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Characterisation of tumour microenvironment remodelling following oncogene inhibition in preclinical studies with imaging mass cytometry.

ABSTRACT: Mouse models are critical in pre-clinical studies of cancer therapy, allowing dissection of mechanisms through chemical and genetic manipulations that are not feasible in the clinical setting. In studies of the tumour microenvironment (TME), multiplexed imaging methods can provide a rich source of information. However, the application of such technologies in mouse tissues is still in its infancy. Here we present a workflow for studying the TME using imaging mass cytometry with a panel of 27 antibodies on frozen mouse tissues. We optimise and validate image segmentation strategies and automate the process in a Nextflow-based pipeline (imcyto) that is scalable and portable, allowing for parallelised segmentation of large multi-image datasets. With these methods we interrogate the remodelling of the TME induced by a KRAS G12C inhibitor in an immune competent mouse orthotopic lung cancer model, highlighting the infiltration and activation of antigen presenting cells and effector cells.

SUBMITTER: van Maldegem F

PROVIDER: S-EPMC8501076 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Characterisation of tumour microenvironment remodelling following oncogene inhibition in preclinical studies with imaging mass cytometry.

van Maldegem Febe F Valand Karishma K Cole Megan M Patel Harshil H Angelova Mihaela M Rana Sareena S Colliver Emma E Enfield Katey K Bah Nourdine N Kelly Gavin G Tsang Victoria Siu Kwan VSK Mugarza Edurne E Moore Christopher C Hobson Philip P Levi Dina D Molina-Arcas Miriam M Swanton Charles C Downward Julian J

Nature communications 20211008 1

Mouse models are critical in pre-clinical studies of cancer therapy, allowing dissection of mechanisms through chemical and genetic manipulations that are not feasible in the clinical setting. In studies of the tumour microenvironment (TME), multiplexed imaging methods can provide a rich source of information. However, the application of such technologies in mouse tissues is still in its infancy. Here we present a workflow for studying the TME using imaging mass cytometry with a panel of 27 anti ...[more]

PMID: 34625563

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Project description:BackgroundTherapeutic modalities including chemo, radiation, immunotherapy, etc. induce PD-L1 expression that facilitates the adaptive immune resistance to evade the antitumour immune response. IFN-γ and hypoxia are some of the crucial inducers of PD-L1 expression in tumour and systemic microenvironment which regulate the expression of PD-L1 via various factors including HIF-1α and MAPK signalling. Hence, inhibition of these factors is crucial to regulate the induced PD-L1 expression and to achieve a durable therapeutic outcome by averting the immunosuppression.MethodsB16-F10 melanoma, 4T1 breast carcinoma, and GL261 glioblastoma murine models were established to investigate the in vivo antitumour efficacy of Ponatinib. Western blot, immunohistochemistry, and ELISA were performed to determine the effect of Ponatinib on the immunomodulation of tumour microenvironment (TME). CTL assay and flow cytometry were such as p-MAPK, p-JNK, p-Erk, and cleaved caspase-3 carried out to evaluate the systemic immunity induced by Ponatinib. RNA sequencing, immunofluorescence and Western blot analysis were used to determine the mechanism of PD-L1 regulation by Ponatinib. Antitumour immunity induced by Ponatinib were compared with Dasatinib.ResultsHere, Ponatinib treatment delayed the growth of tumours by inhibiting PD-L1 and modulating TME. It also downregulated the level of PD-L1 downstream signalling molecules. Ponatinib enhanced the CD8 T cell infiltration, regulated Th1/Th2 ratio and depleted tumour associated macrophages (TAMs) in TME. It induced a favourable systemic antitumour immunity by enhancing CD8 T cell population, tumour specific CTL activity, balancing the Th1/Th2 ratio and lowering PD-L1 expression. Ponatinib inhibited FoxP3 expression in tumour and spleen. RNA sequencing data revealed that Ponatinib treatment downregulated the genes related to transcription including HIF-1α. Further mechanistic studies showed that it inhibited the IFN-γ and hypoxia induced PD-L1 expression via regulating HIF-1α. Dasatinib was used as control to prove that Ponatinib induced antitumour immunity is via PD-L1 inhibition mediated T cell activation.ConclusionsRNA sequencing data along with rigorous in vitro and in vivo studies revealed a novel molecular mechanism by which Ponatinib can inhibit the induced PD-L1 levels via regulating HIF-1α expression which leads to modulation of tumour microenvironment. Thus, our study provides a novel therapeutic insight of Ponatinib for the treatment of solid tumours where it can be used alone or in combination with other drugs which are known to induce PD-L1 expression and generate adaptive resistance.

Project description:BackgroundInterleukin-33 (IL-33) is an effective inducer of pro-inflammatory cytokines regulating innate and adaptive immunity. Inflammation could be a double-edged sword, promoting or inhibiting tumour growth. To date, the roles and mechanisms of IL-33 in tumours remain controversial. Here, we examined the effect of exogenous IL-33 on the biological characteristics of hepatocellular carcinoma (HCC) and the possible mechanism of action.MethodsIn this study, IL-33 expression in the tissues of 69 HCC patients was detected and its relationship with prognosis was evaluated. After establishing a mouse HCC model and IL-33 treatment operation, the infiltration of splenic myeloid-derived suppressor (MDSCs), dendritic (DCs), regulatory T, and natural killer (NK) cells was detected by flow cytometry analysis, and the vascular density of the tumour tissues was detected by immunohistochemistry to reveal the mechanism of IL-33 in HCC proliferation. Finally, the Cancer Genome Atlas database was used to analyse Gene Ontology terms the and Kyoto Encyclopaedia of Genes and Genomes pathway. Moreover, the chi-square test, two-tailed unpaired Student's t-test, and multiple t-tests were performed using SPSS version 23.0 and GraphPad Prism 8.0 software.ResultsThe IL-33 expression level was negatively correlated with the overall survival of HCC patients, suggesting its potential clinical significance in the prognosis of HCC. We found that systemic IL-33 administration significantly promoted the tumour size in vivo. Furthermore, the IL-33-treated mice presented decreased frequencies of tumouricidal NK and CD69+ CD8+ T cells. After IL-33 treatment, the incidence of monocytic MDSCs and conventional DCs increased, while that of granulocytic MDSCs decreased. Moreover, IL-33 promoted the formation of intracellular neovascularization. Therefore, IL-33 accelerated HCC progression by increasing the accumulation of immunosuppressive cells and neovascularization formation. Finally, we found that the transcription of IL-33 was closely related to the PI3K-Akt and MAPK pathways in Gene Set Enrichment Analysis plots, which were involved in the tumourigenesis and pathogenesis of HCC.ConclusionsTaken together, IL-33 may be a key tumour promoter of HCC proliferation and tumourigenicity, an important mediator, and a potential therapeutic target for regulating HCC progression.

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Characterisation of tumour microenvironment remodelling following oncogene inhibition in preclinical studies with imaging mass cytometry.

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Characterisation of tumour microenvironment remodelling following oncogene inhibition in preclinical studies with imaging mass cytometry.

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