In conclusion, our parallel immunohistochemical analysis of 29 HNSCC cell lines suggests, that radioresistance of HNSCC is regulated by stemness-related mechanisms. of cell lines as well as correlating staining results with other cell line characteristics. In addition, CMA-based antibody screening proved an efficient and relatively simple method to identify potential radioresistance biomarkers in HNSCC. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-021-08618-6. value ?0.05 was deemed significant. Results CMA construction For the CMA construction, UT-SCC cell lines with previous data on radiosensitivity were preferentially selected. Altogether 26 UT-SCC cell lines were included, the majority of which were derived from male patients with oral cavity cancer (Table ?(Table1).1). Six cell lines were derived from metastatic samples, and two cell lines from recurring cancers. In addition, PPACK Dihydrochloride three stable CIP2A shRNA-silenced cell lines were included. The cell lines were cultured until a sufficient number of cells was obtained, whereafter cells were pelleted, fixed in formalin and embedded in paraffin. The following formalin-fixed, paraffin-embedded cell blocks were annotated, and a CMA was arrayed. The CMA construction is exhibited in Fig. ?Fig.1A1A and an example of immunohistochemical assessment PPACK Dihydrochloride in Fig. ?Fig.11B. Association of biomarker staining intensities and intrinsic radioresistance Immunohistochemical stains for p53, EGFR, Oct4, CIP2A, and NDFIP1 were analyzed (Fig.?2A-L, Table?2). There were significant correlations between Oct4 and NDFIP1 stains ( 0.46, values are indicated. A p53, B EGFR, and D CIP2A demonstrate no association with radioresistance, whereas C NDFIP1, and E Oct4 have a significant (*) association with the intrinsic radioresistance of the cell lines. F A highly significant conversation CD133 effect between p53 and Oct4 was revealed, implying a predictive role of Oct4 in the absence of p53 mutation/amplification type staining. PPACK Dihydrochloride G The conversation trend between p53 and EGFR did not reach significance Analysis of CIP2A-shRNA-silenced cell lines To test the functionality of CMA in the validation of antibody specificity, and to study protein interactions in genetically modified cancer cell lines, three CIP2A shRNA-silenced cell lines were included in the CMA. CIP2A shRNA-silencing was confirmed by Western blot (Fig. ?(Fig.4A)4A) and is demonstrated by the minimal immunoreactivity of the silenced cell lines compared to the parental non-silenced lines (Fig. ?(Fig.4B-F).4B-F). Parallel immunohistochemistry of the potential radioresistance biomarkers did not reveal significant correlations between CIP2A silencing and p53, EGFR, NDFIP1, or Oct4 expression (Table?3). Table 3 Immunohistochemistry of UT-SCC-14 and UT-SCC-24A and corrresponding CIP2A-shRNA-silenced cell lines thead th rowspan=”2″ colspan=”1″ Cell line /th th colspan=”8″ rowspan=”1″ Immunohistochemistry /th th rowspan=”1″ colspan=”1″ CIP2A /th th rowspan=”1″ colspan=”1″ p53 /th th rowspan=”1″ colspan=”1″ LIMA1 /th th rowspan=”1″ colspan=”1″ EGFR /th th rowspan=”1″ colspan=”1″ NDFIP1 /th th rowspan=”1″ colspan=”1″ Oct4 /th th rowspan=”1″ colspan=”1″ SET /th th rowspan=”1″ colspan=”1″ PME-1 /th /thead Non-treated UT-SCC-142mutated130122CIP2A-shRNA-silenced UT-SCC-14 (plasmid 557)1mutated130111Non-treated UT-SCC-24A3absent231112CIP2A-shRNA-silenced UT-SCC-24A (plasmid 556)1absent230111CIP2A-shRNA-silenced UT-SCC-24A (plasmid 557)1absent231111 Open in a separate window Since CIP2A failed to predict radioresistance in the CMA and since CIP2A silencing did not affect other putative biomarkers expression, we were interested, whether CIP2A would have an association with other PP2A inhibitors and performed immunohistochemistry of two well-established endogenous PP2A inhibitors, PME-1 and SET, which are not pronouncedly identified as radiotherapy biomarkers (Supplemental Physique 1). Surprisingly, in all three silenced cell lines the expression of both PME-1 and SET was reduced as well, suggesting a CIP2A-mediated circuitry leading to a more universal loss of PP2A inhibitor expression (Table ?(Table3).3). However, CIP2A, PME-1 and SET expression levels were not correlated across PPACK Dihydrochloride other UT-SCC cell lines of the CMA. Neither PME-1 nor SET was associated with intrinsic radioresistance of the cell lines. Discussion HNSCC consists of a genetically and behaviorally heterogeneous group of malignancies. The observed genomic instability is due to mutagenic insults around the mucosal lining of the upper aerodigestive tract such as tobacco and alcohol . Accordingly, HNSCC-derived cell lines exhibit highly variable genomic changes [26C29]. The field cancerization phenomenon makes radiotherapy an especially inviting treatment option, and it is included in the treatment plan of nearly one half of head and neck cancer patients . Since no biomarkers can currently be used to identify patients benefitting of different treatment strategies, or to explain the clinical diversity in radiotherapy outcomes, the question of radioresistance identification is usually of tremendous importance for treatment of HNSCC. Prior studies presenting similar methods of cell line array construction have mostly included relatively small amounts of cell lines, and have been constructed with.