# Targeted Protein Profiling of In Vivo NIPP-Treated Tissues Using DigiWest Technology
## 4 Discussion  
To date, only a few individual case reports exist, describing in vivo NIPP treatment of cancer patients [6; 8; 14]; none have been conducted yet on NIPP treatment of precancerous diseases. Recently, we established the electrosurgical argon plasma device VIO3/APC3 (Erbe Elektromedizin, Tubingen, Germany) for the in vivo treatment of patients [9; 30]. Currently, we are performing a prospective, single-armed phase IIb clinical trial (NCT03218436) at the Department for Women's Health, Tubingen, Germany. For this purpose, the molecular examination of tiny cervical punch biopsies obtained from in vivo NIPP-treated patients is becoming more and more crucial. However, although fresh, frozen tissue would be ideal for research purposes, this is not practical for a clinical setting. For histopathological assessment, the morphological structure of tissues must be conserved. Therefore, clinical samples are immediately fixed with formalin and embedded in paraffin wax. Formalin cross-links form a protein grid that preserves the tissue structure and prevents protein degradation [31]. Subsequent paraffin embedding facilitates the handling of samples and sectioning of the tissue into thin slices for staining and microscopical assessment. Furthermore, formalin fixation enables long-term storage at room temperature. As efficient as formalin fixation is for the prevention of tissue degradation, the occurring protein crosslinks disturb most bioanalytical methods. In particular, the separation of molecules based on their molecular size is usually disabled, since the crosslinks cause irreversible protein aggregation, resulting in low amounts of specific proteins. However, DigiWest technology enables Western-blot-like incubation of up to app. 200 antibodies from minimal sample amounts by transferring proteins onto microspheres and miniaturization of the assay system [18]. In the present study, we established the targeted protein analysis from small-sized FFPE tissue sections obtained from cervical punch biopsies utilizing DigiWest technology. This enabled the analysis of molecular tissue effects following in vivo NIPP treatment of CIN. Moreover, we compared the results with the in vitro NIPP-treated human malignant cervical cell line, CSCC. Non-thermally operated NIPP devices lead to the formation of ROS, as shown by previous studies [6; 14; 32]. ROS and RNS are the responsible drivers of NIPP-related anti-neoplastic efficacy in human cervical cancer cells due to cell cycle arrest and apoptosis [9]. Additionally, in this study, NIPP treatment affected central factors in the regulation of apoptosis and cell growth pathways. In particular, NIPP treatment resulted in the transient induction of cell survival factors (CK8/18), accompanied and followed by the downregulation of pro-proliferative factors (here: Akt, p38 MAPK, Src, and RAF) and the upregulation of cell-growth-attenuating pathways. The in vitro cell panel used in this study includes cells from squamous epithelial tumors and adenocarcinoma. SiHa and CaSki cells harbor HPV infections; DoTc2 4510 cells originate from a metastatic CC lesion. Moreover, several well-known mutations of gynecological cancers, such as p53, BRCA2, or PIK3 CA, are represented by these cell lines. In general, we found no evidence for a distinct factor resulting in increased NIPP resistance. This indicates a multifactorial intracellular process initiated by NIPP treatment. Besides its structural function in the cytoskeleton, CK8/18 regulates apoptosis and is released during apoptosis and necrosis [33; 34]. CK18 release can even occur independently of caspase activation [35]. Hence, our findings suggest that NIPP treatment may directly induce a cell survival response, followed by apoptosis. It is likely that NIPP impairs the Akt-driven progression of the G1-S cell cycle phase by inactivating glycogen synthase kinase 3 (GSK-3) and preventing cyclin D1 degradation [36].