RESUMO
Tumor-associated macrophages (TAMs) continuously fine tune their immune modulatory properties, but how gene expression programs coordinate this immune cell plasticity is largely unknown. Selective mRNA translation, controlled by MNK1/MNK2 and mTOR pathways impinging on eIF4E, facilitates reshaping of proteomes without changes in abundance of corresponding mRNAs. Using polysome profiling developed for small samples we show that, during tumor growth, gene expression in TAMs is predominately modulated via mRNA-selective changes in translational efficiencies. These alterations in gene expression paralleled accumulation of antiinflammatory macrophages with augmented phosphorylation of eIF4E, a target of the MNK1 and MNK2 kinases, known to selectively modulate mRNA translation. Furthermore, suppression of the MNK2, but not the mTOR signaling pathway, reprogrammed antiinflammatory macrophages toward a proinflammatory phenotype with the ability to activate CD8+ T cells. Thus, selective changes of mRNA translation depending on MNK2 signaling represents a key node regulating macrophage antiinflammatory functions.
Assuntos
Macrófagos/imunologia , Neoplasias/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Técnicas de Cocultura , Modelos Animais de Doenças , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Naftiridinas/farmacologia , Neoplasias/genética , Neoplasias/patologia , Fosforilação/genética , Fosforilação/imunologia , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Evasão Tumoral/genéticaRESUMO
We describe a lentivirus-encoded chimeric receptor, termed extracellular vesicle (EV)-internalizing receptor (EVIR), which enables the selective uptake of cancer-cell-derived EVs by dendritic cells (DCs). The EVIR enhances DC presentation of EV-associated tumor antigens to CD8+ T cells primarily through MHCI recycling and cross-dressing. EVIRs should facilitate exploring the mechanisms and implications of horizontal transfer of tumor antigens to antigen-presenting cells.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/imunologia , Células Dendríticas/imunologia , Vesículas Extracelulares/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Animais , Células Cultivadas , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Vesículas Extracelulares/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Mannose receptor (MRC1/CD206) has been suggested to mediate allergic sensitization and asthma to multiple glycoallergens, including cockroach allergens. OBJECTIVE: We sought to determine the existence of a protective mechanism through which MRC1 limits allergic inflammation through its intronic miR-511-3p. METHODS: We examined MRC1-mediated cockroach allergen uptake by lung macrophages and lung inflammation using C57BL/6 wild-type (WT) and Mrc1-/- mice. The role of miR-511-3p in macrophage polarization and cockroach allergen-induced lung inflammation in mice transfected with adeno-associated virus (AAV)-miR-511-3p (AAV-cytomegalovirus-miR-511-3p-enhanced green fluorescent protein) was analyzed. Gene profiling of macrophages with or without miR-511-3p overexpression was also performed. RESULTS: Mrc1-/- lung macrophages showed a significant reduction in cockroach allergen uptake compared with WT mice, and Mrc1-/- mice had an exacerbated lung inflammation with increased levels of cockroach allergen-specific IgE and TH2/TH17 cytokines in a cockroach allergen-induced mouse model compared with WT mice. Macrophages from Mrc1-/- mice showed significantly reduced levels of miR-511-3 and an M1 phenotype, whereas overexpression of miR-511-3p rendered macrophages to exhibit a M2 phenotype. Furthermore, mice transfected with AAV-miR-511-3p showed a significant reduction in cockroach allergen-induced inflammation. Profiling of macrophages with or without miR-511-3p overexpression identified 729 differentially expressed genes, wherein expression of prostaglandin D2 synthase (Ptgds) and its product PGD2 were significantly downregulated by miR-511-3p. Ptgds showed a robust binding to miR-511-3p, which might contribute to the protective effect of miR-511-3p. Plasma levels of miR-511-3p were significantly lower in human asthmatic patients compared with nonasthmatic subjects. CONCLUSION: These studies support a critical but previously unrecognized role of MRC1 and miR-511-3p in protection against allergen-induced lung inflammation.
Assuntos
Hipersensibilidade/etiologia , Hipersensibilidade/metabolismo , Lectinas Tipo C/metabolismo , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Lectinas de Ligação a Manose/metabolismo , MicroRNAs/genética , Receptores de Superfície Celular/metabolismo , Alérgenos/imunologia , Animais , Asma/etiologia , Asma/metabolismo , Asma/patologia , Baratas/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Vetores Genéticos/genética , Hipersensibilidade/patologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Receptor de Manose , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Pneumonia/etiologia , Pneumonia/metabolismo , Pneumonia/patologia , Interferência de RNA , Receptores de Superfície Celular/genética , Receptores ImunológicosRESUMO
OBJECTIVE: Perivascular cells, including pericytes, macrophages, smooth muscle cells, and other specialized cell types, like podocytes, participate in various aspects of vascular function. However, aside from the well-established roles of smooth muscle cells and pericytes, the contributions of other vascular-associated cells are poorly understood. Our goal was to ascertain the function of perivascular macrophages in adult tissues under nonpathological conditions. APPROACH AND RESULTS: We combined confocal microscopy, in vivo cell depletion, and in vitro assays to investigate the contribution of perivascular macrophages to vascular function. We found that resident perivascular macrophages are associated with capillaries at a frequency similar to that of pericytes. Macrophage depletion using either clodronate liposomes or antibodies unexpectedly resulted in hyperpermeability. This effect could be rescued when M2-like macrophages, but not M1-like macrophages or dendritic cells, were reconstituted in vivo, suggesting subtype-specific roles for macrophages in the regulation of vascular permeability. Furthermore, we found that permeability-promoting agents elicit motility and eventual dissociation of macrophages from the vasculature. Finally, in vitro assays showed that M2-like macrophages attenuate the phosphorylation of VE-cadherin upon exposure to permeability-promoting agents. CONCLUSIONS: This study points to a direct contribution of macrophages to vessel barrier integrity and provides evidence that heterotypic cell interactions with the endothelium, in addition to those of pericytes, control vascular permeability.
Assuntos
Capilares/metabolismo , Permeabilidade Capilar , Comunicação Celular , Células Endoteliais/metabolismo , Macrófagos Peritoneais/metabolismo , Mesentério/irrigação sanguínea , Peritônio/irrigação sanguínea , Pele/irrigação sanguínea , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Dextranos/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Ovalbumina/metabolismo , Fenótipo , Fosforilação , Rodaminas/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
PHD2 serves as an oxygen sensor that rescues blood supply by regulating vessel formation and shape in case of oxygen shortage. However, it is unknown whether PHD2 can influence arteriogenesis. Here we studied the role of PHD2 in collateral artery growth by using hindlimb ischaemia as a model, a process that compensates for the lack of blood flow in case of major arterial occlusion. We show that Phd2 (also known as Egln1) haplodeficient (Phd2(+/-)) mice displayed preformed collateral arteries that preserved limb perfusion and prevented tissue necrosis in ischaemia. Improved arteriogenesis in Phd2(+/-) mice was due to an expansion of tissue-resident, M2-like macrophages and their increased release of arteriogenic factors, leading to enhanced smooth muscle cell (SMC) recruitment and growth. Both chronic and acute deletion of one Phd2 allele in macrophages was sufficient to skew their polarization towards a pro-arteriogenic phenotype. Mechanistically, collateral vessel preconditioning relied on the activation of canonical NF-κB pathway in Phd2(+/-) macrophages. These results unravel how PHD2 regulates arteriogenesis and artery homeostasis by controlling a specific differentiation state in macrophages and suggest new treatment options for ischaemic disorders.
Assuntos
Artérias/crescimento & desenvolvimento , Extremidades/irrigação sanguínea , Isquemia/prevenção & controle , Macrófagos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/deficiência , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Alelos , Animais , Modelos Animais de Doenças , Extremidades/patologia , Feminino , Heterozigoto , Homeostase , Prolina Dioxigenases do Fator Induzível por Hipóxia , Isquemia/patologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Miócitos de Músculo Liso/citologia , NF-kappa B/metabolismo , Necrose , Fenótipo , Pró-Colágeno-Prolina Dioxigenase/genéticaRESUMO
microRNAs (miRNAs) are short noncoding RNAs, which regulate gene expression post-transcriptionally and play crucial roles in relevant biological and pathological processes. Here, we investigated the putative role of miRNAs in modulating the tumor-initiating potential of mouse medulloblastoma (MB)-derived cancer stem cells (CSCs). We first subjected bona fide highly tumorigenic (HT) CSCs as well as lowly tumorigenic MB CSCs and normal neural stem cells to miRNA profiling, which identified a HT CSC-specific miRNA signature. Next, by cross-checking CSC mRNA/miRNA profiles, we pinpointed miR-135a as a potential tumor suppressor gene, which was strongly downregulated in HT CSCs as well as in the highly malignant experimental tumors derived from them. Remarkably, enforced expression of miR-135a in HT CSCs strongly inhibited tumorigenesis by repressing the miR-135a direct target gene Arhgef6. Considering the upregulation of Arhgef6 in human MBs and its involvement in mediating experimental medulloblastomagenesis, its efficient suppression by miR-135a might make available an effective therapeutic strategy to selectively impair the tumorigenic potential of MB CSCs. Stem Cells 2015;33:1377-1389.
Assuntos
Carcinogênese/patologia , Meduloblastoma/patologia , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Animais , Carcinogênese/genética , Agregação Celular , Transformação Celular Neoplásica/genética , Regulação para Baixo , Perfilação da Expressão Gênica , Meduloblastoma/genética , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Deregulation of microRNAs (miRNAs) can drive oncogenesis, tumor progression, and metastasis by acting cell-autonomously in cancer cells. However, solid tumors are also infiltrated by large amounts of non-neoplastic stromal cells, including macrophages, which express several active miRNAs. Tumor-associated macrophages (TAMs) enhance angiogenic, immunosuppressive, invasive, and metastatic programming of neoplastic tissue and reduce host survival. Here, we review the role of miRNAs (including miR-155, miR-146, and miR-511) in the control of macrophage production and activation, and examine whether reprogramming miRNA activity in TAMs and/or their precursors might be effective for controlling tumor progression.
Assuntos
Diferenciação Celular/imunologia , Macrófagos/imunologia , MicroRNAs/imunologia , Neoplasias/imunologia , Animais , Carcinogênese , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Ativação de MacrófagosRESUMO
The ability of macrophages to promote vascular growth has been associated with the secretion and local delivery of classic proangiogenic factors (e.g., VEGF-A and proteases). More recently, a series of studies have also revealed that physical contact of macrophages with growing blood vessels coordinates vascular fusion of emerging sprouts. Interestingly, the interactions between macrophages and vascular endothelial cells (ECs) appear to be bidirectional, such that activated ECs also support the expansion and differentiation of proangiogenic macrophages from myeloid progenitors. Here, we discuss recent findings suggesting that dynamic angiogenic vascular niches might also exist in vivo, e.g. in tumors, where sprouting blood vessels and immature myeloid cells like monocytes engage in heterotypic interactions that are required for angiogenesis. Finally, we provide an account of emerging mechanisms of cell-to-cell communication that rely on secreted microvesicles, such as exosomes, which can offer a vehicle for the rapid exchange of molecules and genetic information between macrophages and ECs engaged in angiogenesis.
Assuntos
Comunicação Celular , Diferenciação Celular , Endotélio Vascular/citologia , Macrófagos/citologia , Neovascularização Patológica , Neovascularização Fisiológica , Animais , HumanosRESUMO
VisualZoneR is an R-based technique used to analyze spatial transcriptomics data generated by employing Visium or Visium HD technology. Here, we present a protocol to identify compartmental zones from single-cell spatial transcriptomics using VisualZoneR. We describe steps for identifying distinct zones ranging from healthy liver tissue to inner metastatic areas and measuring transcriptomic changes. We then detail procedures for integrating distinct samples and grouping transcriptomic spots into compartmental zones according to their relative distance from the tumor/liver parenchyma boundary.
RESUMO
M1 activation of macrophages promotes inflammation and immunity to intracellular pathogens, whereas M2 macrophage activation promotes resolution of inflammation, wound healing, and tumor growth. These divergent phenotypes are characterized, in part, by the expression of inducible NO synthase and arginase I (Arg1) in M1 versus M2 activated macrophages, respectively. In this study, we demonstrate that the Ron receptor tyrosine kinase tips the balance of macrophage activation by attenuating the M1 phenotype while promoting expression of Arg1 through a Stat6-independent mechanism. Induction of the Arg1 promoter by Ron is mediated by an AP-1 site located 433 bp upstream of the transcription start site. Treatment of primary macrophages with macrophage stimulating protein, the ligand for Ron, induces potent MAPK activation, upregulates Fos, and enhances binding of Fos to the AP-1 site in the Arg1 promoter. In vivo, Arg1 expression in tumor-associated macrophages (TAMs) from Ron(-/-) mice was significantly reduced compared with that in TAMs from control animals. Furthermore, we show that Ron is expressed specifically by Tie2-expressing macrophages, a TAM subset that exhibits a markedly skewed M2 and protumoral phenotype. Decreased Arg1 in TAMs from Ron(-/-) mice was associated with reduced syngeneic tumor growth in these animals. These findings indicate that Ron induces Arg1 expression in macrophages through a previously uncharacterized AP-1 site in the Arg1 promoter and that Ron could be therapeutically targeted in the tumor microenvironment to inhibit tumor growth by targeting expression of Arg1.
Assuntos
Arginase/biossíntese , Regulação da Expressão Gênica/imunologia , Macrófagos/enzimologia , Neoplasias Experimentais/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Arginase/genética , Arginase/imunologia , Separação Celular , Citometria de Fluxo , Expressão Gênica , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/imunologiaRESUMO
Use of autologous cells isolated from elderly patients with multiple comorbidities may account for the modest efficacy of cell therapy in patients with chronic limb threatening ischemia (CLTI). We aimed to determine whether proarteriogenic monocyte/macrophages (Mo/MΦs) from patients with CLTI were functionally impaired and to demonstrate the mechanisms related to any impairment. Proarteriogenic Mo/MΦs isolated from patients with CLTI were found to have an impaired capacity to promote neovascularization in vitro and in vivo compared with those isolated from healthy controls. This was associated with increased expression of human HIV-1 TAT interactive protein-2 (HTATIP2), a transcription factor known to suppress angiogenesis/arteriogenesis. Silencing HTATIP2 restored the functional capacity of CLTI Mo/MΦs, which was associated with increased expression of arteriogenic regulators Neuropilin-1 and Angiopoietin-1, and their ability to enhance angiogenic (endothelial tubule formation) and arteriogenic (smooth muscle proliferation) processes in vitro. In support of the translational relevance of our findings, silencing HTATIP2 in proarteriogenic Mo/MΦs isolated from patients with CLTI rescued their capacity to enhance limb perfusion in the ischemic hindlimb by effecting greater angiogenesis and arteriogenesis. Ex vivo modulation of HTATIP2 may offer a strategy for rescuing the functional impairment of pro-angio/arteriogenic Mo/MΦs prior to autologous delivery and increase the likelihood of clinical efficacy.
Assuntos
Monócitos , Neovascularização Fisiológica , Animais , Camundongos , Humanos , Idoso , Monócitos/metabolismo , Circulação Colateral , Músculo Esquelético/metabolismo , Camundongos Knockout , Isquemia/metabolismo , Fatores de Transcrição , AcetiltransferasesRESUMO
The expression of pro-lymphangiogenic VEGF-C in primary tumors is associated with sentinel lymph node metastasis in most solid cancer types. However, the impact of VEGF-C on distant organ metastasis remains unclear. Perivascular tumor-associated macrophages (TAMs) play a crucial role in guiding hematogenous spread of cancer cells by establishing metastatic pathways within the tumor microenvironment. This process supports breast cancer cell intravasation and metastatic dissemination. We show here that VEGF-C-expressing TAMs reduce the dissemination of mammary cancer cells to the lungs while concurrently increasing lymph node metastasis. These TAMs express podoplanin and interact with normalized tumor blood vessels expressing VEGFR3. Moreover, clinical data suggest inverse association between VEGF-C-expressing TAMs and breast cancer malignancy. Thus, our study elucidates the paradoxical role of VEGF-C-expressing TAMs in redirecting cancer cells to preferentially disseminate to lymph nodes rather than to lungs, partially achieved by normalizing tumor blood vessels and promoting lymphangiogenesis.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Metástase Linfática , Neoplasias da Mama/patologia , Macrófagos Associados a Tumor/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Linfangiogênese , Microambiente TumoralRESUMO
Liver metastases are associated with poor response to current pharmacological treatments, including immunotherapy. We describe a lentiviral vector (LV) platform to selectively engineer liver macrophages, including Kupffer cells and tumor-associated macrophages (TAMs), to deliver type I interferon (IFNα) to liver metastases. Gene-based IFNα delivery delays the growth of colorectal and pancreatic ductal adenocarcinoma liver metastases in mice. Response to IFNα is associated with TAM immune activation, enhanced MHC-II-restricted antigen presentation and reduced exhaustion of CD8+ T cells. Conversely, increased IL-10 signaling, expansion of Eomes CD4+ T cells, a cell type displaying features of type I regulatory T (Tr1) cells, and CTLA-4 expression are associated with resistance to therapy. Targeting regulatory T cell functions by combinatorial CTLA-4 immune checkpoint blockade and IFNα LV delivery expands tumor-reactive T cells, attaining complete response in most mice. These findings support a promising therapeutic strategy with feasible translation to patients with unmet medical need.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias Hepáticas , Humanos , Camundongos , Animais , Antígeno CTLA-4/metabolismo , Microambiente Tumoral/genética , Macrófagos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patologiaRESUMO
OBJECTIVE: The role of semaphorins in tumor progression is still poorly understood. In this study, we aimed at elucidating the regulatory role of semaphorin 3A (SEMA3A) in primary tumor growth and metastatic dissemination. METHODS AND RESULTS: We used 3 different experimental approaches in mouse tumor models: (1) overexpression of SEMA3A in tumor cells, (2) systemic expression of SEMA3A following liver gene transfer in mice, and (3) tumor-targeted release of SEMA3A using gene modified Tie2-expressing monocytes as delivery vehicles. In each of these experimental settings, SEMA3A efficiently inhibited tumor growth by inhibiting vessel function and increasing tumor hypoxia and necrosis, without promoting metastasis. We further show that the expression of the receptor neuropilin-1 in tumor cells is required for SEMA3A-dependent inhibition of tumor cell migration in vitro and metastatic spreading in vivo. CONCLUSIONS: In sum, both systemic and tumor-targeted delivery of SEMA3A inhibits tumor angiogenesis and tumor growth in multiple mouse models; moreover, SEMA3A inhibits the metastatic spreading from primary tumors. These data support the rationale for further investigation of SEMA3A as an anticancer molecule.
Assuntos
Neoplasias da Mama/terapia , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Neoplasias Pulmonares/prevenção & controle , Neovascularização Patológica/prevenção & controle , Semaforina-3A/metabolismo , Células-Tronco/metabolismo , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Hipóxia Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Necrose , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neuropilina-1/metabolismo , Comunicação Parácrina , Interferência de RNA , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Semaforina-3A/genética , Transdução de Sinais , Células Estromais/metabolismo , Fatores de Tempo , Transfecção , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Stem and progenitor cells residing in the intestinal crypts drive the majority of colorectal cancers (CRCs), yet vascular contribution to this niche remains largely unexplored. VEGFA is a key driver of physiological and tumor angiogenesis. Accordingly, current anti-angiogenic cancer therapies target the VEGFA pathway. Here we report that in CRC expansion of the stem/progenitor pool in intestinal crypts requires VEGFA-independent growth and remodeling of blood vessels. Epithelial transformation induced expression of the endothelial peptide apelin, directs migration of distant venous endothelial cells towards progenitor niche vessels ensuring optimal perfusion. In the absence of apelin, loss of injury-inducible PROX1+ epithelial progenitors inhibited both incipient and advanced intestinal tumor growth. Our results establish fundamental principles for the reciprocal communication between vasculature and the intestinal progenitor niche and provide a mechanism for resistance to VEGFA-targeting drugs in CRCs.
RESUMO
Lentiviral vectors (LVs) are increasingly employed in gene and cell therapy. Standard laboratory production of LVs is not easily scalable, and research-grade LVs often contain contaminants that can interfere with downstream applications. Moreover, purified LV production pipelines have been developed mainly for costly, large-scale, clinical-grade settings. Therefore, a standardized and cost-effective process is still needed to obtain efficient, reproducible, and properly executed experimental studies and preclinical development of ex vivo and in vivo gene therapies, as high infectivity and limited adverse reactions are important factors potentially influencing experimental outcomes also in preclinical settings. We describe here an optimized laboratory-scale workflow whereby an LV-containing supernatant is purified and concentrated by sequential chromatographic steps, obtaining biologically active LVs with an infectious titer and specific activity in the order of 109 transducing unit (TU)/mL and 5 × 104 TU/ng of HIV Gag p24, respectively. The purification workflow removes >99% of the starting plasmid, DNA, and protein impurities, resulting in higher gene transfer and editing efficiency in severe combined immunodeficiency (SCID)-repopulating hematopoietic stem and progenitor cells (HSPCs) ex vivo, as well as reduced activation of inflammatory responses ex vivo and in vivo as compared to TU-matched, laboratory-grade vectors. Our results highlight the value of accessible purified LV production for experimental studies and preclinical testing.
RESUMO
Mutations in APC promote colorectal cancer (CRC) progression through uncontrolled WNT signaling. Patients with desmoplastic CRC have a significantly worse prognosis and do not benefit from chemotherapy, but the mechanisms underlying the differential responses of APC-mutant CRCs to chemotherapy are not well understood. We report that expression of the transcription factor prospero homeobox 1 (PROX1) was reduced in desmoplastic APC-mutant human CRCs. In genetic Apc-mutant mouse models, loss of Prox1 promoted the growth of desmoplastic, angiogenic, and immunologically silent tumors through derepression of Mmp14. Although chemotherapy inhibited Prox1-proficient tumors, it promoted further stromal activation, angiogenesis, and invasion in Prox1-deficient tumors. Blockade of vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2) combined with CD40 agonistic antibodies promoted antiangiogenic and immunostimulatory reprogramming of Prox1-deficient tumors, destroyed tumor fibrosis, and unleashed T cell-mediated killing of cancer cells. These results pinpoint the mechanistic basis of chemotherapy-induced hyperprogression and illustrate a therapeutic strategy for chemoresistant and desmoplastic CRCs.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos Imunológicos/farmacologia , Neoplasias Colorretais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imunoterapia , Neovascularização Patológica , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/imunologia , Angiopoietina-2/genética , Angiopoietina-2/imunologia , Animais , Linhagem Celular , Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/terapia , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/imunologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Humanos , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/imunologia , Camundongos , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Neovascularização Patológica/genética , Neovascularização Patológica/imunologia , Neovascularização Patológica/terapia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
Microvascular dysfunction and resulting tissue hypoxia is a major contributor to the pathogenesis and evolution of cardiovascular diseases (CVD). Diverse gene and cell therapies have been proposed to preserve the microvasculature or boost angiogenesis in CVD, with moderate benefit. This study tested in vivo the impact of sequential delivery by bone-marrow (BM) cells of the pro-angiogenic factors vascular endothelial growth factor (VEGFA) and sphingosine-1-phosphate (S1P) in a myocardial infarction model. For that, mouse BM cells were transduced with lentiviral vectors coding for VEGFA or sphingosine kinase (SPHK1), which catalyzes S1P production, and injected them intravenously 4 and 7 days after cardiac ischemia-reperfusion in mice. Sequential delivery by transduced BM cells of VEGFA and S1P led to increased endothelial cell numbers and shorter extravascular distances in the infarct zone, which support better oxygen diffusion 28 days post myocardial infarction, as shown by automated 3D image analysis of the microvasculature. Milder effects were observed in the remote zone, together with increased proportion of capillaries. BM cells delivering VEGFA and S1P also decreased myofibroblast abundance and restricted adverse cardiac remodeling without major impact on cardiac contractility. The results indicate that BM cells engineered to deliver VEGFA/S1P angiogenic factors sequentially may constitute a promising strategy to improve micro-vascularization and oxygen diffusion, thus limiting the adverse consequences of cardiac ischemia.
Assuntos
Células da Medula Óssea/metabolismo , Lisofosfolipídeos/administração & dosagem , Infarto do Miocárdio/genética , Infarto do Miocárdio/terapia , Neovascularização Patológica/genética , Esfingosina/análogos & derivados , Fator A de Crescimento do Endotélio Vascular/genética , Remodelação Ventricular/genética , Animais , Biomarcadores , Terapia Baseada em Transplante de Células e Tecidos , Modelos Animais de Doenças , Terapia Genética , Humanos , Camundongos , Infarto do Miocárdio/diagnóstico , Neovascularização Patológica/tratamento farmacológico , Esfingosina/administração & dosagem , Remodelação Ventricular/efeitos dos fármacosRESUMO
Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca2+-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C+CCR2+ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the pro-metastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy.