Abstract
Diffuse large B-cell lymphomas (DLBCLs) are the most common types of Non-Hodgkin’s lymphomas and are highly heterogeneous in terms of phenotype and treatment response. The natural course of DLBCLs tumor progression is featured by a flow of events leading to the enhancement of proliferative and invasive capabilities and, therefore, towards the establishment of a more aggressive phenotype. Angiogenesis is a constant hallmark of DLBCLs progression, has prognostic potential and promote DLBCLs dissemination. The study of DLBCLs angiogenesis mechanisms, and the tumor endothelium characterization, will allow us to identify new prognostic/predictive biomarkers to proper patient selection to antiangiogenic treatment. In our previous work, by RNAscope technology, we have demonstrated that Janus kinase (Jak) and signal transducer activator of transcription pathway (STAT) is one of the proangiogenic pathways activated in DLBCLs and it drives neoangiogenesis occurred by vasculogenesis mechanism. Here, we describe a detailed protocol to perform RNAscope technology alone and in combination with immunohistochemistry (called dual RNAscope ISH–IHC) in DLBCLs formalin-fixed, paraffin-embedded sections. We propose dual ISH–IHC as an extremely powerful method to study angiogenesis in DLBCLs, because it allows one to answer important biological questions that are difficult to address using other single methods.
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Abbreviations
- ACD:
-
Advanced cell diagnostics
- DLBCLs:
-
Diffuse large B-cell lymphomas
- ECs:
-
Endothelial cells
- FFPE:
-
Formalin-fixed paraffin-embedded
- FVIII:
-
Coagulation factor VIII
- IHC:
-
Immunohistochemistry
- ISH:
-
In situ hybridization
- min:
-
Minute/minutes
- NHLs:
-
Non-Hodgkin lymphomas
- on:
-
Overnight
- R-CHOP:
-
Rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone
- RT:
-
Room temperature
- sec:
-
Seconds
- STAT3:
-
Signal transducer and activator of transcription 3
- TAM:
-
Tumor-associated macrophages
- TRR:
-
Target retrieval reagent
- WB:
-
Washing buffer
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Acknowledgements
This work was supported by Fellowship Fondazione Italiana per la Ricerca sul Cancro e Associazione Italia per la Ricerca sul Cancro (FIRC-AIRC) 1-year fellowship “Laura Bassi” id. 20879 to TA. Associazione “Il Sorriso di Antonio, Corato, Italy, is also acknowledged.
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TA wrote the manuscript with support from RT and SR; TA and MD developed the protocol; EM, GS, and DR supervised the manuscript.
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Annese, T., Tamma, R., De Giorgis, M. et al. RNAscope dual ISH–IHC technology to study angiogenesis in diffuse large B-cell lymphomas. Histochem Cell Biol 153, 185–192 (2020). https://doi.org/10.1007/s00418-019-01834-z
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DOI: https://doi.org/10.1007/s00418-019-01834-z