Hpa021616

IHC was applied to assess HAX1, KIF14, Mieap, and EZR expression in breast tumors (n = 84). Seven-micrometer-thick sections of FFPE tumor samples were deparaffinized, rehydrated, and stained as previously described [57 (link)]. The following antibodies were used: Rabbit anti-HAX1 (NBP1-54800, 1:50, Novusbio, Centennial, CO, USA), rabbit anti-KIF14 (HPA038061, 1:500, Sigma, St. Louis, MO, USA), rabbit anti-Mieap/SPATA18 (HPA036854, 1:100, Sigma, St. Louis, MO, USA), and rabbit anti-EZR (HPA021616, 1:1000, Sigma, St. Louis, MO, USA). The stained sections were assessed for expression of HAX1, KIF14, Mieap, and EZR in different morphological structures of breast tumors. In particular, the assessment included the presence of structures (tubular, alveolar, solid, and trabecular) with positive and negative cells at their periphery (up to 2 layers), the presence of tumor cells with positive and negative expression at the tips (up to 3 rows) of the torpedo-like sprouts connected with solid structures and the torpedo-like structures (not connected with solid structures), positive and negative expression in single tumor cells, etc. The complete study protocol is given in Table S19. In total, 10 to 24 fields of view were analyzed per sample. Expression was counted as positive if it was observed in at least 10 structures of the same type.

Multiplex IHC was used to analyze the co-localization of KIF14, Mieap, and EZR proteins in torpedo-like structures. Multiplex IHC was performed with a Bond RXm system (Leica, Hamburg, Germany) with antibodies against KIF14 (HPA038061, 1:500, Sigma, St. Louis, MO, USA; detected by Opal 520), Mieap/SPATA18 (HPA036854, 1:100, Sigma, St. Louis, MO, USA; Opal 620), EZR (HPA021616, 1:1000, Sigma, St. Louis, MO, USA; Opal 690), and MMP13 (MA5-14238, 1:25, Thermo Fisher Scientific, Waltham, MA, USA). Protein blocking was performed using 3% BSA-PBS (Sigma, St. Louis, MO, USA). TSA visualization was performed with the Opal 520, Opal 620, and Opal 690 (Opal seven-color IHC kit, Perkin Elmer, Waltham, MA, USA). Staining was finished with a DAPI counterstain and slides were enclosed in fluorescence mounting medium (Agilent, Santa-Clara, CA, USA). Slides were scanned using the Vectra 3.0 (PerkinElmer, Waltham, MA, USA). Tissue imaging was performed using inForm Advanced Image Analysis software (inForm 2.1.1 and 2.2.1; Perkin Elmer, Waltham, MA, USA).