# Targeted Protein Profiling of In Vivo NIPP-Treated Tissues Using DigiWest Technology
## 3 Results
### Protein Profiles of Patients and Cell Culture following In Vivo and In Vitro NIPP Treatment  
NIPP treatment induces various biological effects, including antineoplastic efficacy [8]. Therefore, NIPP is a promising tool for the treatment of precancer and cancer. Here, we examined the overall antineoplastic properties by performing both in vitro NIPP treatment of the human malignant cervical cell line, CSCC, as well as in vivo NIPP treatment of patients with histologically confirmed lesions of CIN. First, we analyzed cell pellets from an NIPP-treated CSCC cell culture (\(n=6\)) compared to argon-treated controls (\(n=6\)) harvested after 24 h. We analyzed a total of 132 proteins, covering apoptosis, DDR, and cell cycle control, forty-four of which were matching analytes with the FFPE analysis (Figures 3a and 4a). Generally, the differences in signal intensity in the cell culture sample set was rather low, due to only one analyte showing a mean log2 foldchange greater than 1 or \(-1\) after in vitro NIPP treatment (Figure 3b). Figure 2: DigiWest protein profiles of a control (untreated) as well as 0 h and 24 h after treatment patient samples. Heat map of log2 transformed DigiWest data. Data were median-centered, and hierarchical clustering was performed using complete linkage and Euclidian distance, utilizing the MultiExperiment Viewer (MeV version 4.9.0, ref. [19]) software. Yellow indicates high signal level and blue indicates a low signal level (compared to the median). NIPP-treated CSCC cells showed significantly reduced expression levels of various pro-proliferative factors (Figure 3c). We found that the mitogen-activated protein kinase (MAPK) pathway was targeted by NIPP treatment. This was shown by a significant downregulation of p38 mitogen-activated protein kinases (p38), the RAF proto-oncogene serine/threonine-protein kinase (c-Raf) acting as a kinase cascade initiator [20], as well as the dual specificity mitogen-activated protein kinase kinase 1 (MEK1), being a dual threonine and tyrosine recognition kinase responsible for MAPK phosphorylation and activation [21]. These factors are critically involved in cell growth and apoptosis regulation and can act as oncogenes. Furthermore, the proto-oncogene tyrosine-protein kinase Src was significantly decreased, whereas it showed a slight but statistically significant increase in phosphorylation (pTyr527). As a central proto-oncogene, Src plays an important role in cell survival, proliferation, and invasion, and has been shown to interact with several signaling pathways, including MAPK/MEK1/RAF, Akt, and signal transducer and activator of transcription 3 (STAT3) [22; 23]. Figure 3: Protein expression after in vitro non-invasive physical plasma (NIPP) treatment of the human malignant cervical squamous cell carcinoma cell line (CSCC). (**a**) Heat map of log2-transformed DigiWest data. Yellow indicates a high signal level and blue indicates a low signal level (compared to the median). (**b**) Bar graphs of log2-transformed ratios calculated from the mean expression of NIPP treatment and controls, sorted from the highest positive change to the highest negative change. (**c**) Bar graphs of signals generated from cell culture samples. Shown are the means and standard derivation of NIPP treatment and controls. * indicates a significant difference in expression, n.s. indicates no significant difference in expression (Wilcoxon rank sum test, \(p\) < 0.05). Interestingly, NIPP treatment of CSCC cells caused a significant decrease in the transcription factor STAT3, which plays an important role in many cellular processes, such as cell growth and apoptosis [24]. Furthermore, we found a downregulation of cyclin-dependent kinase inhibitor 1 (p21) being primarily associated with inhibition of cyclin-dependent kinase 2 (CDK2) [25; 26]. Thus, the phosphorylation of CDK2 (CDK2-PT160) was also significantly increased, having an impact on cell growth and cell cycle regulation.