# Targeted Protein Profiling of In Vivo NIPP-Treated Tissues Using DigiWest Technology
## 4 Discussion
CDK2 is a subunit of the cyclin-dependent kinase complex, mainly involved and restricted to the regulation of the G1-S phase of the cell cycle [37]. The fully active CDK2 (in a complex with cyclins) is phosphorylated at threonine 160 (T160) [38], a regulative response which could also be shown after in vitro and in vivo NIPP treatment. Moreover, a downregulation of the CDK2 inhibitor p21 [25; 26] was demonstrated in NIPP-treated CSCC cells. However, CDK2 does not seem to be essential for proceeding or arresting the transition during the G1-S phase [39]. Noticeable, in vitro and in vivo NIPP treatment caused a significantly reduced expression and phosphorylation of the proto-oncogene tyrosine-protein kinase Src, which was shown to be upregulated in about half of the tumors from the colon, liver, lung, breast, and pancreas [40]. Thereby, Src has a central impact on cell survival, proliferation, and invasion. Src has been shown to be involved in further pro-proliferative cell responses and to interact with important regulative factors including MAPK/MEK1/RAF, Akt, and STAT3 [22; 23]. Interestingly, all of these interacting factors have been shown to be reduced after in vitro NIPP treatment of CSCC cells; however, it could be not confirmed after in vivo NIPP treatment of CIN. The results suggest that changes in protein expression observed in cell culture experiments may not be transferable to in vivo treatment. This underlines the importance of performing studies on in vivo NIPP applications and consecutive analysis of the biological effects, some of which differ considerably from in vitro results. Overall, the analysis of cell culture samples is much easier; additionally, here, it resulted in more statistically significant changed analytes than the analysis of FFPE patient samples. However, the changes in expression after treatment were more distinct in the patient samples. This may be because FFPE samples delivered lower absolute intensities of signals compared to cell culture samples, which may be rooted in the differences in sample preparation. However, normalization to the total protein loaded on beads relativizes such effects. Additionally, the smaller samples size in this study may limit the statistical power of the used tests. In conclusion, we demonstrated the molecular efficacy of NIPP treatment within human malignant cervical cell lines and CIN. NIPP effects were primarily based on the inhibitory impact on the cell cycle and cell growth factors. NIPP treatment effects need to be studied more frequently in vivo, or at least in patient-derived cell culture models such as organoids or patient-derived microtumors (PDMs) that mimic the in vivo situation much better [41]. DigiWest technology enables comprehensive protein profiling from very small and FFPE primary tissue biopsies. The following are available online at [[https://www.mdpi.com/article/10.3390/app112311238/s1](https://www.mdpi.com/article/10.3390/app112311238/s1)]([https://www.mdpi.com/article/10.3390/app112311238/s1](https://www.mdpi.com/article/10.3390/app112311238/s1)), Figure S1: Overview of FFPE extraction and DigiWest workflow; Figure S2: Selected DigiWest results of a control (untreated) as well as 0 h and 24 h after treatment FFPE samples; Table S1: Raw and normalized data used for analysis; Table S2: List of used antibodies. Conceptualization, F.R., M.T. and M.W. ; methodology, F.R., M.T. and M.W. ; formal analysis, F.R. and M.W. ; investigation, F.R. and M.W. ; data curation, F.R. and M.W. ; writing--original draft preparation, F.R. and M.W. ; writing--review and editing, M.T., M.H., M.E., H.B., D.W., S.Y.B. and K.S.-L.; supervision, M.T., D.W., S.Y.B. and K.S.-L.; project administration, M.W. All authors have read and agreed to the published version of the manuscript. This study was financially supported by the Faculty of Medicine of the Eberhard Karls University of Tubingen (grant No. 2432-1-0, 417-0-0 to M.W., and IZKF 2018-1-06 to M.W.