Dynamic and nuclear expression of PDGFRα and IGF-1R in alveolar Rhabdomyosarcoma

Abstract

Since the advent of tyrosine kinase inhibitors as targeted therapies in cancer, several receptor tyrosine kinases (RTK) have been identified as operationally important for disease progression. Rhabdomyosarcoma (RMS) is a malignancy in need of new treatment options; therefore, better understanding of the heterogeneity of RTKs would advance this goal. Here, alveolar RMS (aRMS) tumor cells derived from a transgenic mouse model expressing two such RTKs, platelet-derived growth factor (PDGFR)α and insulin-like growth factor (IGF)-1R, were investigated by fluorescence-activated cell sorting (FACS). Sorted subpopulations that were positive or negative for PDGFRα and IGF-1R dynamically altered their cell surface RTK expression profiles as early as the first cell division. Interestingly, a difference in total PDGFRα expression and nuclear IGF-1R expression was conserved in populations. Nuclear IGF-1R expression was greater than cytoplasmic IGF-1R in cells with initially high cell surface IGF-1R, and cells with high nuclear IGF-1R established tumors more efficiently in vivo. RNA interference-mediated silencing of IGF-1R in the subpopulation of cells initially harboring higher cell surface and total IGF-1R resulted in significantly reduced anchorage-independent colony formation as compared with cells with initially lower cell surface and total IGF-1R expression. Finally, in accordance with the findings observed in murine aRMS, human aRMS also had robust expression of nuclear IGF-1R.

Implications: RTK expression status and subcellular localization dynamics are important considerations for personalized medicine.

Figure 1. Murine aRMS isolated by Pdgfrα and Igf1r expression

(A) U23674 (murine aRMS) cell expression profile by FACS of Pdgfrα (APC) and Igf1r (Cy3.5) gated with respect to isotype and secondary only control. (B) Purity of isolated cells by FACS shown. Isolated populations according to cells positive and negative for each receptor designated as hi and lo, respectively. (C) Phase contrast microscopy of sorted populations demonstrating differential morphology of cells grown in culture 7 days post-sort. Scale bar: 200 μm.

Figure 2. Murine aRMS demonstrate dynamic expression of Pdgfrα and Igf1r by FACS in a temporal manner

(A) FACS profile of Pdgfrα and Igf1r expression in U23674 cells at day of sort (Day 0), and 2 and 14 days post sort. Day 7 was similar to Day 14.

Figure 3. Functional Pdgfrα and Igf1r expression post-sort

(A) Immunoblot of sorted populations grown in culture for 1 week post-sort, demonstrating phosphorylated as well as total Pdgfrα, Igf1r and Erk 1/2. (B) Representative data showing mean fluorescence intensity (MFI) of U23674 sorted cells 7 days post sort indicating surface expression of Pdgfrα and Igf1r.

Figure 4. Cells sorted for Igf1r establish more aggressive tumors in vivo

(A) Tumors established from murine aRMS cells sorted for Pdgfrα and Igf1r show a relative dynamic and static expression of each receptor, respectively, reflective of cells in vitro. (B) Pdgfrα^hiIgf1r^lo sorted cells demonstrate a significantly slower growth rate in an in vivo orthotopic allograft mouse model of aRMS compared to Pdgfrα^loIgf1r^hi sorted cells. (C) Pdgfrα^hiIgf1r^hi sorted cells grow at a significantly faster growth rate in an in vivo orthotopic allograft mouse model of aRMS compared to Pdgfrα^loIgf1r^lo sorted cells. (D) Tumor establishment or growth rate of unsorted U23674 cells in vivo do not differ from Pdgfrα^loIgf1r^lo sorted cells.

Figure 5. Nuclear localization of Pdgfrα and Igf1r correlates with cells sorted by FACS for surface expression of the respective receptor

(A) Representative confocal images of U23674 sorted populations showing localization of Pdgfrα and Igf1r (40x) with inset box enlarging image of a single cell to appreciate localization. Scale bar: 100 μm. Larger magnifications of insets are given in Supplemental Figure S3.

Figure 6. Igf1r is present in soluble nuclear fraction of sorted cells and is associated with anchorage-independent colony formation ability

(A) Cytosolic and nuclear fractions of sorted U23674 cells show cells sorted by Igf1r^hi express higher levels of nuclear Igf1r. (B) Cells with higher nuclear Igf1r, show a significantly further decrease in colony formation ability compared to control (non-specific siRNA) when Igf1r is silenced by siRNA. * indicated p <0.05 compared to non-specific siRNA by student’s t-test.

Figure 7. IGF1R localization in human aRMS

(A) Human aRMS sections with chiefly cytosolic IGF1R expression. (B) Images of aRMS sections demonstrating both cytosolic (left) and nuclear (right) IGF1R expression. Scale bar: 100 μM.