Specific detection of OCT3/4 isoform A/B/B1 expression in solid (germ cell) tumours and cell lines: Confirmation of OCT3/4 specificity for germ cell tumours

Background: OCT3/4 (POU5F1) is an established diagnostic immunohistochemical marker for specific histological variants of human malignant germ cell tumours (GCTs), including the seminomatous types and the stem cell component of non-seminomas, known as embryonal carcinoma. OCT3/4 is crucial for the regulation of pluripotency and the self-renewal of normal embryonic stem-and germ cells. Detection of expression of this transcription factor is complicated by the existence of multiple pseudogenes and isoforms. Various claims have been made about OCT3/4 expression in non-GCTs, possibly related to using nonspecific detection methods. False-positive findings undermine the applicability of OCT3/4 as a specific diagnostic tool in a clinical setting. In addition, false-positive findings could result in misinterpretation of pluripotency regulation in solid somatic cancers and their stem cells. Of the three identified isoforms-OCT4A, OCT4B and OCT4B1-only OCT4A proved to regulate pluripotency. Up until now, no convincing nuclear OCT4A protein expression has been shown in somatic cancers or tissues. Methods: This study investigates expression of the various OCT3/4 isoforms in GCTs (both differentiated and undifferentiated) and somatic (non-germ cell) cancers, including representative cell lines and xenografts. Results: Using specific methods, OCT4A and OCT4B1 are shown to be preferentially expressed in undifferentiated GCTs. The OCT4B variant shows no difference in expression between GCTs (either differentiated or undifferentiated) and somatic cancers. In spite of the presence of OCT4A mRNA in somatic cancer-derived cell lines, no OCT3/4

Fig 4 -

Percent duration in motion (A) and motionless (B) by zone across both sexes. Quadrants of the subject tank were labeled by upper/lower sections of the tank and novel/established stimulus shoal proximity. Error bars are +/- SEM. While exact p-values are reported for post-hoc comparisons, p-values smaller than 0.001 are indicated as p < 0.001.</p

Experimental apparatus.

Illustrative example with the subject in the center tank to test preference between an established shoal on the right and a newly-formed shoal on the left. The apparatus consists of three identical 18.9L (5.5 gal.) glass aquaria (each 20.3 cm wide × 25.4 cm high × 40.6 cm long; total width = 121.8 cm) filled with water to a depth of 21 cm.</p

Novel-ExpShoalSSP_PlosONE.

(XLSX)</p

Percent cumulative duration in each quadrant zone across both sexes.

Quadrants are labeled as upper/lower sections of the tank and novel/established stimulus shoal proximity. Error bars are +/- SEM. While exact p-values are reported for post-hoc comparisons, p-values smaller than 0.001 are indicated as p < 0.001.</p

Fig 5 -

Average velocity (A) and velocity SD (B) during movement. Quadrants of the subject tank were labeled by upper/lower sections of the tank and novel/established stimulus shoal proximity. Error bars are +/- SEM. While exact p-values are reported for post-hoc comparisons, p-values smaller than 0.001 are indicated as p < 0.001.</p

Sex differences in inter-quadrant and cross-tank transitions.

Quadrants of the subject tank were labeled by upper/lower sections of the tank and novel/established stimulus shoal proximity. Error bars are +/- SEM. While exact p-values are reported for post-hoc comparisons, p-values smaller than 0.001 are indicated as p < 0.001.</p

Sex differences in behavioral entropy.

H3 measures entropy across three vertical zones while H4 measures entropy across four quadrants. Error bars are +/- SEM. While exact p-values are reported for post-hoc comparisons, p-values smaller than 0.001 are indicated as p < 0.001.</p

Heatmaps collapsed across trials with the same configuration of stimuli.

Warm colors indicate highest intensities of localization while cooler colors indicate lowest intensities of localization.</p