RESUMO
BACKGROUND: Targeting of solid cancers with chimeric antigen receptor (CAR)-T cells is limited by the lack of suitable tumor-specific antigens and the immunosuppressive, desmoplastic tumor microenvironment that impedes CAR-T cell infiltration, activity and persistence. We hypothesized that targeting the endosialin (CD248) receptor, strongly expressed by tumor-associated pericytes and perivascular cancer-associated fibroblasts, would circumvent these challenges and offer an exciting antigen for CAR-T cell therapy due to the close proximity of target cells to the tumor vasculature, the limited endosialin expression in normal tissues and the lack of phenotype observed in endosialin knockout mice. METHODS: We generated endosialin-directed E3K CAR-T cells from three immunocompetent mouse strains, BALB/c, FVB/N and C57BL/6. E3K CAR-T cell composition (CD4+/CD8+ ratio), activity in vitro against endosialin+ and endosialin- cells, and expansion and activity in vivo in syngeneic tumor models as well as in tumor-naive healthy and wounded mice and tumor-bearing endosialin knockout mice was assessed. RESULTS: E3K CAR-T cells were active in vitro against both mouse and human endosialin+, but not endosialin-, cells. Adoptively transferred E3K CAR-T cells exhibited no activity in endosialin knockout mice, tumor-naive endosialin wildtype mice or in wound healing models, demonstrating an absence of off-target and on-target/off-tumor activity. By contrast, adoptive transfer of E3K CAR-T cells into BALB/c, FVB/N or C57BL/6 mice bearing syngeneic breast or lung cancer lines depleted target cells in the tumor stroma resulting in increased tumor necrosis, reduced tumor growth and a substantial impairment in metastatic outgrowth. CONCLUSIONS: Together these data highlight endosialin as a viable antigen for CAR-T cell therapy and that targeting stromal cells closely associated with the tumor vasculature avoids CAR-T cells having to navigate the harsh immunosuppressive tumor microenvironment. Further, the ability of E3K CAR-T cells to recognize and target both mouse and human endosialin+ cells makes a humanized and optimized E3K CAR a promising candidate for clinical development applicable to a broad range of solid tumor types.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Camundongos , Animais , Pericitos/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Linfócitos T/metabolismo , Camundongos Knockout , Microambiente Tumoral , Antígenos de Neoplasias/metabolismo , Antígenos CD/metabolismoRESUMO
Lung cancer is the leading cause of cancer deaths. Its high mortality is associated with high metastatic potential. Here, we show that the RAC1-selective guanine nucleotide exchange factor T cell invasion and metastasis-inducing protein 1 (TIAM1) promotes cell migration and invasion in the most common subtype of lung cancer, non-small-cell lung cancer (NSCLC), through an unexpected nuclear function. We show that TIAM1 interacts with TRIM28, a master regulator of gene expression, in the nucleus of NSCLC cells. We reveal that a TIAM1-TRIM28 complex promotes epithelial-to-mesenchymal transition, a phenotypic switch implicated in cell migration and invasion. This occurs through H3K9me3-induced silencing of protocadherins and by decreasing E-cadherin expression, thereby antagonizing cell-cell adhesion. Consistently, TIAM1 or TRIM28 depletion suppresses the migration of NSCLC cells, while migration is restored by the simultaneous depletion of protocadherins. Importantly, high nuclear TIAM1 in clinical specimens is associated with advanced-stage lung adenocarcinoma, decreased patient survival, and inversely correlates with E-cadherin expression.
Assuntos
Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/genética , Protocaderinas , Carcinoma Pulmonar de Células não Pequenas/genética , Caderinas/genética , Epigênese Genética , Proteína 28 com Motivo Tripartido , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/genéticaRESUMO
Resistance mechanisms to immune checkpoint blockade therapy (ICBT) limit its response duration and magnitude. Paradoxically, Interferon γ (IFNγ), a key cytokine for cellular immunity, can promote ICBT resistance. Using syngeneic mouse tumour models, we confirm that chronic IFNγ exposure confers resistance to immunotherapy targeting PD-1 (α-PD-1) in immunocompetent female mice. We observe upregulation of poly-ADP ribosyl polymerase 14 (PARP14) in chronic IFNγ-treated cancer cell models, in patient melanoma with elevated IFNG expression, and in melanoma cell cultures from ICBT-progressing lesions characterised by elevated IFNγ signalling. Effector T cell infiltration is enhanced in tumours derived from cells pre-treated with IFNγ in immunocompetent female mice when PARP14 is pharmacologically inhibited or knocked down, while the presence of regulatory T cells is decreased, leading to restoration of α-PD-1 sensitivity. Finally, we determine that tumours which spontaneously relapse in immunocompetent female mice following α-PD-1 therapy upregulate IFNγ signalling and can also be re-sensitised upon receiving PARP14 inhibitor treatment, establishing PARP14 as an actionable target to reverse IFNγ-driven ICBT resistance.
Assuntos
Inibidores de Checkpoint Imunológico , Melanoma , Feminino , Humanos , Animais , Camundongos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Receptor de Morte Celular Programada 1 , Interferon gama , Recidiva Local de Neoplasia , Modelos Animais de Doenças , Poli(ADP-Ribose) PolimerasesRESUMO
The impact of radiotherapy on the interaction between immune cells and cancer cells is important not least because radiotherapy can be used alongside immunotherapy as a cancer treatment. Unexpectedly, we found that X-ray irradiation of cancer cells induced significant resistance to natural killer (NK) cell killing. This was true across a wide variety of cancer-cell types as well as for antibody-dependent cellular cytotoxicity. Resistance appeared 72 h postirradiation and persisted for 2 wk. Resistance could also occur independently of radiotherapy through pharmacologically induced cell-cycle arrest. Crucially, multiple steps in NK-cell engagement, synapse assembly, and activation were unaffected by target cell irradiation. Instead, radiotherapy caused profound resistance to perforin-induced calcium flux and lysis. Resistance also occurred to a structurally similar bacterial toxin, streptolysin O. Radiotherapy did not affect the binding of pore-forming proteins at the cell surface or membrane repair. Rather, irradiation instigated a defect in functional pore formation, consistent with phosphatidylserine-mediated perforin inhibition. In vivo, radiotherapy also led to a significant reduction in NK cell-mediated clearance of cancer cells. Radiotherapy-induced resistance to perforin also constrained chimeric antigen receptor T-cell cytotoxicity. Together, these data establish a treatment-induced resistance to lymphocyte cytotoxicity that is important to consider in the design of radiotherapy-immunotherapy protocols.
Assuntos
Citotoxicidade Imunológica , Neoplasias/metabolismo , Radioterapia , Citotoxicidade Celular Dependente de Anticorpos , Proteínas de Bactérias , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Imunoterapia , Células Matadoras Naturais/imunologia , Perforina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , EstreptolisinasRESUMO
Zebrafish embryo tumor transplant models are widely utilized in cancer research. Compared with traditional murine models, the small size and transparency of zebrafish embryos combined with large clutch sizes that increase statistical power and cheap husbandry make them a cost-effective and versatile tool for in vivo drug discovery. However, the lack of a comprehensive analysis of key factors impacting the successful use of these models impedes the establishment of basic guidelines for systematic screening campaigns. Thus, we explored the following crucial factors: (i) user-independent inclusion criteria, focusing on sample homogeneity; (ii) metric definition for data analysis; (iii) tumor engraftment criteria; (iv) image analysis versus quantification of human cancer cells using qPCR (RNA and gDNA); (v) tumor implantation sites; (vi) compound distribution (intratumoral administration versus alternative inoculation sites); and (vii) efficacy (intratumoral microinjection versus compound solution in media). Based on these analyses and corresponding assessments, we propose the first roadmap for systematic drug discovery screening in zebrafish xenograft cancer models using a melanoma cell line as a case study. This study aims to help the wider cancer research community to consider the adoption of this versatile model for cancer drug screening projects.