RESUMEN
Breast cancer (BC) heterogeneity is a key trait of BC tumors with crucial implications on tumorigenesis, diagnosis, and therapeutic modalities. It is influenced by tumor intrinsic features and by the tumor microenvironment (TME) composition of different intra-tumoral regions, which in turn affect cancer progression within patients. In this mini review, we will highlight the mechanisms that generate cancer heterogeneity in BC and how they affect the responses to cancer therapies.
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Neoplasias de la Mama , Microambiente Tumoral , Humanos , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/terapia , Femenino , AnimalesRESUMEN
BACKGROUND: Tertiary Lymphoid Structures (TLS) correlate with positive outcomes in patients with NSCLC and the efficacy of immune checkpoint blockade (ICB) in cancer. The actin regulatory protein hMENA undergoes tissue-specific splicing, producing the epithelial hMENA11a linked to favorable prognosis in early NSCLC, and the mesenchymal hMENAΔv6 found in invasive cancer cells and pro-tumoral cancer-associated fibroblasts (CAFs). This study investigates how hMENA isoforms in tumor cells and CAFs relate to TLS presence, localization and impact on patient outcomes and ICB response. METHODS: Methods involved RNA-SEQ on NSCLC cells with depleted hMENA isoforms. A retrospective observational study assessed tissues from surgically treated N0 patients with NSCLC, using immunohistochemistry for tumoral and stromal hMENA isoforms, fibronectin, and TLS presence. ICB-treated patient tumors were analyzed using Nanostring nCounter and GeoMx spatial transcriptomics. Multiparametric flow cytometry characterized B cells and tissue-resident memory T cells (TRM). Survival and ICB response were estimated in the cohort and validated using bioinformatics pipelines in different datasets. FINDINGS: Findings indicate that hMENA11a in NSCLC cells upregulates the TLS regulator LTßR, decreases fibronectin, and favors CXCL13 production by TRM. Conversely, hMENAΔv6 in CAFs inhibits LTßR-related NF-kB pathway, reduces CXCL13 secretion, and promotes fibronectin production. These patterns are validated in N0 NSCLC tumors, where hMENA11ahigh expression, CAF hMENAΔv6low, and stromal fibronectinlow are associated with intratumoral TLS, linked to memory B cells and predictive of longer survival. The hMENA isoform pattern, fibronectin, and LTßR expression broadly predict ICB response in tumors where TLS indicates an anti-tumor immune response. INTERPRETATION: This study uncovers hMENA alternative splicing as an unexplored contributor to TLS-related Tumor Immune Microenvironment (TIME) and a promising biomarker for clinical outcomes and likely ICB responsiveness in N0 patients with NSCLC. FUNDING: This work is supported by AIRC (IG 19822), ACC (RCR-2019-23669120), CAL.HUB.RIA Ministero Salute PNRR-POS T4, "Ricerca Corrente" granted by the Italian Ministry of Health.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Estructuras Linfoides Terciarias , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Fibronectinas , Inhibidores de Puntos de Control Inmunológico , Proteínas de Microfilamentos/metabolismo , Línea Celular Tumoral , Isoformas de Proteínas , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Microambiente TumoralRESUMEN
The extracellular vesicles (EVs) are carriers that actively transfer functional biomolecules between cells affecting the intercellular communication (Pitt, Kroemer, & Zitvogel, 2016). EV signaling has consequences on the targeted cell behavior impacting multiple processes from health to disease, including cancer (Yates et al., 2022). Radiation treatment (RT) is one of the gold standard and effective treatments for cancer, as curative or palliative (Chandra, Keane, Voncken, & Thomas, 2021). RT induces different release of EVs and their cargo is altered. In addition, the uptake of EVs secreted by irradiated cells is affected. Hence, a deep investigation is required to better understand how RT influence the cell-to-cell communication thought signals shuttle by EVs. Here, detailed methods to study the RT effects on EV size and secretion, EV protein expression, EV uptake will be described. Alterations and adaptions might make the protocols applicable to different cell lines, and with different types of RT and dose exposures.
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Vesículas Extracelulares , Neoplasias , Humanos , Vesículas Extracelulares/metabolismo , Neoplasias/metabolismo , Transducción de Señal , Comunicación Celular , ProteómicaRESUMEN
Radiation therapy (RT) increases tumor response to CTLA-4 inhibition (CTLA4i) in mice and in some patients, yet deep responses are rare. To identify rational combinations of immunotherapy to improve responses we use models of triple negative breast cancer highly resistant to immunotherapy in female mice. We find that CTLA4i promotes the expansion of CD4+ T helper cells, whereas RT enhances T cell clonality and enriches for CD8+ T cells with an exhausted phenotype. Combination therapy decreases regulatory CD4+ T cells and increases effector memory, early activation and precursor exhausted CD8+ T cells. A combined gene signature comprising these three CD8+ T cell clusters is associated with survival in patients. Here we show that targeting additional immune checkpoints expressed by intratumoral T cells, including PD1, is not effective, whereas CD40 agonist therapy recruits resistant tumors into responding to the combination of RT and CTLA4i, indicating the need to target different immune compartments.
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Linfocitos T CD8-positivos , Neoplasias de la Mama Triple Negativas , Femenino , Animales , Ratones , Humanos , Inmunoterapia , Antígenos CD40 , Terapia Combinada , Neoplasias de la Mama Triple Negativas/radioterapiaRESUMEN
Focal radiation therapy (RT) has attracted considerable attention as a combinatorial partner for immunotherapy (IT), largely reflecting a well-defined, predictable safety profile and at least some potential for immunostimulation. However, only a few RT-IT combinations have been tested successfully in patients with cancer, highlighting the urgent need for an improved understanding of the interaction between RT and IT in both preclinical and clinical scenarios. Every year since 2016, ImmunoRad gathers experts working at the interface between RT and IT to provide a forum for education and discussion, with the ultimate goal of fostering progress in the field at both preclinical and clinical levels. Here, we summarize the key concepts and findings presented at the Sixth Annual ImmunoRad conference.
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Neoplasias , Humanos , Terapia Combinada , Neoplasias/radioterapia , Neoplasias/tratamiento farmacológico , InmunoterapiaRESUMEN
Immune checkpoint blockade (ICB) has emerged as a novel therapeutic tool for cancer therapy in the last decade. Unfortunately, a small number of patients benefit from approved immune checkpoint inhibitors (ICIs). Therefore, multiple studies are being conducted to find new ICIs and combination strategies to improve the current ICIs. In this review, we discuss some approved immune checkpoints, such as PD-L1, PD-1, and CTLA-4, and also highlight newer emerging ICIs. For instance, HLA-E, overexpressed by tumor cells, represents an immune-suppressive feature by binding CD94/NKG2A, on NK and T cells. NKG2A blockade recruits CD8+ T cells and activates NK cells to decrease the tumor burden. NKG2D acts as an NK cell activating receptor that can also be a potential ICI. The adenosine A2A and A2B receptors, CD47-SIRPα, TIM-3, LAG-3, TIGIT, and VISTA are targets that also contribute to cancer immunoresistance and have been considered for clinical trials. Their antitumor immunosuppressive functions can be used to develop blocking antibodies. PARPs, mARTs, and B7-H3 are also other potential targets for immunosuppression. Additionally, miRNA, mRNA, and CRISPR-Cas9-mediated immunotherapeutic approaches are being investigated with great interest. Pre-clinical and clinical studies project these targets as potential immunotherapeutic candidates in different cancer types for their robust antitumor modulation.
RESUMEN
Mitophagy is a finely regulated mechanism through which eukaryotic cells selectively dispose of supernumerary, permeabilized or otherwise damaged mitochondria through lysosomal degradation. Dysfunctional mitochondria are prone to release potentially cytotoxic factors including reactive oxygen species (ROS) and caspase activators, such as cytochrome c, somatic (CYCS). Thus, proficient mitophagic responses mediate prominent cytoprotective functions. Moreover, the rapid degradation of permeabilized mitochondria limits the release of mitochondrial components that may drive inflammatory reactions, such as mitochondrial DNA (mtDNA) and transcription factor A, mitochondrial (TFAM), implying that mitophagy also mediates potent anti-inflammatory effects. Here, we detail a simple, flow cytometry-assisted protocol for the specific measurement of mitophagic responses as driven by radiation therapy (RT) in mouse hormone receptor (HR)+ mammary carcinoma TS/A cells. With some variations, this method - which relies on the mitochondria-restricted expression of a fluorescent reporter that is sensitive to pH and hence changes excitation wavelength within lysosomes (mt-mKeima) - can be adapted to a variety of human and mouse cancer cell lines and/or straightforwardly implemented on fluorescence microscopy platforms.
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Mitofagia , Neoplasias , Ratones , Humanos , Animales , Mitofagia/genética , Mitocondrias/metabolismo , Línea Celular , ADN Mitocondrial , Especies Reactivas de Oxígeno/metabolismo , Autofagia , Neoplasias/metabolismoRESUMEN
The tumor immune microenvironment is a determinant of response to cancer immunotherapy and, in many cases, is prognostic for patient survival independently of the type of treatment. Radiation therapy is used in most cancer patients for its direct cytotoxic effects on malignant cells but there is increasing evidence that it also reprograms the tumor immune microenvironment. In this review we discuss the main mechanisms whereby the local inflammatory reaction induced by radiation can reset the cross-talk between the tumor and the immune system. The outcome reflects the balance between immunostimulatory signals that lead to increased tumor antigen presentation and effector T cell activation, and immunosuppressive signals that hinder radiation-induced tumor rejection. The emerging role of small extracellular vesicles (exosomes) in this process will be discussed. Overall, preclinical and early clinical findings support the hypothesis that radiation has the potential to generate an immune-permissive tumor microenvironment. An improved understanding of the pathways involved will enable the design of more effective combinations of radiation and immunotherapy, based on a rationale integration of radiation with other interventions.
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Neoplasias , Microambiente Tumoral , Humanos , Inmunoterapia , Neoplasias/radioterapia , Neoplasias/tratamiento farmacológico , Antígenos de Neoplasias , Linfocitos T/metabolismoRESUMEN
Most patients with non-small cell lung cancer (NSCLC) do not achieve durable clinical responses from immune checkpoint inhibitors, suggesting the existence of additional resistance mechanisms. Nicotinamide adenine dinucleotide (NAD)-induced cell death (NICD) of P2X7 receptor (P2X7R)-expressing T cells regulates immune homeostasis in inflamed tissues. This process is mediated by mono-adenosine 5'-diphosphate (ADP)-ribosyltransferases (ARTs). We found an association between membranous expression of ART1 on tumor cells and reduced CD8 T cell infiltration. Specifically, we observed a reduction in the P2X7R+ CD8 T cell subset in human lung adenocarcinomas. In vitro, P2X7R+ CD8 T cells were susceptible to ART1-mediated ADP-ribosylation and NICD, which was exacerbated upon blockade of the NAD+-degrading ADP-ribosyl cyclase CD38. Last, in murine NSCLC and melanoma models, we demonstrate that genetic and antibody-mediated ART1 inhibition slowed tumor growth in a CD8 T cell-dependent manner. This was associated with increased infiltration of activated P2X7R+CD8 T cells into tumors. In conclusion, we describe ART1-mediated NICD as a mechanism of immune resistance in NSCLC and provide preclinical evidence that antibody-mediated targeting of ART1 can improve tumor control, supporting pursuit of this approach in clinical studies.
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ADP Ribosa Transferasas , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Subgrupos de Linfocitos T , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/metabolismo , Adenosina Difosfato , Animales , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Proteínas Ligadas a GPI/genética , Humanos , Neoplasias Pulmonares/inmunología , RatonesRESUMEN
Tryptophan catabolism is a major metabolic pathway utilized by several professional and non-professional antigen presenting cells to maintain immunological tolerance. Here we report that 3-hydroxy-L-kynurenamine (3-HKA) is a biogenic amine produced via an alternative pathway of tryptophan metabolism. In vitro, 3-HKA has an anti-inflammatory profile by inhibiting the IFN-γ mediated STAT1/NF-κΒ pathway in both mouse and human dendritic cells (DCs) with a consequent decrease in the release of pro-inflammatory chemokines and cytokines, most notably TNF, IL-6, and IL12p70. 3-HKA has protective effects in an experimental mouse model of psoriasis by decreasing skin thickness, erythema, scaling and fissuring, reducing TNF, IL-1ß, IFN-γ, and IL-17 production, and inhibiting generation of effector CD8+ T cells. Similarly, in a mouse model of nephrotoxic nephritis, besides reducing inflammatory cytokines, 3-HKA improves proteinuria and serum urea nitrogen, overall ameliorating immune-mediated glomerulonephritis and renal dysfunction. Overall, we propose that this biogenic amine is a crucial component of tryptophan-mediated immune tolerance.
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Aminas Biogénicas/farmacología , Inmunomodulación/efectos de los fármacos , Quinurenina/análogos & derivados , Animales , Aminas Biogénicas/metabolismo , Aminas Biogénicas/uso terapéutico , Línea Celular Tumoral , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Células Endoteliales , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Inflamación , Interferón gamma/farmacología , Quinurenina/metabolismo , Quinurenina/farmacología , Quinurenina/uso terapéutico , Ratones , FN-kappa B/metabolismo , Nefritis/tratamiento farmacológico , Nefritis/inmunología , Psoriasis/tratamiento farmacológico , Psoriasis/inmunología , Triptófano/metabolismoRESUMEN
Exosomes are bi-layered vesicles secreted by the cells in physiological and pathological conditions. They are involved in cell-cell communication facilitating the transfer of functional macromolecules, including DNA, RNA, proteins and lipids. In this chapter, we will focus on specific class of RNA, the microRNAs, that are shuttled from the exosome-producing cells to the recipient cells where they affect biological processes. We will describe the recent methodologies developed to detect and isolate exosomal microRNAs providing a suitable workflow that contributes to quickly expand the field of exosomes-derived microRNAs and their potential use as biomarkers.
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Exosomas , MicroARNs , Comunicación Celular , ADN , Exosomas/genética , MicroARNs/genética , ProteínasRESUMEN
Deciphering extracellular matrix (ECM) composition and architecture may represent a novel approach to identify diagnostic and therapeutic targets in cancer. Among the ECM components, fibronectin and its fibrillary assembly represent the scaffold to build up the entire ECM structure, deeply affecting its features. Herein we focus on this extraordinary protein starting from its complex structure and defining its role in cancer as prognostic and theranostic marker.
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Fibronectinas/metabolismo , Oncología Médica/métodos , Neoplasias/patología , Humanos , PronósticoRESUMEN
The dynamic interplay between cancer cells and cancer-associated fibroblasts (CAFs) is regulated by multiple signaling pathways, which can lead to cancer progression and therapy resistance. We have previously demonstrated that hMENA, a member of the actin regulatory protein of Ena/VASP family, and its tissue-specific isoforms influence a number of intracellular signaling pathways related to cancer progression. Here, we report a novel function of hMENA/hMENAΔv6 isoforms in tumor-promoting CAFs and in the modulation of pro-tumoral cancer cell/CAF crosstalk via GAS6/AXL axis regulation. LC-MS/MS proteomic analysis reveals that CAFs that overexpress hMENAΔv6 secrete the AXL ligand GAS6, favoring the invasiveness of AXL-expressing pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC) cells. Reciprocally, hMENA/hMENAΔv6 regulates AXL expression in tumor cells, thus sustaining GAS6-AXL axis, reported as crucial in EMT, immune evasion, and drug resistance. Clinically, we found that a high hMENA/GAS6/AXL gene expression signature is associated with a poor prognosis in PDAC and NSCLC. We propose that hMENA contributes to cancer progression through paracrine tumor-stroma crosstalk, with far-reaching prognostic and therapeutic implications for NSCLC and PDAC.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Neoplasias Pancreáticas , Actinas , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Cromatografía Liquida , Humanos , Neoplasias Pulmonares/genética , Proteínas de Microfilamentos , Neoplasias Pancreáticas/genética , Proteómica , Células del Estroma , Espectrometría de Masas en TándemRESUMEN
Tumor infiltration of conventional dendritic cells has been shown to be essential for triggering efficient antitumor immune responses. These findings have generated an increasing demand for reliable methods to accurately identify and quantify specific DC-subpopulations, both in immune monitoring of clinical trial samples as well as in preclinical mouse tumor models. Here, we describe a flow cytometric approach to assess percentages and absolute counts of conventional dendritic cells in solid mouse tumors.
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Células Dendríticas , Neoplasias , Animales , Citometría de Flujo , RatonesRESUMEN
Exosomes are small extracellular vesicles released by prokaryotic and eukaryotic cells with a crucial role in cell-to-cell communication in both physiological and pathological conditions. Exosomes contain and transfer active biomolecules, including nucleic acids, proteins and lipids to target recipient cells. In the last decade, many methodologies have been developed for isolating specific exosomal components. In this chapter, we will detail methods to isolate exosomal DNA, considering the crucial role of exosomal DNA in regulating the behavior of recipient cells in multiple settings, including the response of malignant cells to chemo-, radio- and immunotherapy.
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Exosomas , Vesículas Extracelulares , Comunicación Celular , ADN/genética , ProteínasRESUMEN
The ability of focal radiotherapy to promote priming of tumor-specific CD8+ T cells and increase responses to immunotherapy is dependent on infiltration of the tumor by Batf3-dependent conventional dendritic cell type 1 (cDC1) cells. Such infiltration is driven by radiotherapy-induced IFN type I (IFN-I). Other signals may also modulate cDC1 infiltration of irradiated tumors. Here we found increased expression of adenosine-generating enzymes CD38 and CD73 in irradiated mouse and human breast cancer cells and increased adenosine in mouse tumors following radiotherapy. CD73 blockade alone had no effect. CD73 blockade with radiotherapy restored radiotherapy-induced cDC1 infiltration of tumors in settings where radiotherapy induction of IFN-I was suboptimal. In the absence of radiotherapy-induced IFN-I, blockade of CD73 was required for rejection of the irradiated tumor and for systemic tumor control (abscopal effect) in the context of cytotoxic T-lymphocyte-associated protein 4 blockade. These results suggest that CD73 may be a radiation-induced checkpoint, and that CD73 blockade in combination with radiotherapy and immune checkpoint blockade might improve patient response to therapy.
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5'-Nucleotidasa/antagonistas & inhibidores , Adenosina/metabolismo , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Interferón Tipo I/inmunología , Neoplasias/radioterapia , 5'-Nucleotidasa/inmunología , Animales , Línea Celular Tumoral , Femenino , Humanos , Interferón Tipo I/efectos de la radiación , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Here, we developed an unbiased, functional target-discovery platform to identify immunogenic proteins from primary non-small cell lung cancer (NSCLC) cells that had been induced to apoptosis by cisplatin (CDDP) treatment in vitro, as compared with their live counterparts. Among the multitude of proteins identified, some of them were represented as fragmented proteins in apoptotic tumor cells, and acted as non-mutated neoantigens (NM-neoAgs). Indeed, only the fragmented proteins elicited effective multi-specific CD4+ and CD8+ T cell responses, upon a chemotherapy protocol including CDDP. Importantly, these responses further increased upon anti-PD-1 therapy, and correlated with patients' survival and decreased PD-1 expression. Cross-presentation assays showed that NM-neoAgs were unveiled in apoptotic tumor cells as the result of caspase-dependent proteolytic activity of cellular proteins. Our study demonstrates that apoptotic tumor cells generate a repertoire of immunogenic NM-neoAgs that could be potentially used for developing effective T cell-based immunotherapy across multiple cancer patients.
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Antígenos de Neoplasias/inmunología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Neoplasias Pulmonares/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Anciano , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/aislamiento & purificación , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Estudios de Casos y Controles , Línea Celular Tumoral , Cisplatino/administración & dosificación , Cisplatino/farmacología , Terapia Combinada , Ensayos de Selección de Medicamentos Antitumorales/métodos , Femenino , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunoterapia/métodos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T/fisiologíaRESUMEN
Exosomes are extracellular vesicles secreted by cells and involved in intercellular communications among close and distant cells. Exosomes encapsulate and carry biomolecules as cargo to the recipient cells. They contain nucleic acids (DNA, RNA, microRNA) proteins and lipids. Each exosomal components may be isolated and be studied by specific techniques. In this chapter, different methods will be described to isolate DNA from exosomes, since it is important in shaping the response of the recipient cells following the exosome uptake in multiple scenarios, including physiological and pathological conditions. Moreover, the exosomal DNA may be a novel biomarker for diagnosis, disease progression and patient's treatment response.