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1.
J Pathol ; 260(3): 289-303, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37186300

RESUMEN

Breast cancer invasion and metastasis result from a complex interplay between tumor cells and the tumor microenvironment (TME). Key oncogenic changes in the TME include aberrant synthesis, processing, and signaling of hyaluronan (HA). Hyaluronan-mediated motility receptor (RHAMM, CD168; HMMR) is an HA receptor enabling tumor cells to sense and respond to this aberrant TME during breast cancer progression. Previous studies have associated RHAMM expression with breast tumor progression; however, cause and effect mechanisms are incompletely established. Focused gene expression analysis of an internal breast cancer patient cohort confirmed that increased RHAMM expression correlates with aggressive clinicopathological features. To probe mechanisms, we developed a novel 27-gene RHAMM-related signature (RRS) by intersecting differentially expressed genes in lymph node (LN)-positive patient cases with the transcriptome of a RHAMM-dependent model of cell transformation, which we validated in an independent cohort. We demonstrate that the RRS predicts for poor survival and is enriched for cell cycle and TME-interaction pathways. Further analyses using CRISPR/Cas9-generated RHAMM-/- breast cancer cells provided direct evidence that RHAMM promotes invasion in vitro and in vivo. Immunohistochemistry studies highlighted heterogeneous RHAMM protein expression, and spatial transcriptomics associated the RRS with RHAMM-high microanatomic foci. We conclude that RHAMM upregulation leads to the formation of 'invasive niches', which are enriched in RRS-related pathways that drive invasion and could be targeted to limit invasive progression and improve patient outcomes. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/patología , Ácido Hialurónico/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Receptores de Hialuranos/metabolismo , Microambiente Tumoral
2.
J Immunol ; 204(8): 2295-2307, 2020 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-32179637

RESUMEN

MHC class II (MHCII) expression is usually restricted to APC but can be expressed by cancer cells. We examined the effect of cancer cell-specific MHCII (csMHCII) expression in lung adenocarcinoma on T cell recruitment to tumors and response to anti-PD-1 therapy using two orthotopic immunocompetent murine models of non-small cell lung cancer: CMT167 (CMT) and Lewis lung carcinoma (LLC). We previously showed that CMT167 tumors are eradicated by anti-PD1 therapy, whereas LLC tumors are resistant. RNA sequencing analysis of cancer cells recovered from tumors revealed that csMHCII correlated with response to anti-PD1 therapy, with immunotherapy-sensitive CMT167 cells being csMHCII positive, whereas resistant LLC cells were csMHCII negative. To test the functional effects of csMHCII, MHCII expression was altered on the cancer cells through loss- and gain-of-function of CIITA, a master regulator of the MHCII pathway. Loss of CIITA in CMT167 decreased csMHCII and converted tumors from anti-PD-1 sensitive to anti-PD-1 resistant. This was associated with lower levels of Th1 cytokines, decreased T cell infiltration, increased B cell numbers, and decreased macrophage recruitment. Conversely, overexpression of CIITA in LLC cells resulted in csMHCII in vitro and in vivo. Enforced expression of CIITA increased T cell infiltration and sensitized tumors to anti-PD-1 therapy. csMHCII expression was also examined in a subset of surgically resected human lung adenocarcinomas by multispectral imaging, which provided a survival benefit and positively correlated with T cell infiltration. These studies demonstrate a functional role for csMHCII in regulating T cell infiltration and sensitivity to anti-PD-1.


Asunto(s)
Adenocarcinoma del Pulmón/terapia , Antígenos de Histocompatibilidad Clase II/genética , Neoplasias Pulmonares/terapia , Proteínas Nucleares/genética , Transactivadores/genética , Microambiente Tumoral/genética , Adenocarcinoma del Pulmón/inmunología , Animales , Modelos Animales de Enfermedad , Antígenos de Histocompatibilidad Clase II/inmunología , Neoplasias Pulmonares/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Transactivadores/inmunología , Microambiente Tumoral/inmunología
3.
PLoS Pathog ; 15(6): e1007849, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31166996

RESUMEN

Virus-host interactions are frequently studied in bulk cell populations, obscuring cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at the single-cell level, combining a sensitive and robust fluorescent in situ hybridization platform with multiparameter flow cytometry, to study the expression of gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection and reactivation in vitro. This method allowed robust detection of viral ncRNAs of murine gammaherpesvirus 68 (γHV68), Kaposi's sarcoma associated herpesvirus and Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA regulation during γHV68 infection, we find heterogeneous and asynchronous gene expression during latency and reactivation, with reactivation from latency identified by a distinct gene expression profile within rare cells. Further, during lytic replication with γHV68, we find many cells have limited viral gene expression, with only a fraction of cells showing robust gene expression, dynamic RNA localization, and progressive infection. Lytic viral gene expression was enhanced in primary fibroblasts and by conditions associated with enhanced viral replication, with multiple subpopulations of cells present in even highly permissive infection conditions. These findings, powered by single-cell analysis integrated with automated clustering algorithms, suggest inefficient or abortive γHV infection in many cells, and identify substantial heterogeneity in viral gene expression at the single-cell level.


Asunto(s)
Gammaherpesvirinae/fisiología , Regulación Viral de la Expresión Génica/fisiología , Infecciones por Herpesviridae/metabolismo , ARN Mensajero/biosíntesis , ARN no Traducido/biosíntesis , ARN Viral/biosíntesis , Replicación Viral/fisiología , Animales , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/patología , Humanos , Ratones , Ratones Noqueados , ARN Mensajero/genética , ARN no Traducido/genética , ARN Viral/genética
4.
J Immunother Cancer ; 8(1)2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32312906

RESUMEN

BACKGROUND: Programmed death 1/programmed death ligand 1 (PD-1/PD-L1) targeted immunotherapy affords clinical benefit in ~20% of unselected patients with lung cancer. The factor(s) that determine whether a tumor responds or fails to respond to immunotherapy remains an active area of investigation. We have previously defined divergent responsiveness of two KRAS-mutant cell lines to PD-1/PD-L1 blockade using an orthotopic, immunocompetent mouse model. Responsiveness to PD-1/PD-L1 checkpoint blockade correlates with an interferon gamma (IFNγ)-inducible gene signature and major histocompatibility complex class II (MHC II) expression by cancer cells. In the current study, we aim to identify therapeutic targets that can be manipulated in order to enhance cancer-cell-specific MHC II expression. METHODS: Responsiveness to IFNγ and induction of MHC II expression was assessed after various treatment conditions in mouse and human non-small cell lung cancer (NSCLC) cell lines using mass cytometric and flow cytometric analysis. RESULTS: Single-cell analysis using mass and flow cytometry demonstrated that IFNγ consistently induced PD-L1 and MHC class I (MHC I) across multiple murine and human NSCLC cell lines. In contrast, MHC II showed highly variable induction following IFNγ treatment both between lines and within lines. In mouse models of NSCLC, MHC II induction was inversely correlated with basal levels of phosphorylated extracellular signal-regulated kinase (ERK) 1/2, suggesting potential mitogen-activated protein (MAP) kinase-dependent antagonism of MHC II expression. To test this, cell lines were subjected to varying levels of stimulation with IFNγ, and assessed for MHC II expression in the presence or absence of mitogen-activated protein kinase kinase (MEK) inhibitors. IFNγ treatment in the presence of MEK inhibitors significantly enhanced MHC II induction across multiple lung cancer lines, with minimal impact on expression of either PD-L1 or MHC I. Inhibition of histone deacetylases (HDACs) also enhanced MHC II expression to a more modest extent. Combined MEK and HDAC inhibition led to greater MHC II expression than either treatment alone. CONCLUSIONS: These studies emphasize the active inhibitory role that epigenetic and ERK signaling cascades have in restricting cancer cell-intrinsic MHC II expression in NSCLC, and suggest that combinatorial blockade of these pathways may engender new responsiveness to checkpoint therapies.


Asunto(s)
Antígeno B7-H1/metabolismo , Epigénesis Genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/metabolismo , Neoplasias Pulmonares/patología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Antivirales/farmacología , Antígeno B7-H1/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Interferón gamma/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/genética , Células Tumorales Cultivadas
5.
Immunohorizons ; 3(3): 94-109, 2019 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-31356152

RESUMEN

IL-10 is a potent immunomodulatory cytokine produced by multiple cell types to restrain immune activation. Many herpesviruses use the IL-10 pathway to facilitate infection, but how endogenous IL-10 is regulated during primary infection in vivo remains poorly characterized. In this study, we infected mice with murine gammaherpesvirus 68 (γHV68) and analyzed the production and genetic contribution of IL-10 by mass cytometry (cytometry by time-of-flight) analysis. γHV68 infection elicited a breadth of effector CD4 T cells in the lungs of acutely infected mice, including a highly activated effector subset that coexpressed IFN-γ, TNF-α, and IL-10. By using IL-10 GFP transcriptional reporter mice, we identified that IL-10 was primarily expressed within CD4 T cells during acute infection in the lungs. IL10gfp-expressing CD4 T cells were highly proliferative and characterized by the expression of multiple coinhibitory receptors, including PD-1 and LAG-3. When we analyzed acute γHV68 infection of IL-10-deficient mice, we found that IL-10 limits the frequency of both myeloid and effector CD4 T cell subsets in the infected lung, with minimal changes at a distant mucosal site. These data emphasize the unique insights that high-dimensional analysis can afford in investigating antiviral immunity and provide new insights into the breadth, phenotype, and function of IL-10-expressing effector CD4 T cells during acute virus infection.


Asunto(s)
Gammaherpesvirinae/inmunología , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/metabolismo , Interacciones Huésped-Patógeno , Inmunomodulación , Interleucina-10/metabolismo , Animales , Biomarcadores , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Genes Reporteros , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Interacciones Huésped-Patógeno/inmunología , Inmunofenotipificación , Interleucina-10/genética , Ratones , Ratones Transgénicos
6.
Life Sci Alliance ; 2(3)2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31133614

RESUMEN

Targeting PD-1/PD-L1 is only effective in ∼20% of lung cancer patients, but determinants of this response are poorly defined. We previously observed differential responses of two murine K-Ras-mutant lung cancer cell lines to anti-PD-1 therapy: CMT167 tumors were eliminated, whereas Lewis Lung Carcinoma (LLC) tumors were resistant. The goal of this study was to define mechanism(s) mediating this difference. RNA sequencing analysis of cancer cells recovered from lung tumors revealed that CMT167 cells induced an IFNγ signature that was blunted in LLC cells. Silencing Ifngr1 in CMT167 resulted in tumors resistant to IFNγ and anti-PD-1 therapy. Conversely, LLC cells had high basal expression of SOCS1, an inhibitor of IFNγ. Silencing Socs1 increased response to IFNγ in vitro and sensitized tumors to anti-PD-1. This was associated with a reshaped tumor microenvironment, characterized by enhanced T cell infiltration and enrichment of PD-L1hi myeloid cells. These studies demonstrate that targeted enhancement of tumor-intrinsic IFNγ signaling can induce a cascade of changes associated with increased therapeutic vulnerability.


Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/patología , Interferón gamma/farmacología , Neoplasias Pulmonares/patología , Microambiente Tumoral/efectos de los fármacos , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Biomarcadores de Tumor , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Quimiocina CXCL9/metabolismo , Modelos Animales de Enfermedad , Silenciador del Gen , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Ratones , Terapia Molecular Dirigida , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Proteína 1 Supresora de la Señalización de Citocinas/genética , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo
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