Background

Previous studies have demonstrated MYCN and MYC preferentially regulate the same set of core genes involved in metabolism and cell growth, and while the MYCN allele can functionally replace MYC in murine development (Malynn et al., 2000), MYCN and MYC have separate temporal regulation over organogenesis in early vertebrate development (Hurlin, 2013). MYCN expression is essential for initial establishment of stem and progenitor populations; over the course of organ system development, MYCN expression switches to low MYC expression to support stem and progenitor cell maintenance, and during cell lineage commitment and expansion, elevated MYC levels drive highly proliferative cells until they reach terminal differentiation (Hurlin, 2013).

A similar relationship has been observed in the development of TNBC metastases where MYCN expression is elevated in newly seeded metastatic lesions that expand and differentiate into highly proliferative MYC-expressing tumors (Lawson et al., 2015). Given the near ubiquitous nature of MYC deregulation across tumor tissue types, MYC is the most studied MYC family member. However, due to a correlation between deregulated MYCN expression and a poor prognosis (Beltran, 2014), MYCN has been increasingly studied in both neuronal and non-neuronal cancers, including acute myeloid leukemia (Kawagoe et al., 2007), small-cell lung cancer (Funa et al., 1987), ovarian cancer (Baratta et al., 2015), and now TNBC (Schafer et al., 2020). Therefore, we began to explore the relationship between MYCN and MYC by developing an IF stain that could simultaneously detect both isoforms within the same tumor tissue section. Established protocols that involve the IF detection of MYCN or MYC have been separate stains that primarily recognizes each isoform at highly amplified/overexpressed levels. Given that TNBC and other non-neuronal tissue types exhibit aberrant low-level MYCN expression, we developed a method to detect lower expression of MYCN and MYC using TSA. Further, we have two protocol versions of the method: application of the dual MYC family isoform (MYCN and MYC) TSA-IF to FFPE tumor sections, and a modified version of the stain for evaluation MYC family isoforms expression in cell line cultures fixed in situ after growth as adherent cultures on chambered microscope slides. We anticipate both MYCN and MYC are coexpressed in a variety of cancer tissue types and the advent of these IF methodologies will allow investigators to further characterize MYCN- versus MYC-expressing cells in tumor development and disease etiology.