RESUMEN
Despite the importance of programmed cell death-1 (PD-1) in inhibiting T cell effector activity, the mechanisms regulating its expression remain poorly defined. We found that the chromatin organizer special AT-rich sequence-binding protein-1 (Satb1) restrains PD-1 expression induced upon T cell activation by recruiting a nucleosome remodeling deacetylase (NuRD) complex to Pdcd1 regulatory regions. Satb1 deficienct T cells exhibited a 40-fold increase in PD-1 expression. Tumor-derived transforming growth factor ß (Tgf-ß) decreased Satb1 expression through binding of Smad proteins to the Satb1 promoter. Smad proteins also competed with the Satb1-NuRD complex for binding to Pdcd1 enhancers, releasing Pdcd1 expression from Satb1-mediated repression, Satb1-deficient tumor-reactive T cells lost effector activity more rapidly than wild-type lymphocytes at tumor beds expressing PD-1 ligand (CD274), and these differences were abrogated by sustained CD274 blockade. Our findings suggest that Satb1 functions to prevent premature T cell exhaustion by regulating Pdcd1 expression upon T cell activation. Dysregulation of this pathway in tumor-infiltrating T cells results in diminished anti-tumor immunity.
Asunto(s)
Represión Epigenética/inmunología , Regulación de la Expresión Génica/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Proteínas de Unión a la Región de Fijación a la Matriz/biosíntesis , Receptor de Muerte Celular Programada 1/biosíntesis , Animales , Ensayo de Immunospot Ligado a Enzimas , Humanos , Inmunoprecipitación , Activación de Linfocitos/inmunología , Proteínas de Unión a la Región de Fijación a la Matriz/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/inmunología , Neoplasias/metabolismoRESUMEN
Novel screening techniques for early detection of lung cancer are urgently needed. Profiling circulating tumor cell-free DNA (ctDNA) has emerged as a promising tool for biopsy-free tumor genotyping. However, both the scarcity and short half-life of ctDNA substantially limit the sensitivity and clinical utility of ctDNA detection methodologies. Our discovery that red blood cells (RBCs) sequester mitochondrial DNA opens a new avenue for detecting circulating nucleic acids, as RBCs represent an unrecognized reservoir of circulating nucleic acid. Here, we show that RBCs acquire tumor DNA following coculture with lung cancer cell lines harboring Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR) mutations. RBC-bound tumor DNA is detectable in patients with early-stage non-small cell lung cancer (NSCLC) but not in healthy controls by qPCR. Our results collectively uncover a previously unrecognized yet easily accessible reservoir of tumor DNA, offering a promising foundation for future RBC-based tumor diagnostics.NEW & NOTEWORTHY We present a novel method for lung cancer detection by revealing RBCs as a reservoir for tumor DNA, overcoming the limitations of current circulating tumor ctDNA methodologies. By demonstrating that RBCs can capture tumor DNA, including critical mutations found in lung cancer, we provide a promising, biopsy-free avenue for early cancer diagnostics. This discovery opens up exciting possibilities for developing RBC-based diagnostic tools, significantly enhancing the sensitivity and clinical utility of noninvasive cancer detection.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , ADN Tumoral Circulante , Eritrocitos , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/diagnóstico , Eritrocitos/metabolismo , ADN Tumoral Circulante/genética , ADN Tumoral Circulante/sangre , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Mutación , Línea Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/sangre , Proteínas Proto-Oncogénicas p21(ras)/genética , Masculino , Femenino , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/sangre , ADN de Neoplasias/sangre , ADN de Neoplasias/genéticaRESUMEN
Gene-mediated cytotoxic immunotherapy (GMCI) is an immuno-oncology approach involving local delivery of a replication-deficient adenovirus expressing herpes simplex thymidine kinase (AdV-tk) followed by anti-herpetic prodrug activation that promotes immunogenic tumor cell death, antigen-presenting cell activation, and T cell stimulation. This phase I dose-escalation pilot trial assessed bronchoscopic delivery of AdV-tk in patients with suspected lung cancer who were candidates for surgery. A single intra-tumoral AdV-tk injection in three dose cohorts (maximum 1012 viral particles) was performed during diagnostic staging, followed by a 14-day course of the prodrug valacyclovir, and subsequent surgery 1 week later. Twelve patients participated after appropriate informed consent. Vector-related adverse events were minimal. Immune biomarkers were evaluated in tumor and blood before and after GMCI. Significantly increased infiltration of CD8+ T cells was found in resected tumors. Expression of activation, inhibitory, and proliferation markers, such as human leukocyte antigen (HLA)-DR, CD38, Ki67, PD-1, CD39, and CTLA-4, were significantly increased in both the tumor and peripheral CD8+ T cells. Thus, intratumoral AdV-tk injection into non-small-cell lung cancer (NSCLC) proved safe and feasible, and it effectively induced CD8+ T cell activation. These data provide a foundation for additional clinical trials of GMCI for lung cancer patients with potential benefit if combined with other immune therapies.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Terapia Genética , Inmunoterapia/métodos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/terapia , Adenoviridae/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Citotoxicidad Inmunológica , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Neoplasias Pulmonares/patología , Terapia Neoadyuvante , Timidina Quinasa/genéticaRESUMEN
The accumulation of circulating low-density neutrophils (LDN) has been described in cancer patients and associated with tumor-supportive properties, as opposed to the high-density neutrophils (HDN). Here we aimed to evaluate the clinical significance of circulating LDN in lung cancer patients, and further assessed its diagnostic vs prognostic value. Using mass cytometry (CyTOF), we identified major subpopulations within the circulating LDN/HDN subsets and determined phenotypic modulations of these subsets along tumor progression. LDN were highly enriched in the low-density (LD) fraction of advanced lung cancer patients (median 7.0%; range 0.2%-80%, n = 64), but not in early stage patients (0.7%; 0.05%-6%; n = 35), healthy individuals (0.8%; 0%-3.5%; n = 15), or stable chronic obstructive pulmonary disease (COPD) patients (1.2%; 0.3%-7.4%, n = 13). Elevated LDN (>10%) remarkably related with poorer prognosis in late stage patients. We identified three main neutrophil subsets which proportions are markedly modified in cancer patients, with CD66b+ /CD10low /CXCR4+ /PDL1inter subset almost exclusively found in advanced lung cancer patients. We found substantial variability in subsets between patients, and demonstrated that HDN and LDN retain a degree of inherent spontaneous plasticity. Deep phenotypic characterization of cancer-related circulating neutrophils and their modulation along tumor progression is an important advancement in understanding the role of myeloid cells in lung cancer.
Asunto(s)
Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Neutrófilos/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/inmunología , Antígenos CD/metabolismo , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Femenino , Citometría de Flujo , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Humanos , Neoplasias Pulmonares/patología , Linfocitos/inmunología , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Pronóstico , Enfermedad Pulmonar Obstructiva Crónica/patologíaRESUMEN
Pulmonary lymphangioleiomyomatosis (LAM) is a slow-progressing metastatic disease that is driven by mutations in the tumor suppressor tuberous sclerosis complex 1/2 (TSC1/2). Rapamycin inhibits LAM cell proliferation and is the only approved treatment, but it cannot cause the regression of existing lesions and can only stabilize the disease. However, in other cancers, immunotherapies such as checkpoint blockade against PD-1 and its ligand PD-L1 have shown promise in causing tumor regression and even curing some patients. Thus, we asked whether PD-L1 has a role in LAM progression. In vitro, PD-L1 expression in murine Tsc2-null cells is unaffected by mTOR inhibition with torin but can be upregulated by IFN-γ. Using immunohistochemistry and single-cell flow cytometry, we found increased PD-L1 expression both in human lung tissue from patients with LAM and in Tsc2-null lesions in a murine model of LAM. In this model, PD-L1 is highly expressed in the lung by antigen-presenting and stromal cells, and activated T cells expressing PD-1 infiltrate the affected lung. In vivo treatment with anti-PD-1 antibody significantly prolongs mouse survival in the model of LAM. Together, these data demonstrate that PD-1/PD-L1-mediated immunosuppression may occur in LAM, and suggest new opportunities for therapeutic targeting that may provide benefits beyond those of rapamycin.
Asunto(s)
Antígeno B7-H1/metabolismo , Neoplasias Pulmonares/metabolismo , Pulmón/metabolismo , Linfangioleiomiomatosis/metabolismo , Esclerosis Tuberosa/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Antígeno B7-H1/inmunología , Estudios de Casos y Controles , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Pulmón/efectos de los fármacos , Pulmón/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Linfangioleiomiomatosis/tratamiento farmacológico , Linfangioleiomiomatosis/inmunología , Linfangioleiomiomatosis/patología , Ratones , Ratones Endogámicos C57BL , Esclerosis Tuberosa/tratamiento farmacológico , Esclerosis Tuberosa/inmunología , Esclerosis Tuberosa/patología , Regulación hacia ArribaRESUMEN
Neutrophils accumulate in many types of human and murine tumors and represent a significant portion of tumor-infiltrating myeloid cells. Our current understanding of the role of neutrophils in tumor development has depended primarily on murine models of cancer. However, there are crucial species differences in the evolution of tumors, genetic diversity, immune and inflammatory responses, and intrinsic biology of neutrophils that might have a profound impact on the tumor development and function of neutrophils in mouse versus human tumors. To date, the majority of experimental approaches to study neutrophils in cancer patients have been limited to the examination of circulating blood neutrophils. The phenotype and function of tumor-associated neutrophils (TANs) in humans, particularly in the early stages of tumor development, have not been extensively investigated. Thus, the long-term goal of our work has been to characterize human TANs and determine their specific role in tumor development. Here, we summarize our findings on human TANs obtained from human early stage lung cancer patients. We will describe the phenotypes of different TAN subsets identified in early stage lung tumors, as well as their functional dialog with T cells.
Asunto(s)
Carcinogénesis , Comunicación Celular , Neoplasias Pulmonares/inmunología , Neutrófilos/inmunología , Escape del Tumor , Animales , Diferenciación Celular , Humanos , Inmunidad Celular , Neoplasias Pulmonares/patología , Ratones , Neutrófilos/patología , Fenotipo , Microambiente TumoralRESUMEN
Each year, more than 700,000 people undergo cancer surgery in the United States. However, more than 40% of those patients develop recurrences and have a poor outcome. Traditionally, the medical community has assumed that recurrent tumors arise from selected tumor clones that are refractory to therapy. However, we found that tumor cells have few phenotypical differences after surgery. Thus, we propose an alternative explanation for the resistance of recurrent tumors. Surgery promotes inhibitory factors that allow lingering immunosuppressive cells to repopulate small pockets of residual disease quickly. Recurrent tumors and draining lymph nodes are infiltrated with M2 (CD11b(+)F4/80(hi)CD206(hi) and CD11b(+)F4/80(hi)CD124(hi)) macrophages and CD4(+)Foxp3(+) regulatory T cells. This complex network of immunosuppression in the surrounding tumor microenvironment explains the resistance of tumor recurrences to conventional cancer vaccines despite small tumor size, an intact antitumor immune response, and unaltered cancer cells. Therapeutic strategies coupling antitumor agents with inhibition of immunosuppressive cells potentially could impact the outcomes of more than 250,000 people each year.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Recurrencia Local de Neoplasia/inmunología , Neoplasias/inmunología , Microambiente Tumoral/inmunología , Animales , Antígenos de Diferenciación/inmunología , Antígenos de Diferenciación/metabolismo , Antígeno CD11b/inmunología , Antígeno CD11b/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Humanos , Subunidad alfa del Receptor de Interleucina-4/inmunología , Subunidad alfa del Receptor de Interleucina-4/metabolismo , Estimación de Kaplan-Meier , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/inmunología , Lectinas de Unión a Manosa/metabolismo , Ratones , Ratones Endogámicos C57BL , Neoplasias/patología , Neoplasias/cirugía , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Insuficiencia del Tratamiento , Vacunación/métodosRESUMEN
The mechanisms by which tumor-associated neutrophils (TANs) affect tumor growth are to a large extent unknown. Regulatory T-cells (T-regs) are functionally immune-suppressive subsets of T-cells. Depletion or inhibition of T-regs can enhance antitumor immunity. We demonstrated both by RT-PCR and by ELISA that murine TANs secrete significant amounts of the T-regs chemoattractant, CCL17, much more than circulating or splenic neutrophils, and at a level progressively increasing during tumor development. Migration assays, both in vitro and in vivo, showed recruitment of T-regs by TANs, which was inhibited with anti-CCL17 monoclonal antibodies. Systemic neutrophil depletion in tumor-bearing mice using anti-Ly6G monoclonal antibodies reduced the migration of T-regs into the tumors. We further showed, using flow cytometry, that CCL17 secretion by TANs is not limited to mouse models of cancer but is also relevant to human TANs. Our results suggest a new indirect mechanism by which TANs may inhibit antitumor immune activity, thus promoting tumor growth. We further describe, for the first time, a clear link between TANs and T-regs acting together to impair antitumor immunity.
Asunto(s)
Quimiocina CCL17/inmunología , Neoplasias/inmunología , Neutrófilos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Ly/inmunología , Línea Celular Tumoral , Movimiento Celular/inmunología , Humanos , Activación de Linfocitos/inmunología , Depleción Linfocítica , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Small animal models remain invaluable for the preclinical study of emerging molecular imaging agents. However, the data obtained in this setting are generated in genetically homogenous animals that do not mimic human pathophysiology. The purpose of this study was to prospectively validate precision-cut lung slices (PCLSs) obtained from patients with lung cancer as a translational tool for the development of targeted fluorophores. METHODS: The lung tissue was gently inflated with 2% Low-Melt Agarose (Fisher, 16520050) to avoid lung damage and minimize inflation pressure. The slices were then loaded into specialized cylindrical cartridges and inserted into a compressotome, and slices 150 to 350 µm thick were cut. Samples were incubated with fluorophore conjugates for ex vivo validation and immunohistochemical staining for receptor expression. RESULTS: A total of 184 unique 3-dimensional, architecturally preserved normal lung and non-small cell lung cancer samples were obtained between 2020 and 2022. The median nodule size was 1.1 ± 0.21 cm for benign lesions and 2.1 ± 0.19 cm for malignant nodules. A total of 101 of 135 (74.8%) malignant lesions were adenocarcinoma spectrum lung cancers. The median viability was 9.78 ± 1.86 days, and 1 µM of FAPL-S0456 (high-affinity fibroblast activation protein [FAP] targeting ligand linked to the near-infrared fluorophore S0456, On Target Laboratories)-targeted near-infrared fluorochrome localization demonstrated correlative labeling of FAP-positive tumor areas with a correlation coefficient of +0.94 (P < .01). There was no FAP fluorochrome uptake in normal lungs (r = -1; P < .001). CONCLUSIONS: PCLSs comprise a novel human tissue-based translational model that can be used to validate the efficacy of molecular imaging fluorochromes. PCLSs preserve the tumor microenvironment and parenchymal architecture that closely resemble the interactions of the immune and stromal components in humans.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Animales , Humanos , Carcinoma de Pulmón de Células no Pequeñas/patología , Colorantes Fluorescentes/metabolismo , Neoplasias Pulmonares/patología , Pulmón/patología , Imagen Molecular , Microambiente TumoralRESUMEN
The clinical benefits of tumor-targeting antibodies (tAb) are modest in solid human tumors. The efficacy of many tAbs is dependent on Fc receptor (FcR)-expressing leukocytes that bind Fc fragments of tAb. Tumor-associated macrophages (TAM) and neutrophils (TAN) represent the majority of FcR+ effectors in solid tumors. A better understanding of the mechanisms by which TAMs and TANs regulate tAb response could help improve the efficacy of cancer treatments. Here, we found that myeloid effectors interacting with tAb-opsonized lung cancer cells used antibody-dependent trogocytosis (ADT) but not antibody-dependent phagocytosis. During this process, myeloid cells "nibbled off" tumor cell fragments containing tAb/targeted antigen (tAg) complexes. ADT was only tumoricidal when the tumor cells expressed high levels of tAg and the effectors were present at high effector-to-tumor ratios. If either of these conditions were not met, which is typical for solid tumors, ADT was sublethal. Sublethal ADT, mainly mediated by CD32hiCD64hi TAM, led to two outcomes: (i) removal of surface tAg/tAb complexes from the tumor that facilitated tumor cell escape from the tumoricidal effects of tAb; and (ii) acquisition of bystander tAgs by TAM with subsequent cross-presentation and stimulation of tumor-specific T-cell responses. CD89hiCD32loCD64lo peripheral blood neutrophils (PBN) and TAN stimulated tumor cell growth in the presence of the IgG1 anti-EGFR Ab cetuximab; however, IgA anti-EGFR Abs triggered the tumoricidal activity of PBN and negated the stimulatory effect of TAN. Overall, this study provides insights into the mechanisms by which myeloid effectors mediate tumor cell killing or resistance during tAb therapy. SIGNIFICANCE: The elucidation of the conditions and mechanisms by which human FcR+ myeloid effectors mediate cancer cell resistance and killing during antibody treatment could help develop improved strategies for treating solid tumors.
Asunto(s)
Neoplasias , Neutrófilos , Humanos , Neutrófilos/metabolismo , Macrófagos Asociados a Tumores/metabolismo , Trogocitosis , Citotoxicidad Celular Dependiente de Anticuerpos , Fagocitosis , Neoplasias/patología , Receptores Fc , Antígenos de NeoplasiasRESUMEN
BACKGROUND: Transforming growth factor (TGF)-ß is a potent immunosuppressive cytokine necessary for cancer growth. Animal and human studies have shown that pharmacologic inhibition of TGF-ß slows the growth rate of established tumors and occasionally eradicates them altogether. We observed, paradoxically, that inhibiting TGF-ß before exposing animals to tumor cells increases tumor growth kinetics. We hypothesized that TGF-ß is necessary for the anti-tumor effects of cytotoxic CD8+ T lymphocytes (CTLs) during the early stages of tumor initiation. METHODS: BALB/c mice were pretreated with a blocking soluble TGF-ß receptor (sTGF-ßR, TGF-ß-blockade group, n=20) or IgG2a (Control group, n=20) before tumor inoculation. Tumor size was followed for 6 weeks. In vivo lymphocyte assays and depletion experiments were then performed to investigate the immunological basis of our results. Lastly, animals were pretreated with either sTGF-ßR (n=6) or IgG2a (n=6) prior to immunization with an adenoviral vector encoding the human papillomavirus E7 gene (Ad.E7). One week later, flow cytometry was utilized to measure the number of splenic E7-specific CD8+ T cells. RESULTS: Inhibition of TGF-ß before the injection of tumor cells resulted in significantly larger average tumor volumes on days 11, 17, 22, 26 and 32 post tumor-inoculation (p < 0.05). This effect was due to the inhibition of CTLs, as it was not present in mice with severe combined immunodeficiency (SCID) or those depleted of CD8+ T cells. Furthermore, pretreatment with sTGF-ßR inhibited tumor-specific CTL activity in a Winn Assay. Tumors grew to a much larger size when mixed with CD8+ T cells from mice pretreated with sTGF-ßR than when mixed with CD8+ T cells from mice in the control group: 96 mm3 vs. 22.5 mm3, respectively (p < 0.05). In addition, fewer CD8+ T cells were generated in Ad.E7-immunized mice pretreated with sTGF-ßR than in mice from the control group: 0.6% total CD8+ T cells vs. 1.9%, respectively (p < 0.05). CONCLUSIONS: These studies provide the first in vivo evidence that TGF-ß may be necessary for anti-tumor immune responses in certain cancers. This finding has important implications for our understanding of anti-tumor immune responses, the role of TGF-ß in the immune system, and the future development of TGF-ß inhibiting drugs.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Epítopos/inmunología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Antígeno B7-2/metabolismo , Recuento de Células , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inmunización , Inmunoglobulina G , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Depleción Linfocítica , Ratones , Metástasis de la Neoplasia , Neoplasias/inmunología , Neoplasias/patología , Proteínas E7 de Papillomavirus/inmunología , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Transducción de Señal/inmunología , Solubilidad , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Intraoperative molecular imaging (IMI)-guided resections have been shown to improve oncologic outcomes for patients undergoing surgery for solid malignancies. The technology utilizes fluorescent tracers targeting cancer cells without the use of any ionizing radiation. However, currently available targeted IMI tracers are effective only for tumors with a highly specific receptor expression profile, and there is an unmet need for IMI tracers to label a broader range of tumor types. Here, we describe the development and testing of a novel tracer (CR)-S0456) targeted to the sodium multivitamin transporter (SMVT). METHODS: Preclinical models of fibrosarcoma (HT-1080), lung (A549), breast (4T1), and renal cancers (HEK-293 T) in vitro and in vivo were used for assessment of (CR)-S0456 specific tumor labeling via sodium-mediated SMVT uptake in dipotassium phosphate or choline chloride-containing media buffer. Additionally, pharmacologic inhibition of multiple intracellular coenzyme-R obligate signaling pathways, including holocarboxylase synthetase (sulconazole nitrate), PI3K/AKT/mTOR (omipalisib), and calmodulin-dependent phosphatase (calmidazolium), were investigated to assess (CR)-S0456 uptake kinetics. Human fibrosarcoma-bearing xenografts in athymic nude mice were used for tumor and metabolic-specific labeling. Novel NIR needle confocal laser endomicroscopic (nCLE) intratumoral sampling was performed to demonstrate single-cell specific labeling by CR-S0456. RESULTS: CR-S0456 localization in vitro correlated with highly proliferative cell lines (MTT) and doubling time (p < 0.05) with the highest microscopic fluorescence detected in aggressive human fibrosarcomas (HT-1080). Coenzyme-R-specific localization was demonstrated to be SMVT-specific after competitive inhibition of internal localization with excess administration of pantothenic acid. Inhibiting the activity of SMVT by affecting sodium ion hemostasis prevented the complete uptake of CR-S0456. In vivo validation demonstrated (CR)-S0456 localization to xenograft models with accurate identification of primary tumors as well as margin assessment down to 1 mm3 tumor volume. Systemic treatment of xenograft-bearing mice with a dual PI3K/mTOR inhibitor suppressed intratumoral cell signaling and (CR)-S0456 uptake via a reduction in SMVT expression. Novel analysis of in vivo intratumoral cytologic fluorescence using near-infrared confocal laser endomicroscopy demonstrated the absence of coenzyme-R-mediated NIR fluorescence but not fibroblast activation protein (FAP)-conjugated fluorochrome, indicating specific intracellular inhibition of coenzyme-R obligate pathways. CONCLUSION: These findings suggest that a SMVT-targeted NIR contrast agent can be a suitable tracer for imaging a wide range of malignancies as well as evaluating metabolic response to systemic therapies, similar to PET imaging with immune checkpoint inhibitors.
Asunto(s)
Fibrosarcoma , Simportadores , Humanos , Animales , Ratones , Colorantes Fluorescentes , Sodio/metabolismo , Sodio/farmacología , Células HEK293 , Ratones Desnudos , Fosfatidilinositol 3-Quinasas/metabolismo , Biotina/metabolismo , Transducción de Señal , Fibrosarcoma/diagnóstico por imagen , Fibrosarcoma/tratamiento farmacológicoRESUMEN
BACKGROUND: Intraoperative molecular imaging (IMI) uses tumor-targeted optical contrast agents to improve identification and clearance of cancer during surgery. Recently, pH-activatable contrast agents have been developed but none has been tested in lung cancer. Here, we report the successful clinical translation of pegsitacianine (ONM-100), a pH-activatable nanoprobe, for fluorescence-guided lung cancer resection. METHODS: We first characterized the pH setpoint for pegsitacianine fluorescence activation in vitro. We then optimized the specificity, dosing, and timing of pegsitacianine in murine flank xenograft models of lung adenocarcinoma and squamous cell carcinoma. Finally, we tested pegsitacianine in humans undergoing lung cancer surgery as part of an ongoing phase 2 trial. RESULTS: We found that the fluorescence activation of pegsitacianine occurred below physiologic pH in vitro. Using preclinical models of lung cancer, we found that the probe selectively labeled both adenocarcinoma and squamous cell carcinoma xenografts (mean tumor-to-background ratio [TBR] > 2.0 for all cell lines). In the human pilot study, we report cases in which pegsitacianine localized pulmonary adenocarcinoma and pulmonary squamous cell carcinoma (TBRs= 2.7 and 2.4) in real time to illustrate its successful clinical translation and potential to improve surgical management. CONCLUSIONS: This translational study demonstrates the feasibility of pegsitacianine as an IMI probe to label the two most common histologic subtypes of human lung cancer.
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Adenocarcinoma del Pulmón , Adenocarcinoma , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Humanos , Ratones , Animales , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/cirugía , Medios de Contraste , Proyectos Piloto , Colorantes Fluorescentes/química , Carcinoma de Células Escamosas/cirugía , Adenocarcinoma/diagnóstico por imagen , Adenocarcinoma/cirugía , Concentración de Iones de HidrógenoRESUMEN
Both cancer-related inflammation and tumor-induced immune suppression are associated with expansion of myeloid cell subsets including myeloid-derived suppressor cells. However, little known regarding characteristics of myeloid cells in patients with bladder cancer. In this study, we analyzed myeloid cells from peripheral blood (PBMC) and tumor tissue that were collected from patients with superficial noninvasive and invasive urothelial carcinomas. Our results demonstrate that PBMC from bladder cancer patients contain two major CD11b myeloid cell subsets: granulocyte-type CD15(high) CD33(low) cells and monocyte-type CD15(low) CD33(high) cells. The number of circulating granulocytic but not monocytic myeloid cells in cancer patients was markedly increased when compared to healthy individuals. Both myeloid cell subsets from cancer patients were highly activated and produced substantial amounts of proinflammatory chemokines/cytokines including CCL2, CCL3, CCL4, G-CSF, IL-8 and IL-6. Granulocytic myeloid cells were able to inhibit in vitro T cell proliferation through induction of CD4(+) Foxp3(+) T regulatory cells. Analysis of bladder cancer tissues revealed that tumors were infiltrated with monocyte-macrophage CD11b(+) HLA-DR(+) and granulocytic CD11b(+) CD15(+) HLA-DR(-) myeloid cells. Collectively, this study identifies myeloid cell subsets in patients with bladder cancer. We demonstrate that these highly activated inflammatory myeloid cells represent a source of multiple chemokines/cytokines and may contribute to inflammation and immune dysfunction in bladder cancer.
Asunto(s)
Células Mieloides/inmunología , Neoplasias de la Vejiga Urinaria/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos CD11/metabolismo , Citocinas/metabolismo , Granulocitos/inmunología , Humanos , Tolerancia Inmunológica , Antígeno Lewis X/metabolismo , Activación de Linfocitos , Monocitos/inmunología , Lectina 3 Similar a Ig de Unión al Ácido SiálicoRESUMEN
Aberrant expression of protein kinase C (PKC) isozymes is a hallmark of cancer. The different members of the PKC family control cellular events associated with cancer development and progression. Whereas the classical/conventional PKCα isozyme has been linked to tumor suppression in most cancer types, here we demonstrate that this kinase is required for the mitogenic activity of aggressive human prostate cancer cells displaying aberrantly high PKCα expression. Immunohistochemical analysis showed abnormal up-regulation of PKCα in human primary prostate tumors. Interestingly, silencing PKCα expression from aggressive prostate cancer cells impairs cell cycle progression, proliferation and invasion, as well as their tumorigenic activity in a mouse xenograft model. Mechanistic analysis revealed that PKCα exerts a profound control of gene expression, particularly over genes and transcriptional networks associated with cell cycle progression and E2F transcription factors. PKCα RNAi depletion from PC3 prostate cancer cells led to a reduction in the expression of pro-inflammatory cytokine and epithelial-to-mesenchymal transition (EMT) genes, as well as a prominent down-regulation of the immune checkpoint ligand PD-L1. This PKCα-dependent gene expression profile was corroborated in silico using human prostate cancer databases. Our studies established PKCα as a multifunctional kinase that plays pleiotropic roles in prostate cancer, particularly by controlling genetic networks associated with tumor growth and progression. The identification of PKCα as a pro-tumorigenic kinase in human prostate cancer provides strong rationale for the development of therapeutic approaches towards targeting PKCα or its effectors.
Asunto(s)
Neoplasias de la Próstata , Proteína Quinasa C-alfa , Masculino , Humanos , Ratones , Animales , Proteína Quinasa C-alfa/genética , Redes Reguladoras de Genes , Proteína Quinasa C/genética , División Celular , Neoplasias de la Próstata/genética , Isoenzimas/genéticaRESUMEN
Activities of dendritic cells (DCs) that present tumor antigens are often suppressed in tumors. Here we report that this suppression is induced by tumor microenvironment-derived factors, which activate the activating transcription factor-3 (ATF3) transcription factor and downregulate cholesterol 25-hydroxylase (CH25H). Loss of CH25H in antigen presenting cells isolated from human lung tumors is associated with tumor growth and lung cancer progression. Accordingly, mice lacking CH25H in DCs exhibit an accelerated tumor growth, decreased infiltration and impaired activation of intratumoral CD8+ T cells. These mice do not establish measurable long-term immunity against malignant cells that undergo chemotherapy-induced immunogenic cell death. Mechanistically, downregulation of CH25H stimulates membrane fusion between endo-phagosomes and lysosomes, accelerates lysosomal degradation and restricts cross-presentation of tumor antigens in the intratumoral DCs. Administration of STING agonist MSA-2 reduces the lysosomal activity in DCs, restores antigen cross presentation, and increases therapeutic efficacy of PD-1 blockade against tumour challenge in a CH25H-dependent manner. These studies highlight the importance of downregulation of CH25H in DCs for tumor immune evasion and resistance to therapy.
Asunto(s)
Reactividad Cruzada , Neoplasias Pulmonares , Ratones , Humanos , Animales , Antígenos de Neoplasias , Linfocitos T CD8-positivos , Células Dendríticas , Neoplasias Pulmonares/metabolismo , Lisosomas , Ratones Endogámicos C57BL , Microambiente TumoralRESUMEN
PURPOSE: Fluorescence-guided surgery using tumor-targeted contrast agents has been developed to improve the completeness of oncologic resections. Quenched activity-based probes that fluoresce after covalently binding to tumor-specific enzymes have been proposed to improve specificity, but none have been tested in humans. Here, we report the successful clinical translation of a cathepsin activity-based probe (VGT-309) for fluorescence-guided surgery. EXPERIMENTAL DESIGN: We optimized the specificity, dosing, and timing of VGT-309 in preclinical models of lung cancer. To evaluate clinical feasibility, we conducted a canine study of VGT-309 during pulmonary tumor resection. We then conducted a randomized, double-blind, dose-escalation study in healthy human volunteers receiving VGT-309 to evaluate safety. Finally, we tested VGT-309 in humans undergoing lung cancer surgery. RESULTS: In preclinical models, we found highly specific tumor cell labeling that was blocked by a broad spectrum cathepsin inhibitor. When evaluating VGT-309 for guidance during resection of canine tumors, we found that the probe selectively labeled tumors and demonstrated high tumor-to-background ratio (TBR; range: 2.15-3.71). In the Phase I human study, we found that VGT-309 was safe at all doses studied. In the ongoing Phase II trial, we report two cases in which VGT-309 localized visually occult, non-palpable tumors (TBRs = 2.83 and 7.18) in real time to illustrate its successful clinical translation and potential to improve surgical management. CONCLUSIONS: This first-in-human study demonstrates the safety and feasibility of VGT-309 to label human pulmonary tumors during resection. These results may be generalizable to other cancers due to cathepsin overexpression in many solid tumors.
Asunto(s)
Neoplasias Pulmonares , Cirugía Asistida por Computador , Animales , Catepsinas/metabolismo , Medios de Contraste , Perros , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/cirugía , Ensayos Clínicos Controlados Aleatorios como Asunto , Cirugía Asistida por Computador/métodosRESUMEN
Neutrophils are the first responders to infection and inflammation and are thus a critical component of innate immune defense. Understanding the behavior of neutrophils as they act within various inflammatory contexts has provided insights into their role in sterile and infectious diseases; however, the field of neutrophils in cancer is comparatively young. Here, we summarize key concepts and current knowledge gaps related to the diverse roles of neutrophils throughout cancer progression. We discuss sources of neutrophil heterogeneity in cancer and provide recommendations on nomenclature for neutrophil states that are distinct in maturation and activation. We address discrepancies in the literature that highlight a need for technical standards that ought to be considered between laboratories. Finally, we review emerging questions in neutrophil biology and innate immunity in cancer. Overall, we emphasize that neutrophils are a more diverse population than previously appreciated and that their role in cancer may present novel unexplored opportunities to treat cancer.
Asunto(s)
Neoplasias , Neutrófilos , Humanos , Inmunidad Innata , Inflamación , Neoplasias/genética , FenotipoRESUMEN
Many cancers are known to produce high amounts of PGE(2), which is involved in both tumor progression and tumor-induced immune dysfunction. The key enzyme responsible for the biological inactivation of PGE(2) in tissue is NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). It is well established that cancer cells frequently show down-regulated expression of 15-PGDH, which plays a major role in catabolism of the PGE(2). Here we demonstrate that tumor-infiltrated CD11b cells are also deficient for the 15-PGDH gene. Targeted adenovirus-mediated delivery of 15-PGDH gene resulted in substantial inhibition of tumor growth in mice with implanted CT-26 colon carcinomas. PGDH-mediated antitumor effect was associated with attenuated tumor-induced immune suppression and substantially reduced secretion of immunosuppressive mediators and cytokines such as PGE(2), IL-10, IL-13, and IL-6 by intratumoral CD11b cells. We show also that introduction of 15-PGDH gene in tumor tissue is sufficient to redirect the differentiation of intratumoral CD11b cells from immunosuppressive M2-oriented F4/80(+) tumor-associated macrophages (TAM) into M1-oriented CD11c(+) MHC class II-positive myeloid APCs. Notably, the administration of the 15-PGDH gene alone demonstrated a significant therapeutic effect promoting tumor eradication and long-term survival in 70% of mice with preestablished tumors. Surviving mice acquired antitumor T cell-mediated immune response. This study for the first time demonstrates an important role of the 15-PGDH in regulation of local antitumor immune response and highlights the potential to be implemented to enhance the efficacy of cancer therapy and immunotherapy.
Asunto(s)
Antígeno CD11b/inmunología , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Células Mieloides/enzimología , Células Mieloides/inmunología , Neoplasias/enzimología , Neoplasias/inmunología , Escape del Tumor/inmunología , Adenoviridae/genética , Animales , Células Presentadoras de Antígenos/enzimología , Células Presentadoras de Antígenos/inmunología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Citocinas/inmunología , Citocinas/metabolismo , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Hidroxiprostaglandina Deshidrogenasas/genética , Ganglios Linfáticos/enzimología , Ganglios Linfáticos/inmunología , Masculino , Ratones , Trasplante de Neoplasias , Neoplasias/genética , Neoplasias/patología , Tasa de SupervivenciaRESUMEN
Despite the undisputable role of the small GTPase Rac1 in the regulation of actin cytoskeleton reorganization, the Rac guanine-nucleotide exchange factors (Rac-GEFs) involved in Rac1-mediated motility and invasion in human lung adenocarcinoma cells remain largely unknown. Here, we identify FARP1, ARHGEF39, and TIAM2 as essential Rac-GEFs responsible for Rac1-mediated lung cancer cell migration upon EGFR and c-Met activation. Noteworthily, these Rac-GEFs operate in a non-redundant manner by controlling distinctive aspects of ruffle dynamics formation. Mechanistic analysis reveals a leading role of the AXL-Gab1-PI3K axis in conferring pro-motility traits downstream of EGFR. Along with the positive association between the overexpression of Rac-GEFs and poor lung adenocarcinoma patient survival, we show that FARP1 and ARHGEF39 are upregulated in EpCam+ cells sorted from primary human lung adenocarcinomas. Overall, our study reveals fundamental insights into the complex intricacies underlying Rac-GEF-mediated cancer cell motility signaling, hence underscoring promising targets for metastatic lung cancer therapy.