the vicious circles are processes allowing tumor cells to adapt to an underoxygenated tumor milieu. These processes play an important role in tumor progression, which reflects a specific type of evolution of cancer cells. Late effects of this evolution include appearance of highly aggressive, chemo- and radiotherapy resistant neoplastic cells. Increased adaptation capabilities of such cancer cells have a negative influence on the therapeutic process. Effective therapeutic strategies should not be directed against single cancer cell markers; instead, they should be targeted so as to break both vicious cycles. Herein we discuss several such strategies. In our opinion, effective therapeutic approaches must include a combination of several agents that recognize and simultaneously break both vicious cycles, i.e. vascularization and invasiveness. Also, agents that decrease hypoxia in cancer cells, for example drugs inhibiting activity of HIF-1α, might also prove therapeutically effective in such approaches.
Asunto(s)
Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/patología , Glioblastoma/irrigación sanguínea , Glioblastoma/patología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Progresión de la Enfermedad , Glioblastoma/tratamiento farmacológico , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Invasividad Neoplásica/patología , Invasividad Neoplásica/fisiopatología , Neovascularización PatológicaRESUMEN
HMGB1 is an evolutionarily conserved protein with a wide spectrum of action. Its main receptors are RAGE and TLR found on the surface of immune system cells as well as endothelial cells. Although signaling pathways for both receptor groups are different, ultimately they both activate NFκB transcription factor which, in turn, activates genes encoding adhesion proteins, proinflammatory cytokines and proangiogenic factors. Inside cells, HMGB1 is found mainly in the cell nucleus, where it participates in replication, recombination, transcription and DNA repair processes. Following release into the extracellular space, HMGB1 becomes a proinflammatory cytokine which stimulates formation of new blood microvessels, enhances cell migration, activates the inflammatory condition and affects cell proliferation. HMGB1 protein also takes part in regeneration of damaged tissues and stimulates autophagy. HMGB1 plays a potential role in anticancer therapy. Increased amounts of HMGB1 in cancer cells and elevated levels in the bloodstream are noted among patients afflicted with various cancers. HMGB1 protects cells from apoptosis, as it affects telomere stability. HMGB1 also stimulates a number of proteins involved in proliferation of cancer cells and inhibits signals that control cell growth. Ability to arrest HMGB1 release from cells or to inhibit its activity appears to be a promising therapeutic approach. At present, several inhibitors of HMGB1 are known and can be used in anticancer therapy.
Asunto(s)
Proteína HMGB1/antagonistas & inhibidores , Proteína HMGB1/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Apoptosis , Autofagia , Ciclo Celular , Movimiento Celular , Proliferación Celular , Células Endoteliales/metabolismo , Humanos , FN-kappa B/metabolismo , Neoplasias/irrigación sanguínea , Neoplasias/patología , Transducción de SeñalRESUMEN
One of the preconditions of effective anticancer therapy is efficient transfer of the therapeutic agent (chemotherapeutic) to tumor cells. Fundamental barriers making drug delivery and action difficult include underoxygenation, elevated interstitial pressure, poor and abnormal tumor blood vascular network and acidic tumor milieu. In this study we aimed at developing an optimized scheme of administering a combination of an angiogenesis-inhibiting drug (vasostatin) and a chemotherapeutic (cyclophosphamide) in the therapeutic treatment of mice bearing experimental B16-F10 melanoma tumors. We report that the strongest tumor growth inhibition was observed in mice that received two, three or four vasostatin doses in combination with one injection of cyclophosphamide (i.e., V2 + CTX, V3 + CTX or V4 + CTX schemes). Double administration of vasostatin increases oxygenation of B16-F10 tumors. On the other hand, its five-fold administration lowers tumor oxygenation, breaks down tumor vascular network (increasing hypoxia) and leads in consequence to death of cancer cells and appearance of necrotic areas in the tumor. A decreased cyclophosphamide dose in combination with two doses of vasostatin (V2 + CTX scheme) inhibits tumor growth similarly to a larger dose of cyclophosphamide alone.
Asunto(s)
Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Calreticulina/uso terapéutico , Ciclofosfamida/uso terapéutico , Melanoma Experimental/tratamiento farmacológico , Fragmentos de Péptidos/uso terapéutico , Animales , Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/normas , Calreticulina/administración & dosificación , Hipoxia de la Célula , Línea Celular Tumoral , Ciclofosfamida/administración & dosificación , Femenino , Proteína HMGB1/metabolismo , Inmunohistoquímica , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Oxígeno/metabolismo , Fragmentos de Péptidos/administración & dosificaciónRESUMEN
Neoplastic cells which co-form tumors are usually subjected to various stress factors, mainly hypoxia and shortage of nutrient factors. Such cells employ different strategies that permit their survival under such conditions. Experiments in vitro are usually carried out in the presence of 21% oxygen and medium supplemented with 10% FBS. Altering these parameters can approximate the in vivo conditions found within tumor mass. The present paper reports certain properties (especially ability to metastasize) of B16-F10 cells able to grow upon exposure to altered growth conditions (medium supplemented with 0.06% FBS or presence of 1% oxygen for 24 or 72 hours). These properties were compared with those of control cells cultured in the presence of 21% oxygen and in medium supplemented with 10% FBS. Some properties of the cells exposed to medium supplemented with 0.06% FBS differ from those of cells cultured under low oxygenation conditions (ability to form metastases, to migrate, or to express various proteins). Only the partial deprivation of oxygen did increase both the number of migrating cells and the number of metastases formed. Serum deficiency enhanced only the cell ability to metastasize, but not to migrate. It appears that cultured B16-F10 cells employ different adaptation strategies under conditions of oxygen shortage and those of serum deficiency. Under oxygen deprivation, such cells most likely undergo an epithelial-mesenchymal transition, whereas serum deficiency ("starvation"), while increasing the tumorigenicity of B16-F10 cells, does not induce the epithelial-mesenchymal transition.