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Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
Loss of expression of TIMP3 in clear cell renal cell carcinoma
Damien Masson1, Nathalie Rioux-Leclercq, Patricia Fergelot
1CNRS UMR 6061, Institut de Génétique et Développement, Université Rennes 1, 35043 Rennes, France.
Aims:
In clear cell renal cell carcinoma (CCRCC), vascular endothelial growth factor (VEGF) represents the central positive mediator of tumour angiogenesis while VEGF receptor (VEGFR) is the primary target of anti-angiogenic therapies. TIMP3 is a physiological VEGFR-2 antagonist and thus could be considered as an anti-angiogenic factor. We therefore determined the status of this physiological inhibitor in CCRCC.
Patients And Methods:
Archival tumour from 105 patients was studied. TIMP3 expression was analysed using immuno-histochemistry and real-time RT-PCR. Results were correlated with clinicopathological variables. To analyse the mechanisms of gene silencing involved, we performed Multiplex Ligation-dependent Probe Amplification (MLPA) and methylation-specific MLPA (MS-MLPA). At last, we evaluated the main upstream pathway described implicating TGFbetaRII, which induces TIMP3 expression.
Results:
A down-expression of TIMP3, determined by immunohistochemistry, affected 100/105 renal cancers (95.2%). TIMP3 mRNA levels were significantly lower in high-grade tumours. Loss of heterozygosity of the TIMP3 gene was observed in 8 tumours (7.6%) and the 5'CpG island of the TIMP3 promoter was found to be methylated in 25 tumours (23.8%). A down-expression of TGFbetaRII was found in 85/105 CCRCCs (80.9%). A significant correlation was found between TIMP3 expression and TGFbetaRII expression.
Conclusions:
This is the first demonstration that the loss of TIMP3 expression is observed in almost all CCRCCs. This loss of expression is a common molecular event in CCRCC. It may be an important initiation step for tumour development in a complex process implicating loss of heterozygosity on chromosome 22q, promoter hyper-methylation and inactivation of the TGFbetaRII pathway.
Insights
Loss of TIMP3 expression is a common molecular event in clear cell renal cell carcinoma (CCRCC), potentially initiating tumor development. This involves gene silencing mechanisms and the TGFbetaRII pathway.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Genetics
Background:
- Clear cell renal cell carcinoma (CCRCC) involves tumor angiogenesis mediated by vascular endothelial growth factor (VEGF).
- VEGF receptor (VEGFR) is a primary target for anti-angiogenic therapies.
- TIMP3 acts as a physiological VEGFR-2 antagonist, suggesting potential anti-angiogenic properties.
Purpose of the Study:
- To investigate the expression status of TIMP3, a natural VEGFR-2 antagonist, in CCRCC.
- To determine the molecular mechanisms underlying TIMP3 dysregulation in CCRCC.
Main Methods:
- Immunohistochemistry and real-time RT-PCR were used to analyze TIMP3 expression in 105 archival CCRCC tumors.
- Multiplex Ligation-dependent Probe Amplification (MLPA) and methylation-specific MLPA (MS-MLPA) were employed to assess gene silencing mechanisms.
- The expression of TGFbetaRII, an upstream pathway regulating TIMP3, was also evaluated.
Main Results:
- Down-expression of TIMP3 was observed in 95.2% of CCRCC cases.
- Lower TIMP3 mRNA levels correlated with higher tumor grade.
- Gene silencing mechanisms included loss of heterozygosity (7.6%) and promoter hyper-methylation (23.8%) of TIMP3.
- Down-expression of TGFbetaRII was found in 80.9% of CCRCCs, with a significant correlation to TIMP3 expression.
Conclusions:
- Loss of TIMP3 expression is a frequent molecular event in CCRCC, potentially serving as an early step in tumorigenesis.
- This loss is associated with genetic alterations (LOH, hyper-methylation) and inactivation of the TGFbetaRII pathway.
- Understanding TIMP3 regulation in CCRCC may offer insights for novel anti-angiogenic therapeutic strategies.
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