RNA transcripts are bound and regulated by RNA-binding proteins (RBPs). Current methods for identifying in vivo targets of a RBP are imperfect and not amenable to examining small numbers of cells. To address these issues, we developed TRIBE (Targets of RNA-binding proteins Identified By Editing), a technique that couples an RBP to the catalytic domain of the Drosophila RNA editing enzyme ADAR and expresses the fusion protein in vivo. RBP targets are marked with novel RNA editing events and identified by sequencing RNA. We have used TRIBE to identify the targets of three RBPs (Hrp48, dFMR1 and NonA). TRIBE compares favorably to other methods, including CLIP, and we have identified RBP targets from as little as 150 specific fly neurons. TRIBE can be performed without an antibody and in small numbers of specific cells.

Programmed cell death 1 ligand 1 (PD-L1, B7H1) is a cell-surface protein that suppresses the cytotoxic CD8+ T cell-mediated immune response. PD-L1 expression and its clinical relevance in sarcomas are not well understood. Therefore, we sought to measure RNA expression levels for PD-L1 in 38 clinically annotated osteosarcoma tumor samples, and aimed to determine if PD-L1 expression correlates with clinical features and tumor-infiltrating T-lymphocytes (TILs). Quantitative real-time RT-PCR for PD-L1 was optimized in 18 cell lines, of which 5 were osteosarcoma-derived. qRT-PCR results were validated via flow cytometry and immunohistochemistry (IHC) in select cell lines. Total RNA was isolated from 38 human osteosarcoma samples for qRT-PCR analysis. Clinical data were sorted and significance was determined by Student t-test. TILs were examined in patient samples by tissue microarray (TMA) hematoxylin-eosin (HE) staining. We confirmed the constitutive PD-L1 mRNA expression in cell lines by qRT-PCR, flow cytometry, and IHC. Across human osteosarcoma samples, PD-L1 mRNA gene expression ranged over four-log (>5000-fold difference). Relative expression levels were evaluated against clinical factors such as age/gender, metastasis, recurrence, chemotherapy, percent necrosis, and survival; no significant associations were identified. The presence of TILs was associated with high PD-L1 expression (R2=0.37, P=0.01). In summary, we developed an RNA-based assay to determine PD-L1 expression levels, and we show for the first time that high levels of PD-L1 are expressed in a subset of osteosarcoma, and PD-L1 expression is positively correlated with TILs. There are multiple agents targeting PD-1/PD-L1 in clinical development, and this may be a novel immunotherapeutic strategy for osteosarcoma clinical trials.

The clinical significance of Cluster of Differentiation 44 (CD44) remains controversial in human ovarian cancer. The aim of this study is to evaluate the clinical significance of CD44 expression by using a unique tissue microarray, and then to determine the biological functions of CD44 in ovarian cancer. In this study, a unique ovarian cancer tissue microarray (TMA) was constructed with paired primary, metastatic, and recurrent tumor tissues from 26 individual patients. CD44 expression in TMA was assessed by immunohistochemistry. Both the metastatic and recurrent ovarian cancer tissues expressed higher level of CD44 than the patient-matched primary tumor. A significant association has been shown between CD44 expression and both the disease free survival and overall survival. A strong increase of CD44 was found in the tumor recurrence of mouse model. Finally, when CD44 was knocked down, proliferation, migration/invasion activity, and spheroid formation were significantly suppressed, while drug sensitivity was enhanced. Thus, up-regulation of CD44 represents a crucial event in the development of metastasis, recurrence, and drug resistance to current treatments in ovarian cancer. Developing strategies to target CD44 may prevent metastasis, recurrence, and drug resistance in ovarian cancer.