Reverse phase protein microarray (RPPA) projects

Figure 1: RPPA antibodies were chosen to quantify the activation state of 12 cancer-relevant signaling nodes (figure inspired by B. Vogelstein lecture, AACR, 2010).
© dkfz.de

Antibody validation for RPPA

Antibody selection for RPPA focusses on probing the activation state of known cancer-relevant signaling pathways (figure 1). All antibodies are initially screened by Western blot on a cell line panel, and antibodies suitable for RPPA must show an explainable band (pattern). Only those antibodies resulting in a correlation factor of >0.7, when comparing Western blot signals and RPPA signals, are employed in routine RPPA applications, with few exceptions comprising mainly antibodies against large proteins that can not be analyzed by Western blot, e.g. Ki-67. In this instance, immunohistochemistry data are used for validation. For RPPA antibody validation we follow key steps described on the website of the RPPA Core Facility, MD Anderson Cancer Center.

Ongoing RPPA projects

Current systems biology projects focussed on identifying molecular mechanisms of drugs targeting EGFR, ERBB2, as well as c-MET. RPPA was also employed to profile a panel of NSCLC cell lines. Combining transcriptomics and targeted proteomics yielded new insights into a stabilization of the transcription factor GLI1 as consequence of the crosstalk between EGF- and Hedgehog-signaling.

We have also employed RPPA for risk classification for patients suffering hormone receptor-positive breast cancer and have identified a protein biomarker signature that can predict the need of chemotherapy (PCT/EP 2013/052541).

The RPPA technology is used throughout several third-party funded projects:

In MetastaSys, for example, we plan to carry out proteomic profiling of brain and liver metastasis to contribute to elucidating the systems biology of cancer metastasis.

As part of the e.BIO project SysMetBC, we plan to gain insights into crosstalk between metabolic pathways, commonly deregulated in cancer, and canonical signaling pathways. This project focuses on human breast cancer and employs clinical samples covering all major molecular subtypes of human breast cancer as well as established breast cancer cell line models.

As part of the MycNet consortium, funded as CancerSys (BMBF) iniative, a systematic proteomic profiling of neuroblastoma and glioblastoma cell lines will be carried out.

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