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BACKGROUND AND OBJECTIVE: TP53 loss-of-function (TP53LOF) mutations might be a driver of poor prognosis and chemoresistance in both human papillomavirus (HPV)-independent (HPV-) and HPV-associated (HPV+) penile squamous cell carcinoma (PSCC). Here, we aim to describe transcriptomic differences in the PSCC microenvironment stratified by TP53LOF and HPV status. METHODS: We used single-cell RNA sequencing (scRNA-seq) and T-cell receptor sequencing to obtain a comprehensive atlas of the cellular architecture of PSCC. TP53LOF and HPV status were determined by targeted next-generation sequencing and sequencing HPV-DNA reads. Six HPV+ TP53 wild type (WT), six HPV- TP53WT, and four TP53LOF PSCC samples and six controls were included. Immunohistochemistry and hematoxylin-eosin confirmed the morphological context of the observed signatures. Prognostic differences between patient groups were validated in 541 PSCC patients using Kaplan-Meier survival estimates. KEY FINDINGS AND LIMITATIONS: Patients with aberrant p53 staining fare much worse than patients with either HPV- or HPV+ tumors and WT p53 expression. Using scRNA-seq, we revealed 65 cell subtypes within 83 682 cells. TP53LOF tumors exhibit a partial epithelial-to-mesenchymal transition, immune-excluded, angiogenic, and morphologically invasive environment, underlying their aggressive phenotype. HPV- TP53WT tumors show stemness and immune exhaustion. HPV+ TP53WT tumors mirror normal epithelial maturation with upregulation of antibody-drug-conjugate targets and activation of innate immunity. Inherent to the scRNA-seq analysis, low sample size is a limitation and validation of signatures in large PSCC cohorts is needed. CONCLUSIONS AND CLINICAL IMPLICATIONS: This first scRNA-seq atlas offers unprecedented in-depth insights into PSCC biology underlying prognostic differences based on TP53 and HPV status. Our findings provide clues for testing novel biomarker-driven therapies in PSCC. PATIENT SUMMARY: Here, we analyzed tissues of penile cancer at the level of individual cells, which helps us understand why patients who harbor a deactivating mutation in the TP53 gene do much worse than patients lacking such a mutation. Such an analysis may help us tailor future therapies based on TP53 gene mutations and human papillomavirus status of these tumors.
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Mutación , Infecciones por Papillomavirus , Neoplasias del Pene , Fenotipo , Análisis de la Célula Individual , Proteína p53 Supresora de Tumor , Humanos , Masculino , Neoplasias del Pene/genética , Neoplasias del Pene/virología , Neoplasias del Pene/patología , Proteína p53 Supresora de Tumor/genética , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/virología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virología , Carcinoma de Células Escamosas/patología , Medicina de Precisión , Persona de Mediana Edad , Papillomaviridae/genética , Pronóstico , Microambiente Tumoral/genética , Anciano , Virus del Papiloma HumanoRESUMEN
Chemotherapy using temozolomide is the standard treatment for patients with glioblastoma. Despite treatment, prognosis is still poor largely due to the emergence of temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. To induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells' molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell RNA sequencing on four of the cell cultures to track the evolution of temozolomide resistance. The induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as "adaptive" (ADA) or "non-adaptive" (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. The adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumor's ability to survive. Depending on the tumor's adaptability potential, subpopulations with acquired resistance mechanisms may arise.
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Neoplasias Encefálicas , Glioblastoma , Humanos , Temozolomida/farmacología , Temozolomida/uso terapéutico , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Dacarbazina/farmacología , Dacarbazina/uso terapéutico , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Línea Celular Tumoral , Fenotipo , Genómica , Resistencia a Antineoplásicos/genética , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Regulación Neoplásica de la Expresión GénicaRESUMEN
Tumor endothelial cells (TECs) actively repress inflammatory responses and maintain an immune-excluded tumor phenotype. However, the molecular mechanisms that sustain TEC-mediated immunosuppression remain largely elusive. Here, we show that autophagy ablation in TECs boosts antitumor immunity by supporting infiltration and effector function of T-cells, thereby restricting melanoma growth. In melanoma-bearing mice, loss of TEC autophagy leads to the transcriptional expression of an immunostimulatory/inflammatory TEC phenotype driven by heightened NF-kB and STING signaling. In line, single-cell transcriptomic datasets from melanoma patients disclose an enriched InflammatoryHigh /AutophagyLow TEC phenotype in correlation with clinical responses to immunotherapy, and responders exhibit an increased presence of inflamed vessels interfacing with infiltrating CD8+ T-cells. Mechanistically, STING-dependent immunity in TECs is not critical for the immunomodulatory effects of autophagy ablation, since NF-kB-driven inflammation remains functional in STING/ATG5 double knockout TECs. Hence, our study identifies autophagy as a principal tumor vascular anti-inflammatory mechanism dampening melanoma antitumor immunity.
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Melanoma , Humanos , Ratones , Animales , Melanoma/patología , Células Endoteliales/metabolismo , Linfocitos T CD8-positivos , FN-kappa B/metabolismo , Autofagia , Inmunoterapia , Microambiente TumoralRESUMEN
The combination of atezolizumab plus bevacizumab (atezo/bev) has dramatically changed the treatment landscape of advanced HCC (aHCC), achieving durable responses in some patients. Using single-cell transcriptomics, we characterize the intra-tumoural and peripheral immune context of patients with aHCC treated with atezo/bev. Tumours from patients with durable responses are enriched for PDL1+ CXCL10+ macrophages and, based on cell-cell interaction analysis, express high levels of CXCL9/10/11 and are predicted to attract peripheral CXCR3+ CD8+ effector-memory T cells (CD8 TEM) into the tumour. Based on T cell receptor sharing and pseudotime trajectory analysis, we propose that CD8 TEM preferentially differentiate into clonally-expanded PD1- CD45RA+ effector-memory CD8+ T cells (CD8 TEMRA) with pronounced cytotoxicity. In contrast, in non-responders, CD8 TEM remain frozen in their effector-memory state. Finally, in responders, CD8 TEMRA display a high degree of T cell receptor sharing with blood, consistent with their patrolling activity. These findings may help understand the possible mechanisms underlying response to atezo/bev in aHCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Linfocitos T CD8-positivos , Carcinoma Hepatocelular/tratamiento farmacológico , Bevacizumab/farmacología , Bevacizumab/uso terapéutico , Receptor de Muerte Celular Programada 1 , Células T de Memoria , Neoplasias Hepáticas/tratamiento farmacológico , Antígenos Comunes de Leucocito , Macrófagos , Receptores de Antígenos de Linfocitos T , Quimiocina CXCL10RESUMEN
BACKGROUND: Patient-derived glioma stem-like cells (GSCs) have become the gold-standard in neuro-oncological research; however, it remains to be established whether loss of in situ microenvironment affects the clinically-predictive value of this model. We implemented a GSC monolayer system to investigate in situ-in vitro molecular correspondence and the relationship between in vitro and patient response to temozolomide (TMZ). METHODS: DNA/RNA-sequencing was performed on 56 glioblastoma tissues and 19 derived GSC cultures. Sensitivity to TMZ was screened across 66 GSC cultures. Viability readouts were related to clinical parameters of corresponding patients and whole-transcriptome data. RESULTS: Tumour DNA and RNA sequences revealed strong similarity to corresponding GSCs despite loss of neuronal and immune interactions. In vitro TMZ screening yielded three response categories which significantly correlated with patient survival, therewith providing more specific prediction than the binary MGMT marker. Transcriptome analysis identified 121 genes related to TMZ sensitivity of which 21were validated in external datasets. CONCLUSION: GSCs retain patient-unique hallmark gene expressions despite loss of their natural environment. Drug screening using GSCs predicted patient response to TMZ more specifically than MGMT status, while transcriptome analysis identified potential biomarkers for this response. GSC drug screening therefore provides a tool to improve drug development and precision medicine for glioblastoma.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Temozolomida/farmacología , Temozolomida/uso terapéutico , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Dacarbazina/farmacología , Dacarbazina/uso terapéutico , Evaluación Preclínica de Medicamentos , Biomarcadores , ADN/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Resistencia a Antineoplásicos/genética , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Línea Celular Tumoral , Microambiente TumoralRESUMEN
BACKGROUND: Glioblastoma (GBM) is an aggressive brain cancer that typically results in death in the first 15 months after diagnosis. There have been limited advances in finding new treatments for GBM. In this study, we investigated molecular differences between patients with extremely short (≤ 9 months, Short term survivors, STS) and long survival (≥ 36 months, Long term survivors, LTS). METHODS: Patients were selected from an in-house cohort (GLIOTRAIN-cohort), using defined inclusion criteria (Karnofsky score > 70; age < 70 years old; Stupp protocol as first line treatment, IDH wild type), and a multi-omic analysis of LTS and STS GBM samples was performed. RESULTS: Transcriptomic analysis of tumour samples identified cilium gene signatures as enriched in LTS. Moreover, Immunohistochemical analysis confirmed the presence of cilia in the tumours of LTS. Notably, reverse phase protein array analysis (RPPA) demonstrated increased phosphorylated GAB1 (Y627), SRC (Y527), BCL2 (S70) and RAF (S338) protein expression in STS compared to LTS. Next, we identified 25 unique master regulators (MR) and 13 transcription factors (TFs) belonging to ontologies of integrin signalling and cell cycle to be upregulated in STS. CONCLUSION: Overall, comparison of STS and LTS GBM patients, identifies novel biomarkers and potential actionable therapeutic targets for the management of GBM.
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Neoplasias Encefálicas , Glioblastoma , Humanos , Anciano , Glioblastoma/patología , Pronóstico , Neoplasias Encefálicas/patología , Encéfalo/patología , SobrevivientesRESUMEN
Glioblastoma is a highly lethal grade of astrocytoma with very low median survival. Despite extensive efforts, there is still a lack of alternatives that might improve these prospects. We uncovered that the chemotherapeutic agent temozolomide impinges on fatty acid synthesis and desaturation in newly diagnosed glioblastoma. This response is, however, blunted in recurring glioblastoma from the same patient. Further, we describe that disrupting cellular fatty acid homeostasis in favor of accumulation of saturated fatty acids such as palmitate synergizes with temozolomide treatment. Pharmacological inhibition of SCD and/or FADS2 allows palmitate accumulation and thus greatly augments temozolomide efficacy. This effect was independent of common GBM prognostic factors and was effective against cancer cells from recurring glioblastoma. In summary, we provide evidence that intracellular accumulation of saturated fatty acids in conjunction with temozolomide based chemotherapy induces death in glioblastoma cells derived from patients.
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Glioblastomas are aggressive primary brain cancers that recur as therapy-resistant tumors. Myeloid cells control glioblastoma malignancy, but their dynamics during disease progression remain poorly understood. Here, we employed single-cell RNA sequencing and CITE-seq to map the glioblastoma immune landscape in mouse tumors and in patients with newly diagnosed disease or recurrence. This revealed a large and diverse myeloid compartment, with dendritic cell and macrophage populations that were conserved across species and dynamic across disease stages. Tumor-associated macrophages (TAMs) consisted of microglia- or monocyte-derived populations, with both exhibiting additional heterogeneity, including subsets with conserved lipid and hypoxic signatures. Microglia- and monocyte-derived TAMs were self-renewing populations that competed for space and could be depleted via CSF1R blockade. Microglia-derived TAMs were predominant in newly diagnosed tumors, but were outnumbered by monocyte-derived TAMs following recurrence, especially in hypoxic tumor environments. Our results unravel the glioblastoma myeloid landscape and provide a framework for future therapeutic interventions.
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Neoplasias Encefálicas/inmunología , Glioblastoma/inmunología , Macrófagos Asociados a Tumores/citología , Macrófagos Asociados a Tumores/inmunología , Animales , Humanos , Ratones , Análisis de la Célula IndividualRESUMEN
Glioblastoma (GBM), a highly invasive and vascular malignancy is shown to rapidly develop resistance and evolve to a more invasive phenotype following bevacizumab (Bev) therapy. Rho Guanine Nucleotide Exchange Factor proteins (RhoGEFs) are mediators of key components in Bev resistance pathways, GBM and Bev-induced invasion. To identify GEFs with enhanced mRNA expression in the leading edge of GBM tumours, a cohort of GEFs was assessed using a clinical dataset. The GEF ßPix/COOL-1 was identified, and the functional effect of gene depletion assessed using 3D-boyden chamber, proliferation, and colony formation assays in GBM cells. Anti-angiogenic effects were assessed in endothelial cells using tube formation and wound healing assays. In vivo effects of ßPix/COOL-1-siRNA delivered via RGD-Nanoparticle in combination with Bev was studied in an invasive model of GBM. We found that siRNA-mediated knockdown of ßPix/COOL-1 in vitro decreased cell invasion, proliferation and increased apoptosis in GBM cell lines. Moreover ßPix/COOL-1 mediated endothelial cell migration in vitro. Mice treated with ßPix/COOL-1 siRNA-loaded RGD-Nanoparticle and Bev demonstrated a trend towards improved median survival compared with Bev monotherapy. Our hypothesis generating study suggests that the RhoGEF ßPix/COOL-1 may represent a target of vulnerability in GBM, in particular to improve Bev efficacy.
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Targeting HER2 is effective in 24% of ERBB2 amplified metastatic colorectal cancer; however, secondary resistance occurs in most of the cases. We studied the evolution of individual metastases during treatment to discover spatially resolved determinants of resistance. Circulating tumor DNA (ctDNA) analysis identified alterations associated with resistance in the majority of refractory patients. ctDNA profiles and lesion-specific radiographic reports revealed organ- or metastasis-private evolutionary patterns. When radiologic assessments documented progressive disease in target lesions, response to HER2 blockade was retained in other metastases. Genomic and functional analyses on samples and cell models from eight metastases of a patient co-recruited to a postmortem study unveiled lesion-specific evolutionary trees and pharmacologic vulnerabilities. Lesion size and contribution of distinct metastases to plasma ctDNA were correlated.