Supplemental Figure 1. Ror1 expression and phenotypic outcomes upon Ror2 loss. (a) qRT-PCR and (b) Western blotting of Ror1 and Ror2 expression in shLUC vs. shRor2 tumor cells showing the specificity of Ror2 shRNA to Ror2 and unchanged levels of Ror1 in tumor cells upon Ror2 loss. (c) H&E staining of tumors derived from the shRor2-2 hairpin demonstrating reproducible phenotypes to the shRor2-1 hairpin. (d) Immunohistochemistry of 7xTCF-eGFP within T1 showing enhanced Wnt activity within areas of squamous differentiation within shRor2 tumors relative to shLUC control tumors. (e) Loricrin immunofluorescence (red), illustrating the presence of this epidermal differentiation marker in shRor2 T1 tumors.
Supplemental Figure 2. Ror2 overexpression inhibits Wnt/-catenin dependent canonical activity in TP53-null 2225L tumors in vivo. 2225L tumor cells were co-transduced with Wnt reporter lentivirus and either iG2-eGFP control or iG2-Ror2 lentivirus. (a) Western blot for Ror2 protein demonstrating the ovexpression of Ror2 protein above endogenous levels in iG2-Ror2 tumors compared to iG2-eGFP empty vector and untransduced control tumors. (b) Bioluminescence imaging of untransduced, iG2-eGFP control and iG2-Ror2 tumors showing decreased 7x-TCF-Luciferase activity upon Ror2 overexpression compared to iG2-eGFP controls. (c) Quantitation of total flux (photons/second) from (b) demonstrating less Wnt/-catenin activity upon Ror2 overexpression.
Supplemental Figure 3. qRT-PCR validation of genes up- and down-regulated in shRor2 T1, T2, and 2225L tumors relative to shLUC control tumors from microarray. (a) qRT-PCR plots of genes downregulated in T1, T2, and 2225L upon Ror2 depletion relative to shLUC controls. Genes include Ptpn7, Art3, and Tnfrsf18. (b) qRT-PCR plots of genes commonly upregulated in T1, T2, and 2225L tumors upon Ror2 depletion relative to shLUC controls. Genes include S100b, Dnajc6, and Kcnk1. n=3 replicates for each sorted shLUC and shRor2 group (based on tdTomato transduction) from T1, T2, and 2225L tumors. Plotted values represent means ± SD (error bars).
Supplemental Figure 4. Characterization of T1 and T2 TP53-null in vitro organoids and downstream signaling. (a) Brightfield DIC images of shLUC vs. shRor2 organoids across T1 and T2 TP53-null models. Arrows denote areas of squamous differentiation by brightfield compared to control shLUC organoids. Scale 50µm. (b) H&E staining of shLUC vs. shRor2 organoids revealing squamous features (arrows) within the centers of shRor2 T1 and T2 organoids, with no evidence of these features within shLUC T1 and T2 organoids. Scale 50µm. (c,d) Western blotting for Dvl2 and Rho pathway components in (c) T1 organoids and (d) T2 organoids. Dvl2 phosphorylation is impaired only in T2 organoids upon Ror2 loss.
Supplemental Figure 5. Ror2 silencing in vivo changes the patterning of pERM staining among tumor cell populations within 2225L. (a) pERM (green) and K8 (red) immunofluorescence in shLUC and shRor2 2225L tumors. Dotted lines signify tumor-stromal interface. (b) Single channel images for pERM staining in shLUC vs. shRor2 tumors depicting the change in pERM landscape upon Ror2 depletion in 2225L tumors.
Supplemental Table 1. Primer sequences for qRT-PCR.