RESUMEN
Exosomes are nano-sized extracellular vesicles that contain DNA, RNA, proteins, and lipids. Exosomes likely participate in facilitating intercellular communication and tumor growth. In this issue of Immunity, Zhang et al. (2019) report on the metabolic activity of B cell-derived exosomes in facilitating the suppression of cytotoxic T cells.
Asunto(s)
Exosomas , Neoplasias , Linfocitos B , Linfocitos T CD8-positivos , Comunicación Celular , HumanosRESUMEN
Skin wound repair is essential for organismal survival and failure of which leads to non-healing wounds, a leading health issue worldwide. However, mechanistic understanding of chronic wounds remains a major challenge due to lack of appropriate genetic mouse models. αSMA+ myofibroblasts, a unique class of dermal fibroblasts, are associated with cutaneous wound healing but their precise function remains unknown. We demonstrate that genetic depletion of αSMA+ myofibroblasts leads to pleiotropic wound healing defects, including lack of reepithelialization and granulation, dampened angiogenesis, and heightened hypoxia, hallmarks of chronic non-healing wounds. Other wound-associated FAP+ and FSP1+ fibroblasts do not exhibit such dominant functions. While type I collagen (COL1) expressing cells play a role in the repair process, COL1 produced by αSMA+ myofibroblasts is surprisingly dispensable for wound repair. In contrast, we show that ß1 integrin from αSMA+ myofibroblasts, but not TGFßRII, is essential for wound healing, facilitating contractility, reepithelization, and vascularization. Collectively, our study provides evidence for the functions of myofibroblasts in ß1 integrin-mediated wound repair with potential implications for treating chronic non-healing wounds.
Asunto(s)
Colágeno Tipo I , Miofibroblastos , Cicatrización de Heridas , Animales , Colágeno Tipo I/genética , Fibroblastos , Integrina beta1/genética , Ratones , PielRESUMEN
Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers1-10 and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity11-15, although these have been less well-studied in ICB treatment16. A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling17 that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter18) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.
Asunto(s)
Linfocitos B/inmunología , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/inmunología , Inmunoterapia , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Estructuras Linfoides Terciarias/inmunología , Linfocitos B/citología , Linfocitos B/metabolismo , Biomarcadores de Tumor/análisis , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/cirugía , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/inmunología , Células Clonales/citología , Células Clonales/inmunología , Células Clonales/metabolismo , Células Dendríticas Foliculares/citología , Células Dendríticas Foliculares/inmunología , Regulación Neoplásica de la Expresión Génica , Humanos , Memoria Inmunológica/inmunología , Espectrometría de Masas , Melanoma/patología , Melanoma/cirugía , Metástasis de la Neoplasia/genética , Fenotipo , Pronóstico , RNA-Seq , Receptores Inmunológicos/inmunología , Análisis de la Célula Individual , Linfocitos T/citología , Linfocitos T/inmunología , TranscriptomaRESUMEN
BACKGROUND: Type IV collagen is an abundant component of basement membranes in all multicellular species and is essential for the extracellular scaffold supporting tissue architecture and function. Lower organisms typically have two type IV collagen genes, encoding α1 and α2 chains, in contrast with the six genes in humans, encoding α1-α6 chains. The α chains assemble into trimeric protomers, the building blocks of the type IV collagen network. The detailed evolutionary conservation of type IV collagen network remains to be studied. RESULTS: We report on the molecular evolution of type IV collagen genes. The zebrafish α4 non-collagenous (NC1) domain, in contrast with its human ortholog, contains an additional cysteine residue and lacks the M93 and K211 residues involved in sulfilimine bond formation between adjacent protomers. This may alter α4 chain interactions with other α chains, as supported by temporal and anatomic expression patterns of collagen IV chains during the zebrafish development. Despite the divergence between zebrafish and human α3 NC1 domain (endogenous angiogenesis inhibitor, Tumstatin), the zebrafish α3 NC1 domain exhibits conserved antiangiogenic activity in human endothelial cells. CONCLUSIONS: Our work supports type IV collagen is largely conserved between zebrafish and humans, with a possible difference involving the α4 chain.
Asunto(s)
Colágeno Tipo IV , Pez Cebra , Animales , Humanos , Colágeno Tipo IV/genética , Células Endoteliales , Subunidades de Proteína/análisis , Subunidades de Proteína/metabolismo , Membrana Basal/metabolismoRESUMEN
The mutant form of the GTPase KRAS is a key driver of pancreatic cancer but remains a challenging therapeutic target. Exosomes are extracellular vesicles generated by all cells, and are naturally present in the blood. Here we show that enhanced retention of exosomes, compared to liposomes, in the circulation of mice is likely due to CD47-mediated protection of exosomes from phagocytosis by monocytes and macrophages. Exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry short interfering RNA or short hairpin RNA specific to oncogenic KrasG12D, a common mutation in pancreatic cancer. Compared to liposomes, the engineered exosomes (known as iExosomes) target oncogenic KRAS with an enhanced efficacy that is dependent on CD47, and is facilitated by macropinocytosis. Treatment with iExosomes suppressed cancer in multiple mouse models of pancreatic cancer and significantly increased overall survival. Our results demonstrate an approach for direct and specific targeting of oncogenic KRAS in tumours using iExosomes.
Asunto(s)
Exosomas/metabolismo , Silenciador del Gen , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Proteínas Proto-Oncogénicas p21(ras)/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Animales , Antígeno CD47/metabolismo , Modelos Animales de Enfermedad , Exosomas/inmunología , Femenino , Terapia Genética , Liposomas/inmunología , Ratones , Monocitos/citología , Monocitos/inmunología , Metástasis de la Neoplasia/prevención & control , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/patología , Pinocitosis , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Tasa de SupervivenciaRESUMEN
The Stimulator of Interferon Genes (STING) pathway is implicated in the innate immune response and is important in both oncogenesis and cancer treatment. Specifically, activation of the cytosolic DNA sensor STING in antigen-presenting cells (APCs) induces a type I interferon response and cytokine production that facilitates antitumor immune therapy. However, use of STING agonists (STINGa) as a cancer therapeutic has been limited by unfavorable pharmacological properties and targeting inefficiency due to rapid clearance and limited uptake into the cytosol. Exosomes, a class of extracellular vesicles shed by all cells are under consideration for their use as effective carriers of drugs owing to their innate ability to be taken up by cells and their biocompatibility for optimal drug biodistribution. Therefore, we engineered exosomes to deliver the STING agonist cyclic GMP-AMP (iExoSTINGa), to exploit their favorable pharmacokinetics and pharmacodynamics. Selective targeting of the STING pathway in APCs with iExoSTINGa was associated with superior potency compared with STINGa alone in suppressing B16F10 tumor growth. Moreover, iExoSTINGa showed superior uptake of STINGa into dendritic cells compared with STINGa alone, which led to increased accumulation of activated CD8+ T-cells and an antitumor immune response. Our study highlights the potential of exosomes in general, and iExoSTINGa specifically, in enhancing cancer therapy outcomes.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Exosomas/metabolismo , Inmunidad Innata/inmunología , Melanoma Experimental/inmunología , Proteínas de la Membrana/agonistas , Nucleótidos Cíclicos/farmacología , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Inmunidad Innata/efectos de los fármacos , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Melanoma Experimental/prevención & control , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
Hepatic fibrosis is a wound healing response that results in excessive extracellular matrix (ECM) accumulation in response to chronic hepatic injury. Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor associated with the pathogenesis of liver fibrosis. Though a promising potential therapeutic target, there are no specific drug candidates for STAT3. Exosomes are extracellular vesicles generated by all cell types with a capacity to efficiently enter cells across different biological barriers. Here, we utilize exosomes as delivery conduit to specifically target STAT3 in liver fibrosis. Exosomes derived from clinical grade fibroblast-like mesenchymal stem cells (MSCs) were engineered to carry siRNA or antisense oligonucleotide (ASO) targeting STAT3 (iExosiRNA-STAT3 or iExomASO-STAT3 ). Compared to scrambled siRNA control, siRNA-STAT3, or ASO-STAT3, iExosiRNA-STAT3 or iExomASO-STAT3 showed enhanced STAT3 targeting efficiency. iExosiRNA-STAT3 or iExomASO-STAT3 treatments suppressed STAT3 levels and ECM deposition in established liver fibrosis in mice, and significantly improved liver function. iExomASO-Stat3 restored liver function more efficiently when compared to iExosiRNA-STAT3 . Our results identify a novel anti-fibrotic approach for direct targeting of STAT3 with exosomes with immediate translational potential.
Asunto(s)
Exosomas/genética , Regulación de la Expresión Génica , Cirrosis Hepática/terapia , Oligonucleótidos Antisentido/farmacología , ARN Interferente Pequeño/genética , Factor de Transcripción STAT3/antagonistas & inhibidores , Animales , Tetracloruro de Carbono/toxicidad , Femenino , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Ratones , Ratones Endogámicos BALB C , Transducción de SeñalRESUMEN
The inherent plasticity and resiliency of fibroblasts make this cell type a conventional tool for basic research. But where do they come from, are all fibroblasts the same, and how do they function in disease? The first fibroblast lineages in mammalian development emerge from the ooze of primary mesenchyme during gastrulation. They are cells that efficiently create and negotiate the extracellular matrix of the mesoderm in order to migrate and meet their developmental fate. Mature fibroblasts in epithelial tissues live in the interstitial spaces between basement membranes that spatially delimit complex organ structures. While the function of resident fibroblasts in healthy tissues is largely conjecture, the accumulation of fibroblasts in pathologic lesions offers insight into biologic mechanisms that control their function; fibroblasts are poised to coordinate fibrogenesis in tissue injury, neoplasia, and aging. Here, we examine the developmental origin and plasticity of fibroblasts, their molecular and functional definitions, the epigenetic control underlying their identity and activation, and the evolution of their immune regulatory functions. These topics are reviewed through the lens of fate mapping using genetically engineered mouse models and from the perspective of single-cell RNA sequencing. Recent observations suggest dynamic and heterogeneous functions for fibroblasts that underscore their complex molecular signatures and utility in injured tissues.
Asunto(s)
Fibroblastos/metabolismo , Fibroblastos/fisiología , Envejecimiento/fisiología , Animales , Epigénesis Genética/genética , Humanos , Análisis de Secuencia de ARNRESUMEN
Metastatic dissemination employs both the blood and lymphatic vascular systems. Solid tumors dynamically remodel and generate both vessel types during cancer progression. Lymphatic vessel invasion and cancer cells in the tumor-draining lymph nodes (LNs) are prognostic markers for breast cancer metastasis and patient outcome, and tumor-induced lymphangiogenesis likely influences metastasis. Deregulated tumor tissue fluid homeostasis and immune trafficking associated with tumor lymphangiogenesis may contribute to metastatic spreading; however, the precise functional characterization of lymphatic endothelial cells (LECs) in tumors is challenged by the lack of specific reagents to decipher their rate-limiting role in metastasis. Therefore, we generated novel transgenic mice (PDPN promoter-driven Cre recombinase transgene [PDPN-Cre] and PDPN promoter-driven thymidine kinase transgene [PDPN-tk]) that allow for the identification and genetically controlled depletion of proliferating podoplanin (Pdpn)-expressing LECs. We demonstrate that suppression of lymphangiogenesis is successfully achieved in lymphangioma lesions induced in the PDPN-tk mice. In multiple metastatic breast cancer mouse models, we identified distinct roles for LECs in primary and metastatic tumors. Our findings support the functional contribution of primary tumor lymphangiogenesis in controlling metastasis to axillary LNs and lung parenchyma. Reduced lymphatic vessel density enhanced primary tumor lymphedema and increased the frequency of intratumoral macrophages but was not associated with a significant impact on primary tumor growth despite a marked reduction in metastatic dissemination. Our findings identify the rate-limiting contribution of the breast tumor lymphatic vessels for lung metastasis.
Asunto(s)
Neoplasias de la Mama/metabolismo , Glicoproteínas de Membrana/fisiología , Animales , Neoplasias de la Mama/fisiopatología , Movimiento Celular , Células Endoteliales/patología , Células Endoteliales/fisiología , Femenino , Humanos , Ganglios Linfáticos/patología , Linfangiogénesis/genética , Linfangiogénesis/fisiología , Sistema Linfático/fisiología , Vasos Linfáticos/patología , Macrófagos , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Metástasis de la Neoplasia/fisiopatología , Timidina Quinasa/genéticaRESUMEN
Diagnosis of pancreatic ductal adenocarcinoma (PDAC) is associated with a dismal prognosis despite current best therapies; therefore new treatment strategies are urgently required. Numerous studies have suggested that epithelial-to-mesenchymal transition (EMT) contributes to early-stage dissemination of cancer cells and is pivotal for invasion and metastasis of PDAC. EMT is associated with phenotypic conversion of epithelial cells into mesenchymal-like cells in cell culture conditions, although such defined mesenchymal conversion (with spindle-shaped morphology) of epithelial cells in vivo is rare, with quasi-mesenchymal phenotypes occasionally observed in the tumour (partial EMT). Most studies exploring the functional role of EMT in tumours have depended on cell-culture-induced loss-of-function and gain-of-function experiments involving EMT-inducing transcription factors such as Twist, Snail and Zeb1 (refs 2, 3, 7-10). Therefore, the functional contribution of EMT to invasion and metastasis remains unclear, and genetically engineered mouse models to address a causal connection are lacking. Here we functionally probe the role of EMT in PDAC by generating mouse models of PDAC with deletion of Snail or Twist, two key transcription factors responsible for EMT. EMT suppression in the primary tumour does not alter the emergence of invasive PDAC, systemic dissemination or metastasis. Suppression of EMT leads to an increase in cancer cell proliferation with enhanced expression of nucleoside transporters in tumours, contributing to enhanced sensitivity to gemcitabine treatment and increased overall survival of mice. Collectively, our study suggests that Snail- or Twist-induced EMT is not rate-limiting for invasion and metastasis, but highlights the importance of combining EMT inhibition with chemotherapy for the treatment of pancreatic cancer.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Transición Epitelial-Mesenquimal , Metástasis de la Neoplasia/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Masculino , Ratones , Invasividad Neoplásica/patología , Proteínas de Transporte de Nucleósidos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Factores de Transcripción de la Familia Snail , Análisis de Supervivencia , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína 1 Relacionada con Twist/deficiencia , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo , GemcitabinaRESUMEN
Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer-cell-derived exosomes in the circulation is currently lacking. Using mass spectrometry analyses, we identify a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer-cell-derived exosomes. GPC1(+) circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of patients and mice with cancer. GPC1(+) crExos were detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Levels of GPC1(+) crExos correlate with tumour burden and the survival of pre- and post-surgical patients. GPC1(+) crExos from patients and from mice with spontaneous pancreatic tumours carry specific KRAS mutations, and reliably detect pancreatic intraepithelial lesions in mice despite negative signals by magnetic resonance imaging. GPC1(+) crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy.
Asunto(s)
Exosomas/metabolismo , Glipicanos , Neoplasias Pancreáticas/diagnóstico , Animales , Biomarcadores/sangre , Línea Celular Tumoral , Exosomas/genética , Femenino , Glipicanos/sangre , Glipicanos/metabolismo , Células HCT116 , Humanos , Células MCF-7 , Masculino , Ratones , Células 3T3 NIH , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas p21(ras) , Proteínas ras/metabolismoRESUMEN
Structures similar to blood vessels in location, morphology, flexibility, and transparency have been recovered after demineralization of multiple dinosaur cortical bone fragments from multiple specimens, some of which are as old as 80 Ma. These structures were hypothesized to be either endogenous to the bone (i.e., of vascular origin) or the result of biofilm colonizing the empty osteonal network after degradation of original organic components. Here, we test the hypothesis that these structures are endogenous and thus retain proteins in common with extant archosaur blood vessels that can be detected with high-resolution mass spectrometry and confirmed by immunofluorescence. Two lines of evidence support this hypothesis. First, peptide sequencing of Brachylophosaurus canadensis blood vessel extracts is consistent with peptides comprising extant archosaurian blood vessels and is not consistent with a bacterial, cellular slime mold, or fungal origin. Second, proteins identified by mass spectrometry can be localized to the tissues using antibodies specific to these proteins, validating their identity. Data are available via ProteomeXchange with identifier PXD001738.
Asunto(s)
Vasos Sanguíneos/anatomía & histología , Vasos Sanguíneos/metabolismo , Dinosaurios/anatomía & histología , Dinosaurios/metabolismo , Fósiles/anatomía & histología , Actinas/genética , Actinas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Vasos Sanguíneos/microbiología , Huesos/irrigación sanguínea , Pollos , Dinosaurios/genética , Técnica del Anticuerpo Fluorescente/métodos , Espectrometría de Masas , Modelos Biológicos , Datos de Secuencia Molecular , Miosinas/genética , Miosinas/aislamiento & purificación , Filogenia , Proteómica/métodos , Alineación de Secuencia , Especificidad de la Especie , Struthioniformes , Tropomiosina/genética , Tropomiosina/aislamiento & purificación , Tubulina (Proteína)/genética , Tubulina (Proteína)/aislamiento & purificaciónRESUMEN
Increased numbers of S100A4(+) cells are associated with poor prognosis in patients who have cancer. Although the metastatic capabilities of S100A4(+) cancer cells have been examined, the functional role of S100A4(+) stromal cells in metastasis is largely unknown. To study the contribution of S100A4(+) stromal cells in metastasis, we used transgenic mice that express viral thymidine kinase under control of the S100A4 promoter to specifically ablate S100A4(+) stromal cells. Depletion of S100A4(+) stromal cells significantly reduced metastatic colonization without affecting primary tumor growth. Multiple bone marrow transplantation studies demonstrated that these effects of S100A4(+) stromal cells are attributable to local non-bone marrow-derived S100A4(+) cells, which are likely fibroblasts in this setting. Reduction in metastasis due to the loss of S100A4(+) fibroblasts correlated with a concomitant decrease in the expression of several ECM molecules and growth factors, particularly Tenascin-C and VEGF-A. The functional importance of stromal Tenascin-C and S100A4(+) fibroblast-derived VEGF-A in metastasis was established by examining Tenascin-C null mice and transgenic mice expressing Cre recombinase under control of the S100A4 promoter crossed with mice carrying VEGF-A alleles flanked by loxP sites, which exhibited a significant decrease in metastatic colonization without effects on primary tumor growth. In particular, S100A4(+) fibroblast-derived VEGF-A plays an important role in the establishment of an angiogenic microenvironment at the metastatic site to facilitate colonization, whereas stromal Tenascin-C may provide protection from apoptosis. Our study demonstrates a crucial role for local S100A4(+) fibroblasts in providing the permissive "soil" for metastatic colonization, a challenging step in the metastatic cascade.
Asunto(s)
Proteínas S100/metabolismo , Células del Estroma/metabolismo , Tenascina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Línea Celular Tumoral , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Ganciclovir/farmacología , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Metástasis de la Neoplasia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína de Unión al Calcio S100A4 , Proteínas S100/genética , Células del Estroma/efectos de los fármacos , Tenascina/genética , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Microambiente Tumoral/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Hypoxia is associated with tissue injury and fibrosis but its functional role in fibroblast activation and tissue repair/regeneration is unknown. Using kidney injury as a model system, we demonstrate that injured epithelial cells produce an increased number of exosomes with defined genetic information to activate fibroblasts. Exosomes released by injured epithelial cells promote proliferation, α-smooth muscle actin expression, F-actin expression, and type I collagen production in fibroblasts. Fibroblast activation is dependent on exosomes delivering TGF-ß1 mRNA among other yet to be identified moieties. This study suggests that TGF-ß1 mRNA transported by exosomes constitutes a rapid response to initiate tissue repair/regenerative responses and activation of fibroblasts when resident parenchyma is injured. The results also inform potential utility of exosome-targeted therapies to control tissue fibrosis.
Asunto(s)
Riñón/lesiones , Riñón/fisiopatología , Regeneración/fisiología , Factor de Crecimiento Transformador beta1/fisiología , Animales , Hipoxia de la Célula/fisiología , Células Cultivadas , Células Epiteliales/fisiología , Exosomas/fisiología , Fibroblastos/fisiología , Fibrosis , Humanos , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Células 3T3 NIH , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regeneración/genética , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
mRNA incorporated in lipid nanoparticles (LNPs) became a new class of vaccine modality for induction of immunity against COVID-19 and ushered in a new era in vaccine development. Here, we report a novel, easy-to-execute, and cost effective engineered extracellular vesicles (EVs)-based combined mRNA and protein vaccine platform (EVX-M+P vaccine) and explore its utility in proof-of-concept immunity studies in the settings of cancer and infectious disease. As a first example, we engineered EVs to contain ovalbumin mRNA and protein (EVOvaM+P) to serve as cancer vaccine against ovalbumin-expressing melanoma tumors. EVOvaM+P administration to mice with established melanoma tumors resulted in tumor regression associated with effective humoral and adaptive immune responses. As a second example, we generated engineered EVs, natural nanoparticle carriers shed by all cells, that contain mRNA and protein Spike (S) protein to serve as a combined mRNA and protein vaccine (EVSpikeM+P vaccine) against SARS-CoV-2 infection. EVSpikeM+P vaccine administration in mice and baboons elicited robust production of neutralizing IgG antibodies against RBD (receptor binding domain) of S protein and S protein specific T cell responses. Our proof-of-concept study describes a new platform with an ability for rapid development of combination mRNA and protein vaccines employing EVs for deployment against cancer and other diseases.
RESUMEN
Glomerular filtration relies on the type IV collagen (ColIV) network of the glomerular basement membrane, namely, in the triple helical molecules containing the α3, α4, and α5 chains of ColIV. Loss of function mutations in the genes encoding these chains (Col4a3, Col4a4, and Col4a5) is associated with the loss of renal function observed in Alport syndrome (AS). Precise understanding of the cellular basis for the patho-mechanism remains unknown and a specific therapy for this disease does not currently exist. Here, we generated a novel allele for the conditional deletion of Col4a3 in different glomerular cell types in mice. We found that podocytes specifically generate α3 chains in the developing glomerular basement membrane, and that its absence is sufficient to impair glomerular filtration as seen in AS. Next, we show that horizontal gene transfer, enhanced by TGFß1 and using allogenic bone marrow-derived mesenchymal stem cells and induced pluripotent stem cells, rescues Col4a3 expression and revive kidney function in Col4a3-deficient AS mice. Our proof-of-concept study supports that horizontal gene transfer such as cell fusion enables cell-based therapy in Alport syndrome.
Asunto(s)
Nefritis Hereditaria , Podocitos , Ratones , Animales , Nefritis Hereditaria/genética , Nefritis Hereditaria/metabolismo , Podocitos/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Membrana Basal Glomerular/metabolismo , Células Madre/metabolismoRESUMEN
Extracellular vesicles (EVs) are generated by all cells and systemic administration of allogenic EVs derived from epithelial and mesenchymal cells have been shown to be safe, despite carrying an array of functional molecules, including thousands of proteins. To address whether epithelial cells derived EVs can be modified to acquire the capacity to induce immune response, we engineered 293T EVs to harbor the immunomodulatory CD80, OX40L and PD-L1 molecules. We demonstrated abundant levels of these proteins on the engineered cells and EVs. Functionally, the engineered EVs efficiently elicit positive and negative co-stimulation in human and murine T cells. In the setting of cancer and auto-immune hepatitis, the engineered EVs modulate T cell functions and alter disease progression. Moreover, OX40L EVs provide additional benefit to anti-CTLA-4 treatment in melanoma-bearing mice. Our work provides evidence that epithelial cell derived EVs can be engineered to induce immune responses with translational potential to modulate T cell functions in distinct pathological settings.
RESUMEN
Oncogenic KRASG12D (KRAS∗) is critical for the initiation and maintenance of pancreatic ductal adenocarcinoma (PDAC) and is a known repressor of tumor immunity. Conditional elimination of KRAS∗ in genetic mouse models of PDAC leads to the reactivation of FAS, CD8+ T cell-mediated apoptosis, and complete eradication of tumors. KRAS∗ elimination recruits activated CD4+ and CD8+ T cells and promotes the activation of antigen-presenting cells. Mechanistically, KRAS∗-mediated immune evasion involves the epigenetic regulation of Fas death receptor in cancer cells, via methylation of its promoter region. Furthermore, analysis of human RNA sequencing identifies that high KRAS expression in PDAC tumors shows a lower proportion of CD8+ T cells and demonstrates shorter survival compared with tumors with low KRAS expression. This study highlights the role of CD8+ T cells in the eradication of PDAC following KRAS∗ elimination and provides a rationale for the combination of KRAS∗ targeting with immunotherapy to control PDAC.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas p21(ras) , Animales , Humanos , Ratones , Apoptosis , Carcinoma Ductal Pancreático/genética , Linfocitos T CD8-positivos , Epigénesis Genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is associated with mutations in Kras, a known oncogenic driver of PDAC; and the KRAS G12D mutation is present in nearly half of PDAC patients. Recently, a non-covalent small molecule inhibitor (MRTX1133) was identified with specificity to the Kras G12D mutant protein. Here we explore the impact of Kras G12D inhibition by MRTX1133 on advanced PDAC and its influence on the tumor microenvironment. Employing different orthotopic xenograft and syngeneic tumor models, eight different PDXs, and two different autochthonous genetic models, we demonstrate that MRTX1133 reverses early PDAC growth, increases intratumoral CD8 + effector T cells, decreases myeloid infiltration, and reprograms cancer associated fibroblasts. Autochthonous genetic mouse models treated with MRTX1133 leads to regression of both established PanINs and advanced PDAC. Regression of advanced PDAC requires CD8 + T cells and immune checkpoint blockade therapy (iCBT) synergizes with MRTX1133 to eradicate PDAC and prolong overall survival. Mechanistically, inhibition of mutant Kras in advanced PDAC and human patient derived organoids (PDOs) induces Fas expression in cancer cells and facilitates CD8 + T cell mediated death. These results demonstrate the efficacy of MRTX1133 in different mouse models of PDAC associated with reprogramming of stromal fibroblasts and a dependency on CD8 + T cell mediated tumor clearance. Collectively, this study provides a rationale for a synergistic combination of MRTX1133 with iCBT in clinical trials.