RESUMEN
The antitumor immunity can be enhanced through the synchronized codelivery of antigens and immunostimulatory adjuvants to antigen-presenting cells, particularly dendritic cells (DCs), using nanovaccines (NVs). To study the influence of intracellular vaccine cargo release kinetics on the T cell activating capacities of DCs, we compared stimuli-responsive to nonresponsive polymersome NVs. To do so, we employed "AND gate" multiresponsive (MR) amphiphilic block copolymers that decompose only in response to the combination of chemical cues present in the environment of the intracellular compartments in antigen cross-presenting DCs: low pH and high reactive oxygen species (ROS) levels. After being unmasked by ROS, pH-responsive side chains are exposed and can undergo a charge shift within a relevant pH window of the intracellular compartments in antigen cross-presenting DCs. NVs containing the model antigen Ovalbumin (OVA) and the iNKT cell activating adjuvant α-Galactosylceramide (α-Galcer) were fabricated using microfluidics self-assembly. The MR NVs outperformed the nonresponsive NV in vitro, inducing enhanced classical- and cross-presentation of the OVA by DCs, effectively activating CD8+, CD4+ T cells, and iNKT cells. Interestingly, in vivo, the nonresponsive NVs outperformed the responsive vaccines. These differences in polymersome vaccine performance are likely linked to the kinetics of cargo release, highlighting the crucial chemical requirements for successful cancer nanovaccines.
Asunto(s)
Nanovacunas , Vacunas , Animales , Ratones , Especies Reactivas de Oxígeno , Linfocitos T CD8-positivos , Células Dendríticas , Antígenos/química , Adyuvantes Inmunológicos/farmacología , Vacunas/química , Ovalbúmina , Concentración de Iones de Hidrógeno , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: While immune checkpoint inhibitors such as anti-PD-L1 antibodies have revolutionized cancer treatment, only subgroups of patients show durable responses. Insight in the relation between clinical response, PD-L1 expression and intratumoral localization of PD-L1 therapeutics could improve patient stratification. Therefore, we present the modular synthesis of multimodal antibody-based imaging tools for multiscale imaging of PD-L1 to study intratumoral distribution of PD-L1 therapeutics. RESULTS: To introduce imaging modalities, a peptide containing a near-infrared dye (sulfo-Cy5), a chelator (DTPA), an azide, and a sortase-recognition motif was synthesized. This peptide and a non-fluorescent intermediate were used for site-specific functionalization of c-terminally sortaggable mouse IgG1 (mIgG1) and Fab anti-PD-L1. To increase the half-life of the Fab fragment, a 20 kDa PEG chain was attached via strain-promoted azide-alkyne cycloaddition (SPAAC). Biodistribution and imaging studies were performed with 111In-labeled constructs in 4T1 tumor-bearing mice. Comparing our site-specific antibody-conjugates with randomly conjugated antibodies, we found that antibody clone, isotype and method of DTPA conjugation did not change tumor uptake. Furthermore, addition of sulfo-Cy5 did not affect the biodistribution. PEGylated Fab fragment displayed a significantly longer half-life compared to unPEGylated Fab and demonstrated the highest overall tumor uptake of all constructs. PD-L1 in tumors was clearly visualized by SPECT/CT, as well as whole body fluorescence imaging. Immunohistochemistry staining of tumor sections demonstrated that PD-L1 co-localized with the fluorescent and autoradiographic signal. Intratumoral localization of the imaging agent could be determined with cellular resolution using fluorescent microscopy. CONCLUSIONS: A set of molecularly defined multimodal antibody-based PD-L1 imaging agents were synthesized and validated for multiscale monitoring of PD-L1 expression and localization. Our modular approach for site-specific functionalization could easily be adapted to other targets.
Asunto(s)
Inmunoconjugados , Neoplasias , Animales , Antígeno B7-H1/metabolismo , Línea Celular Tumoral , Humanos , Inmunoconjugados/metabolismo , Inmunohistoquímica , Ratones , Neoplasias/diagnóstico por imagen , Distribución TisularRESUMEN
Plasticity of cancer invasion and metastasis depends on the ability of cancer cells to switch between collective and single-cell dissemination, controlled by cadherin-mediated cell-cell junctions. In clinical samples, E-cadherin-expressing and -deficient tumours both invade collectively and metastasize equally, implicating additional mechanisms controlling cell-cell cooperation and individualization. Here, using spatially defined organotypic culture, intravital microscopy of mammary tumours in mice and in silico modelling, we identify cell density regulation by three-dimensional tissue boundaries to physically control collective movement irrespective of the composition and stability of cell-cell junctions. Deregulation of adherens junctions by downregulation of E-cadherin and p120-catenin resulted in a transition from coordinated to uncoordinated collective movement along extracellular boundaries, whereas single-cell escape depended on locally free tissue space. These results indicate that cadherins and extracellular matrix confinement cooperate to determine unjamming transitions and stepwise epithelial fluidization towards, ultimately, cell individualization.
Asunto(s)
Neoplasias de la Mama/patología , Adhesión Celular/fisiología , Invasividad Neoplásica/patología , Uniones Adherentes/patología , Animales , Línea Celular , Línea Celular Tumoral , Regulación hacia Abajo/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Células HEK293 , Humanos , Uniones Intercelulares/patología , Células MCF-7 , Ratones Endogámicos BALB CRESUMEN
Progression of epithelial cancers predominantly proceeds by collective invasion of cell groups with coordinated cell-cell junctions and multicellular cytoskeletal activity. Collectively invading breast cancer cells express the gap junction protein connexin-43 (Cx43), yet whether Cx43 regulates collective invasion remains unclear. We here show that Cx43 mediates gap-junctional coupling between collectively invading breast cancer cells and, via hemichannels, adenosine nucleotide/nucleoside release into the extracellular space. Using molecular interference and rescue strategies, we identify that Cx43 hemichannel function, but not intercellular communication, induces leader cell activity and collective migration through the engagement of the adenosine receptor 1 (ADORA1) and AKT signaling. Accordingly, pharmacological inhibition of ADORA1 or AKT signaling caused leader cell collapse and halted collective invasion. ADORA1 inhibition further reduced local invasion of orthotopic mammary tumors in vivo, and joint up-regulation of Cx43 and ADORA1 in breast cancer patients correlated with decreased relapse-free survival. This identifies autocrine purinergic signaling, through Cx43 hemichannels, as a critical pathway in leader cell function and collective invasion.
Asunto(s)
Neoplasias de la Mama/genética , Conexina 43/genética , Invasividad Neoplásica/genética , Receptores Purinérgicos P1/genética , Adenosina Trifosfato/genética , Neoplasias de la Mama/patología , Comunicación Celular/genética , Línea Celular Tumoral , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Uniones Comunicantes/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Uniones Intercelulares/genética , Invasividad Neoplásica/patología , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/genéticaRESUMEN
The tumour microenvironment (TME) is composed of extracellular matrix and non-mutated cells supporting tumour growth and development. Tumour-associated macrophages (TAMs) are among the most abundant immune cells in the TME and are responsible for the onset of a smouldering inflammation. TAMs play a pivotal role in oncogenic processes as tumour proliferation, angiogenesis and metastasis, and they provide a barrier against the cytotoxic effector function of T lymphocytes and natural killer (NK) cells. However, TAMs are highly plastic cells that can adopt either pro- or anti-inflammatory roles in response to environmental cues. Consequently, TAMs represent an attractive target to recalibrate immune responses in the TME. Initial TAM-targeted strategies, such as macrophage depletion or disruption of TAM recruitment, have shown beneficial effects in preclinical models and clinical trials. Alternatively, reprogramming TAMs towards a proinflammatory and tumouricidal phenotype has become an attractive strategy in immunotherapy. This work summarises the molecular wheelwork of macrophage biology and presents an overview of molecular strategies to repolarise TAMs in immunotherapy.
Asunto(s)
Inmunoterapia/métodos , Macrófagos/metabolismo , Neoplasias/inmunología , Neoplasias/terapia , Microambiente Tumoral/inmunología , Animales , HumanosRESUMEN
Cancer invasion programs are adaptive by switching between metastatic collective and single-cell dissemination; however, current intravital microscopy models for epithelial cancer in mice fail to reliably recreate such invasion plasticity. Using microimplantation of breast cancer spheroids into the murine mammary fat pad and live-cell monitoring, we show microenvironmental conditions and cytoskeletal adaptation during collective to single-cell transition in vivo E-cadherin-expressing 4T1 and E-cadherin-negative MMT tumors both initiated collective invasion along stromal structures, reflecting invasion patterns in 3D organotypic culture and human primary ductal and lobular carcinoma. Collectively invading cells developed weakly oscillatory actin dynamics, yet provided zones for single-cell transitions with accentuated, more chaotic actin fluctuations. This identifies collective invasion in vivo as a dynamic niche and efficient source for single-cell release.
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Plasticidad de la Célula , Microscopía Intravital , Neoplasias Mamarias Animales/diagnóstico por imagen , Neoplasias Mamarias Animales/patología , Actinas/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Femenino , Células HEK293 , Humanos , Imagenología Tridimensional , Neoplasias Mamarias Animales/irrigación sanguínea , Ratones Endogámicos BALB C , Invasividad Neoplásica , Metástasis de la Neoplasia , Neovascularización Patológica/patología , Células del Estroma/patologíaRESUMEN
Breast cancer undergoes collective tissue invasion and, in experimental models, can collectively metastasize. The prevalence of collective invasion and its contribution to distant metastasis in clinical disease, however, remains poorly defined. We here scored the adipose tissue invasion of primary invasive ductal carcinoma (IDC), expressing E-cadherin, and E-cadherin negative invasive lobular carcinoma (ILC) and identified predominantly collective invasion patterns (86/86 samples) in both carcinoma types. Whereas collective invasion in IDC lesions retained adherens junctions, multicellular clusters and "Indian files" in ILC, despite the absence of adherens junctions (AJ) proteins E-cadherin and ß-catenin, retained CD44 at cell-cell contacts. By histomorphological scoring and semi-automated image analysis, we show that the extent of collective invasion into the adipose tissue correlated with decreased distant metastasis-free survival (5-year follow-up; hazard ratio: 2.32 and 2.29, respectively). Thus, collective invasion represents the predominant invasion mode in breast cancer, develops distinct junctional subtypes in IDC and ILC, and associates with distant metastasis, suggesting a critical role in systemic dissemination.
Asunto(s)
Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Carcinoma Lobular/patología , Invasividad Neoplásica/patología , Adulto , Anciano , Neoplasias de la Mama/mortalidad , Carcinoma Ductal de Mama/mortalidad , Carcinoma Lobular/mortalidad , Supervivencia sin Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Persona de Mediana EdadRESUMEN
Cancer metastases arise from a multi-step process that requires metastasizing tumor cells to adapt to signaling input from varying tissue environments [1]. As an early metastatic event, cancer cell dissemination occurs through different migration programs, including multicellular, collective, and single-cell mesenchymal or amoeboid migration [2-4]. Migration modes can interconvert based on changes in cell adhesion, cytoskeletal mechanotransduction [5], and/or proteolysis [6], most likely under the control of transcriptional programs such as the epithelial-to-mesenchymal transition (EMT) [7, 8]. However, how plasticity of tumor cell migration and EMT is spatiotemporally controlled and connected upon challenge by the tumor microenvironment remains unclear. Using 3D cultures of collectively invading breast and head and neck cancer spheroids, here we identify hypoxia, a hallmark of solid tumors [9], as an inducer of the collective-to-amoeboid transition (CAT), promoting the dissemination of amoeboid-moving single cells from collective invasion strands. Hypoxia-induced amoeboid detachment was driven by hypoxia-inducible factor 1 (HIF-1), followed the downregulation of E-cadherin, and produced heterogeneous cell subsets whose phenotype and migration were dependent (â¼30%) or independent (â¼70%) of Twist-mediated EMT. EMT-like and EMT-independent amoeboid cell subsets showed stable amoeboid movement over hours as well as leukocyte-like traits, including rounded morphology, matrix metalloproteinase (MMP)-independent migration, and nuclear deformation. Cancer cells undergoing pharmacological stabilization of HIFs retained their constitutive ability for early metastatic seeding in an experimental model of lung metastasis, indicating that hypoxia-induced CAT enhances cell release rather than early organ colonization. Induced by metabolic challenge, amoeboid movement may thus constitute a common endpoint of both EMT-dependent and EMT-independent cancer dissemination programs.
Asunto(s)
Neoplasias de la Mama/patología , Transición Epitelial-Mesenquimal , Neoplasias de Cabeza y Cuello/patología , Factor 1 Inducible por Hipoxia/metabolismo , Mecanotransducción Celular , Neoplasias de la Mama/metabolismo , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Femenino , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Metástasis de la Neoplasia , Proteínas Nucleares/metabolismo , Hipoxia Tumoral , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
Key features of asthma include bronchial hyperresponsiveness (BHR), eosinophilic airway inflammation, and bronchial remodeling, characterized by subepithelial collagen deposition, airway fibrosis, and increased bronchial smooth muscle (BSM) mass. The calcium-activated K(+) channel K(Ca)3.1 is expressed by many cells implicated in the pathogenesis of asthma, and is involved in both inflammatory and remodeling responses in a number of tissues. The specific K(Ca)3.1 blocker 5-[(2-chlorophenyl)(diphenyl)methyl]-1H-pyrazole (TRAM-34) attenuates BSM cell proliferation, and both mast cell and fibrocyte recruitment in vitro. We aimed to examine the effects of K(Ca)3.1 blockade on BSM remodeling, airway inflammation, and BHR in a murine model of chronic asthma. BALB/c mice were sensitized with intraperitoneal ovalbumin (OVA) on Days 0 and 14, and then challenged with intranasal OVA during Days 14-75. OVA-sensitized/challenged mice received TRAM-34 (120 mg/kg/day, subcutaneous) from Days -7 to 75 (combined treatment), Days -7 to 20 (preventive treatment), or Days 21 to 75 (curative treatment). Untreated mice received daily injections of vehicle (n = 8 per group). Bronchial remodeling was assessed by histological and immunohistochemical analyses. Inflammation was evaluated using bronchoalveolar lavage and flow cytometry. We also determined BHR in both conscious and anesthetized mice via plethysmography. We demonstrated that curative treatment with TRAM-34 abolishes BSM remodeling and subbasement collagen deposition, and attenuates airway eosinophilia. Although curative treatment alone did not significantly reduce BHR, the combined treatment attenuated nonspecific BHR to methacholine. This study indicates that K(Ca)3.1 blockade could provide a new therapeutic strategy in asthma.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Modelos Animales de Enfermedad , Eosinofilia/prevención & control , Canales de Potasio Calcio-Activados/antagonistas & inhibidores , Pirazoles/farmacología , Animales , Citocinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB CRESUMEN
Cancer cell invasion into healthy tissues develops preferentially along pre-existing tracks of least resistance, followed by secondary tissue remodelling and destruction. The tissue scaffolds supporting or preventing guidance of invasion vary in structure and molecular composition between organs. In the brain, the guidance is provided by myelinated axons, astrocyte processes, and blood vessels which are used as invasion routes by glioma cells. In the human breast, containing interstitial collagen-rich connective tissue, disseminating breast cancer cells preferentially invade along bundled collagen fibrils and the surface of adipocytes. In both invasion types, physical guidance prompted by interfaces and space is complemented by molecular guidance. Generic mechanisms shared by most, if not all, tissues include (i) guidance by integrins towards fibrillar interstitial collagen and/or laminins and type IV collagen in basement membranes decorating vessels and adipocytes, and, likely, CD44 engaging with hyaluronan; (ii) haptotactic guidance by chemokines and growth factors; and likely (iii) physical pushing mechanisms. Tissue-specific, resticted guidance cues include ECM proteins with restricted expression (tenascins, lecticans), cell-cell interfaces, and newly secreted matrix molecules decorating ECM fibres (laminin-332, thrombospondin-1, osteopontin, periostin). We here review physical and molecular guidance mechanisms in interstitial tissue and brain parenchyma and explore shared principles and organ-specific differences, and their implications for experimental model design and therapeutic targeting of tumour cell invasion.
Asunto(s)
Neoplasias Encefálicas/patología , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Glioma/patología , Tejido Adiposo/patología , Vasos Sanguíneos/patología , Cadherinas/fisiología , Moléculas de Adhesión Celular/fisiología , Movimiento Celular/fisiología , Colágeno/fisiología , Distroglicanos/fisiología , Matriz Extracelular/patología , Femenino , Humanos , Receptores de Hialuranos/fisiología , Inmunoglobulina G/fisiología , Integrinas/fisiología , Glándulas Mamarias Humanas/patología , Invasividad Neoplásica/patología , Fibras Nerviosas Mielínicas/patología , Receptores de Superficie Celular/fisiología , Sindecanos/fisiologíaRESUMEN
Cancer invasion into an extracellular matrix (ECM) results from a biophysical reciprocal interplay between the expanding cancer lesion and tissue barriers imposed by the adjacent microenvironment. In vivo, connective tissue provides both densely packed ECM barriers adjacent to channel/track-like spaces and loosely organized zones, both of which may impact cancer invasion mode and efficiency; however little is known about how three-dimensional (3D) spaces and aligned tracks present in interstitial tissue guide cell invasion. We here describe a two-photon laser ablation procedure to generate 3D microtracks in dense 3D collagen matrices that support and guide collective cancer cell invasion. Whereas collective invasion of mammary tumor (MMT) breast cancer cells into randomly organized collagen networks required matrix metalloproteinase (MMP) activity for cell-derived collagen breakdown, re-alignment and track generation, preformed tracks supported MMP-independent collective invasion down to a track caliber of 3 µm. Besides contact guidance along the track of least resistance and initial cell deformation (squeezing), MMP-independent collective cell strands led to secondary track expansion by a pushing mechanism. Thus, two-photon laser ablation is useful to generate barrier-free microtracks in a 3D ECM which guide collective invasion independently of pericellular proteolysis.