RESUMEN
Enhanced blood vessel (BV) formation is thought to drive tumor growth through elevated nutrient delivery. However, this observation has overlooked potential roles for mural cells in directly affecting tumor growth independent of BV function. Here we provide clinical data correlating high percentages of mural-ß3-integrin-negative tumor BVs with increased tumor sizes but no effect on BV numbers. Mural-ß3-integrin loss also enhances tumor growth in implanted and autochthonous mouse tumor models with no detectable effects on BV numbers or function. At a molecular level, mural-cell ß3-integrin loss enhances signaling via FAK-p-HGFR-p-Akt-p-p65, driving CXCL1, CCL2, and TIMP-1 production. In particular, mural-cell-derived CCL2 stimulates tumor cell MEK1-ERK1/2-ROCK2-dependent signaling and enhances tumor cell survival and tumor growth. Overall, our data indicate that mural cells can control tumor growth via paracrine signals regulated by ß3-integrin, providing a previously unrecognized mechanism of cancer growth control.
Asunto(s)
Integrina beta3/metabolismo , Neoplasias/metabolismo , Carga Tumoral/fisiología , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Femenino , Humanos , Masculino , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/fisiologíaRESUMEN
ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination. Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206+CD163+ tumor-associated macrophages and vessels. Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages. Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression. Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation. Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth. We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.
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Movimiento Celular/fisiología , Miosina Tipo II/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular/inmunología , Proteínas del Citoesqueleto , Femenino , Humanos , Interleucina-1alfa/metabolismo , Masculino , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Persona de Mediana Edad , FN-kappa B/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Fosforilación , Proteómica , Receptor Cross-Talk/fisiología , Transducción de Señal , Microambiente Tumoral/inmunologíaRESUMEN
Rho GTPases are a family of small G proteins that regulate a wide array of cellular processes related to their key roles controlling the cytoskeleton. Cancer is a multistep disease caused by the accumulation of genetic mutations and epigenetic alterations, from the initial stages of cancer development when cells in normal tissues undergo transformation, to the acquisition of invasive and metastatic traits, responsible for a large number of cancer related deaths. In this review, we discuss the role of Rho GTPase signaling in cancer in every step of disease progression. Rho GTPases contribute to tumor initiation and progression, by regulating proliferation and apoptosis, but also metabolism, senescence, and cancer cell stemness. Rho GTPases play a major role in cell migration and in the metastatic process. They are also involved in interactions with the tumor microenvironment and regulate inflammation, contributing to cancer progression. After years of intensive research, we highlight the importance of relevant models in the Rho GTPase field, and we reflect on the therapeutic opportunities arising for cancer patients.
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Transformación Celular Neoplásica/metabolismo , Neoplasias/tratamiento farmacológico , Microambiente Tumoral/fisiología , Proteínas de Unión al GTP rho/metabolismo , Animales , Movimiento Celular/fisiología , Transformación Celular Neoplásica/inmunología , Humanos , Transducción de Señal/genéticaRESUMEN
In a recent study published in Science, Albrengues et al. (2018) unveil an intriguing mechanism whereby the release of neutrophil extra-cellular traps during chronic lung inflammation awakens dormant malignant cells and contributes to cancer progression.
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Trampas Extracelulares , Neoplasias , Neumonía , Animales , Inflamación , Ratones , NeutrófilosRESUMEN
Tumor cells exhibit two different modes of individual cell movement. Mesenchymal-type movement is characterized by an elongated cellular morphology and requires extracellular proteolysis. In amoeboid movement, cells have a rounded morphology, are less dependent on proteases, and require high Rho-kinase signaling to drive elevated levels of actomyosin contractility. These two modes of cell movement are interconvertible. We show that mesenchymal-type movement in melanoma cells is driven by activation of the GTPase Rac through a complex containing NEDD9, a recently identified melanoma metastasis gene, and DOCK3, a Rac guanine nucleotide exchange factor. Rac signals through WAVE2 to direct mesenchymal movement and suppress amoeboid movement through decreasing actomyosin contractility. Conversely, in amoeboid movement, Rho-kinase signaling activates a Rac GAP, ARHGAP22, that suppresses mesenchymal movement by inactivating Rac. We demonstrate tight interplay between Rho and Rac in determining different modes of tumor cell movement, revealing how tumor cells switch between different modes of movement.
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Movimiento Celular , Melanoma/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Actomiosina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular Tumoral , Quimerina 1/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/metabolismo , Transducción de Señal , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Abnormal cell migration and invasion underlie metastatic dissemination, one of the major challenges for cancer treatment. Melanoma is one of the deadliest and most aggressive forms of skin cancer due in part to its migratory and metastatic potential. Cancer cells use a variety of migratory strategies regulated by cytoskeletal remodelling. In particular, we discuss the importance of amoeboid invasive melanoma strategies, since they have been identified at the edge of human melanomas. We hypothesize that the presence of amoeboid melanoma cells will favour tumor progression since they are invasive and metastatic; they support immunosuppression; they harbour cancer stem cell properties and they are involved in therapy resistance. The Rho-ROCK-Myosin II pathway is key to maintain amoeboid melanoma invasion but this pathway is further regulated by pro-tumorigenic/pro-metastatic/pro-survival signalling pathways such as JAK-STAT3, TGFß-SMAD, NF-κB, Wnt11/5-FDZ7 and BRAFV600E -MEK-ERK. These pathways support amoeboid behaviour and are actionable in the clinic. After melanoma wide surgical margin removal, we propose that possible remaining melanoma cells should be eradicated using anti-amoeboid therapies.
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Melanoma/terapia , Neoplasias Cutáneas/terapia , Movimiento Celular , Terapia Combinada , Humanos , Melanoma/secundario , Neoplasias Cutáneas/patología , Microambiente TumoralRESUMEN
BACKGROUND: Metastasis is a hallmark of cancer and responsible for most cancer deaths. Migrastatics were defined as drugs interfering with all modes of cancer cell invasion and thus cancers' ability to metastasise. First anti-metastatic treatments have recently been approved. METHODS: We used bioinformatic analyses of publicly available melanoma databases. Experimentally, we performed in vitro target validation (including 2.5D cell morphology analysis and mass spectrometric analysis of RhoA binding partners), developed a new traceable spontaneously metastasising murine melanoma model for in vivo validation, and employed histology (haematoxylin/eosin and phospho-myosin II staining) to confirm drug action in harvested tumour tissues. RESULTS: Unbiased and targeted bioinformatic analyses identified the Rho kinase (ROCK)-myosin II pathway and its various components as potentially relevant targets in melanoma. In vitro validation demonstrated redundancy of several RhoGEFs upstream of RhoA and confirmed ROCK as a druggable target downstream of RhoA. The anti-metastatic effects of two ROCK inhibitors were demonstrated through in vivo melanoma metastasis tracking and inhibitor effects also confirmed ex vivo by digital pathology. CONCLUSIONS: We proposed a migrastatic drug development pipeline. As part of the pipeline, we provide a new traceable spontaneous melanoma metastasis model for in vivo quantification of metastasis and anti-metastatic effects by non-invasive imaging.
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Biología Computacional/métodos , Melanoma/tratamiento farmacológico , Miosina Tipo II/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Humanos , Masculino , Espectrometría de Masas , Melanoma/metabolismo , Ratones , Metástasis de la Neoplasia , Mapas de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Coronary microvascular dysfunction combined with maladaptive cardiomyocyte morphology and energetics is a major contributor to heart failure advancement. Thus, dually enhancing cardiac angiogenesis and targeting cardiomyocyte function to slow, or reverse, the development of heart failure is a logical step towards improved therapy. We present evidence for the potential to repurpose a former anti-cancer Arg-Gly-Asp (RGD)-mimetic pentapeptide, cilengitide, here used at low doses. Cilengitide targets αvß3 integrin and this protein is upregulated in human dilated and ischaemic cardiomyopathies. Treatment of mice after abdominal aortic constriction (AAC) surgery with low-dose cilengitide (ldCil) enhances coronary angiogenesis and directly affects cardiomyocyte hypertrophy with an associated reduction in disease severity. At a molecular level, ldCil treatment has a direct effect on cardiac endothelial cell transcriptomic profiles, with a significant enhancement of pro-angiogenic signalling pathways, corroborating the enhanced angiogenic phenotype after ldCil treatment. Moreover, ldCil treatment of Angiotensin II-stimulated AngII-stimulated cardiomyocytes significantly restores transcriptomic profiles similar to those found in normal human heart. The significance of this finding is enhanced by transcriptional similarities between AngII-treated cardiomyocytes and failing human hearts. Taken together, our data provide evidence supporting a possible new strategy for improved heart failure treatment using low-dose RGD-mimetics with relevance to human disease. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Cardiomegalia/tratamiento farmacológico , Fármacos Cardiovasculares/farmacología , Reposicionamiento de Medicamentos , Insuficiencia Cardíaca/tratamiento farmacológico , Integrina alfaVbeta3/antagonistas & inhibidores , Miocitos Cardíacos/efectos de los fármacos , Venenos de Serpiente/farmacología , Angiotensina II/farmacología , Animales , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatología , Estudios de Casos y Controles , Células Cultivadas , Modelos Animales de Enfermedad , Fibrosis , Regulación de la Expresión Génica , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Humanos , Integrina alfaVbeta3/metabolismo , Masculino , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Neovascularización Fisiológica/efectos de los fármacos , Recuperación de la Función , Transducción de Señal , TranscriptomaRESUMEN
The Transforming Growth Factor-beta (TGFß) pathway mediates a broad spectrum of cellular processes and is involved in several diseases, including cancer. TGFß has a dual role in tumours, acting as a tumour suppressor in the early phase of tumorigenesis and as a tumour promoter in more advanced stages. In this review, we discuss the effects of TGFß-driven transcription on all stages of tumour progression, with special focus on lung cancer. Since some TGFß target genes are specifically involved in promoting metastasis, we speculate that these genes might be good targets to block tumour progression without compromising the tumour suppressor effects of the TGFß pathway.
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Transformación Celular Neoplásica/genética , Neoplasias/genética , Transcripción Genética , Factor de Crecimiento Transformador beta/genética , Genes Supresores de Tumor , Humanos , Estadificación de Neoplasias , Neoplasias/patología , Transducción de Señal/genéticaRESUMEN
Neural precursor expressed, developmentally down-regulated 9 (NEDD9), a member of the Cas family of signal transduction molecules, is amplified at the genetic level in melanoma, and elevated expression levels have been shown to correlate with melanoma progression and metastasis. NEDD9 interacts with the guanine nucleotide exchange factor DOCK3 to promote Rac activation and the elongated, mesenchymal-type of tumour cell invasion, but the molecular mechanisms through which NEDD9 promotes melanoma metastasis are not fully understood. We show that signalling through increased NEDD9 levels requires integrin ß3 signalling, which leads to elevated phosphorylation of integrin ß3. This results in increased Src and FAK but decreased ROCK signalling to drive elongated, mesenchymal-type invasion in environments that contain vitronectin. NEDD9 overexpression does not affect ROCK signalling through activation of RhoA but decreases ROCKII signalling through Src-dependent phosphorylation of a negative regulatory site Tyr722. In NEDD9-overexpressing melanoma cells, inhibition of Src with dasatinib results in a switch from Rac-driven elongated, mesenchymal-type invasion to ROCK-dependent rounded, amoeboid invasion. These findings brings into question whether dasatinib would work as a therapeutic agent to block melanoma invasion and metastasis. On the basis of the in vitro data presented here, a combination treatment of dasatinib and a ROCK inhibitor might be a better alternative in order to inhibit both elongated, mesenchymal-type and rounded, amoeboid motility.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Movimiento Celular/genética , Integrina beta3/metabolismo , Melanoma/genética , Melanoma/patología , Mesodermo/metabolismo , Fosfoproteínas/metabolismo , Familia-src Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Humanos , Metástasis de la Neoplasia/genética , Fosfoproteínas/genética , Fosforilación , Transducción de Señal/genética , Células Tumorales CultivadasRESUMEN
Glioblastoma (GBM) is associated with infiltration of peritumoral (PT) parenchyma by isolated tumor cells that leads to tumor regrowth. Recently, GBM stem-like or initiating cells (GICs) have been identified in the PT area, but whether these GICs have enhanced migratory and invasive capabilities compared with GICs from the tumor mass (TM) is presently unknown. We isolated GICs from the infiltrated PT tissue and the TM of three patients and found that PT cells have an advantage over TM cells in two-dimensional and three-dimensional migration and invasion assays. Interestingly, PT cells display a high plasticity in protrusion formation and cell shape and their migration is insensitive to substrate stiffness, which represent advantages to infiltrate microenvironments of different rigidity. Furthermore, mouse and chicken embryo xenografts revealed that only PT cells showed a dispersed distribution pattern, closely associated to blood vessels. Consistent with cellular plasticity, simultaneous Rac and RhoA activation are required for the enhanced invasive capacity of PT cells. Moreover, Rho GTPase signaling modulators αVß3 and p27 play key roles in GIC invasiveness. Of note, p27 is upregulated in TM cells and inhibits RhoA activity. Gene silencing of p27 increased the invasive capacity of TM GICs. Additionally, ß3 integrin is upregulated in PT cells. Blockade of dimeric integrin αVß3, a Rac activator, reduced the invasive capacity of PT GICs in vitro and abrogated the spreading of PT cells into chicken embryos. Thus, our results describe the invasive features acquired by a unique subpopulation of GICs that infiltrate neighboring tissue.
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Neoplasias Encefálicas/patología , Movimiento Celular/fisiología , Glioblastoma/patología , Células Madre Neoplásicas/patología , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular , Línea Celular Tumoral , Movimiento Celular/genética , Embrión de Pollo , Regulación hacia Abajo , Femenino , Glioblastoma/genética , Glioblastoma/metabolismo , Xenoinjertos , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica , Células Madre Neoplásicas/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , Regulación hacia Arriba , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Cell migration plays a pivotal role in various biological processes including cancer dissemination and successful metastasis, where the role of mechanical signals is increasingly acknowledged. This review focuses on the intricate mechanisms through which cancer cells modulate their migratory strategies via organelle adaptations in response to the extracellular matrix (ECM). Specifically, the nucleus and mitochondria emerge as pivotal mediators in this process. These organelles serve as sensors, translating mechanical stimuli into rapid metabolic alterations that sustain cell migration. Importantly, prolonged exposure to such stimuli can induce transcriptional or epigenetic changes, ultimately enhancing metastatic traits. Deciphering the intricate interplay between ECM properties and organelle adaptations not only advances our understanding of cytoskeletal dynamics but also holds promise for the development of innovative anti-metastatic therapeutic strategies.
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Matriz Extracelular , Neoplasias , Orgánulos , Animales , Humanos , Movimiento Celular , Matriz Extracelular/metabolismo , Mecanotransducción Celular , Mitocondrias/metabolismo , Metástasis de la Neoplasia , Neoplasias/patología , Neoplasias/metabolismo , Orgánulos/metabolismoRESUMEN
INTRODUCTION: Despite the success of immunotherapies for melanoma in recent years, there remains a significant proportion of patients who do not yet derive benefit from available treatments. Immunotherapies currently licensed for clinical use target the adaptive immune system, focussing on Tcell interactions and functions. However, the most prevalent immune cells within the tumor microenvironment (TME) of melanoma are macrophages, a diverse immune cell subset displaying high plasticity, to which no current therapies are yet directly targeted. Macrophages have been shown not only to activate the adaptive immune response, and enhance cancer cell killing, but, when influenced by factors within the TME of melanoma, these cells also promote melanoma tumorigenesis and metastasis. AREAS COVERED: We present a review of the most up-to-date literatureavailable on PubMed, focussing on studies from within the last 10 years. We also include data from ongoing and recent clinical trials targeting macrophages in melanoma listed on clinicaltrials.gov. EXPERT OPINION: Understanding the multifaceted role of macrophages in melanoma, including their interactions with immune and cancer cells, the influence of current therapies on macrophage phenotype and functions and how macrophages could be targeted with novel treatment approaches, are all critical for improving outcomes for patients with melanoma.
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Inmunoterapia , Melanoma , Microambiente Tumoral , Macrófagos Asociados a Tumores , Humanos , Melanoma/inmunología , Melanoma/terapia , Melanoma/patología , Microambiente Tumoral/inmunología , Inmunoterapia/métodos , Inmunoterapia/tendencias , Macrófagos Asociados a Tumores/inmunología , Animales , Terapia Molecular Dirigida , Ensayos Clínicos como Asunto , Macrófagos/inmunologíaRESUMEN
ROCK belongs to the AGC family of Ser/Thr protein kinases that are involved in many cellular processes. ROCK-driven actomyosin contractility regulates cytoskeletal dynamics underpinning cell migration, proliferation, and survival in many cancer types. ROCK1/2 play key protumorigenic roles in several subtypes and stages of cancer development. Therefore, successfully targeting ROCK and its downstream effectors presents an interesting avenue for cancer treatment. Because local use of ROCK inhibitors will reduce the side effects of systemic administration, we propose different therapeutic strategies and latest-generation ROCK inhibitors for use in the clinic.
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Neoplasias , Humanos , Neoplasias/metabolismo , Quinasas Asociadas a rho/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto de Actina/metabolismo , Movimiento CelularRESUMEN
Metastasis involves dissemination of cancer cells away from a primary tumour and colonization at distal sites. During this process, the mechanical properties of the nucleus must be tuned since they pose a challenge to the negotiation of physical constraints imposed by the microenvironment and tissue structure. We discovered increased expression of the inner nuclear membrane protein LAP1 in metastatic melanoma cells, at the invasive front of human primary melanoma tumours and in metastases. Human cells express two LAP1 isoforms (LAP1B and LAP1C), which differ in their amino terminus. Here, using in vitro and in vivo models that recapitulate human melanoma progression, we found that expression of the shorter isoform, LAP1C, supports nuclear envelope blebbing, constrained migration and invasion by allowing a weaker coupling between the nuclear envelope and the nuclear lamina. We propose that LAP1 renders the nucleus highly adaptable and contributes to melanoma aggressiveness.
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Melanoma , Membrana Nuclear , Humanos , Isoformas de Proteínas/metabolismo , Movimiento Celular , Membrana Nuclear/metabolismo , Melanoma/genética , Melanoma/metabolismo , Microambiente TumoralRESUMEN
Cell migration is crucial for cancer dissemination. We find that AMP-activated protein kinase (AMPK) controls cell migration by acting as an adhesion sensing molecular hub. In 3-dimensional matrices, fast-migrating amoeboid cancer cells exert low adhesion/low traction linked to low ATP/AMP, leading to AMPK activation. In turn, AMPK plays a dual role controlling mitochondrial dynamics and cytoskeletal remodelling. High AMPK activity in low adhering migratory cells, induces mitochondrial fission, resulting in lower oxidative phosphorylation and lower mitochondrial ATP. Concurrently, AMPK inactivates Myosin Phosphatase, increasing Myosin II-dependent amoeboid migration. Reducing adhesion or mitochondrial fusion or activating AMPK induces efficient rounded-amoeboid migration. AMPK inhibition suppresses metastatic potential of amoeboid cancer cells in vivo, while a mitochondrial/AMPK-driven switch is observed in regions of human tumours where amoeboid cells are disseminating. We unveil how mitochondrial dynamics control cell migration and suggest that AMPK is a mechano-metabolic sensor linking energetics and the cytoskeleton.
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Proteínas Quinasas Activadas por AMP , Dinámicas Mitocondriales , Neoplasias , Humanos , Adenosina Trifosfato/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Adhesión Celular , Movimiento Celular/fisiología , Miosina Tipo II/metabolismo , Fosforilación Oxidativa , FosforilaciónRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis because of its high propensity to metastasize and its immunosuppressive microenvironment. Using a panel of pancreatic cancer cell lines, three-dimensional (3D) invasion systems, microarray gene signatures, microfluidic devices, mouse models, and intravital imaging, we demonstrate that ROCK-Myosin II activity in PDAC cells supports a transcriptional program conferring amoeboid invasive and immunosuppressive traits and in vivo metastatic abilities. Moreover, we find that immune checkpoint CD73 is highly expressed in amoeboid PDAC cells and drives their invasive, metastatic, and immunomodulatory traits. Mechanistically, CD73 activates RhoA-ROCK-Myosin II downstream of PI3K. Tissue microarrays of human PDAC biopsies combined with bioinformatic analysis reveal that rounded-amoeboid invasive cells with high CD73-ROCK-Myosin II activity and their immunosuppressive microenvironment confer poor prognosis to patients. We propose targeting amoeboid PDAC cells as a therapeutic strategy.
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Adenocarcinoma , Amoeba , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Humanos , Ratones , Adenocarcinoma/patología , Amoeba/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Movimiento Celular/fisiología , Proteínas del Citoesqueleto , Terapia de Inmunosupresión , Miosina Tipo II/metabolismo , Neoplasias Pancreáticas/patología , Microambiente TumoralRESUMEN
Here we describe a multiplex chromogenic immunohistochemistry platform to stain and analyze two markers in paraffin tissue sections from mouse or human. The basis of the protocol is a series of stripping and re-probing steps with subsequent image analysis, which allows the user to perform multiplex imaging in a reliable and affordable manner. Here, we describe specific usage to assess the levels of PD-L1 in tumor-associated macrophages. We have used different antibodies and assessed this protocol for up to five consecutive antibodies per slide. For complete details on the use and execution of this protocol, please refer to Orgaz et al. (2020).1.
Asunto(s)
Colorantes , Parafina , Humanos , Animales , Ratones , Inmunohistoquímica , Procesamiento de Imagen Asistido por Computador , AnticuerposRESUMEN
Tumor-associated macrophages (TAMs) are key contributors to antitumor immunity. Here, we present a protocol to drive human monocyte-macrophage differentiation using tumor-derived conditioned media, followed by phenotypic and functional characterization of TAMs in vitro. We describe CD14+ cell isolation from healthy human blood, and detail the procedure to induce macrophage polarization. Finally, we outline morphological assessment of macrophages, and validation of their functional behaviors with a tumor cell killing assay. This translatable-based approach can be applied to different cancer cell types. For complete details on the use and execution of this protocol, please refer to Georgouli et al. (2019).
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Macrófagos , Monocitos , Humanos , Medios de Cultivo Condicionados/farmacología , Línea Celular Tumoral , Macrófagos/metabolismo , Separación CelularRESUMEN
Cell migration is crucial for efficient immune responses and is aberrantly used by cancer cells during metastatic dissemination. Amoeboid migrating cells use myosin II-powered blebs to propel themselves, and change morphology and direction. Immune cells use amoeboid strategies to respond rapidly to infection or tissue damage, which require quick passage through several barriers, including blood, lymph and interstitial tissues, with complex and varied environments. Amoeboid migration is also used by metastatic cancer cells to aid their migration, dissemination and survival, whereby key mechanisms are hijacked from professionally motile immune cells. We explore important parallels observed between amoeboid immune and cancer cells. We also consider key distinctions that separate the lifespan, state and fate of these cell types as they migrate and/or fulfil their function. Finally, we reflect on unexplored areas of research that would enhance our understanding of how tumour cells use immune cell strategies during metastasis, and how to target these processes.