GPCRs and Fibroblast Heterogeneity in Fibroblast-Associated Diseases

G protein-coupled receptors (GPCRs) are the largest and most diverse class of signaling receptors. GPCRs regulate many functions in the human body and have earned the title of most targeted receptors . About one-third of the commercially available drugs for various diseases target the GPCRs. Fibroblasts lay the architectural skeleton of the body, and play a key role in supporting the growth, maintenance, and repair of almost all tissues by responding to the cellular cues via diverse and intricate GPCR signaling pathways. This review discusses the dynamic architecture of the GPCRs and their intertwined signaling in pathological conditions such as idiopathic pulmonary fibrosis, cardiac fibrosis, pancreatic fibrosis, hepatic fibrosis, and cancer as opposed to the GPCR signaling of fibroblasts in physiological conditions. Understanding the dynamics of GPCR signaling in fibroblasts with disease progression can help in the recognition of the complex interplay of different GPCR subtypes in fibroblast-mediated diseases. This review highlights the importance of designing and adaptation of next-generation strategies such as GPCR-omics, focused target identification, polypharmacology, and effective personalized medicine approaches to achieve better therapeutic outcomes for fibrosis and fibrosis associated malignancies

Investigating Immune Profiles in Differentiated Thyroid Cancer by Multiplex Immunofluorescence

BACKGROUND: As the most common endocrine malignancy in the United States (U.S.), differentiated thyroid cancer (DTC) accounts for 3.8% of all cancers in the U.S., with roughly 10% of cases progressing to distant metastatic DTC, which is associated with a poor five year survival outcome despite conventional management, including surgery and radioactive iodine ablation. Recently, novel immunotherapies have garnered attention as a viable therapeutic resource for patients with advanced DTC. However, the response to therapy has been variable and unpredictable, which may be associated with an immune suppressive circulating phenotype. Nonetheless, the intra-tumoral immune infiltrate remains to be elucidated, demonstrating a critical need to address the gap in understanding in order to better prognosticate the disease. OBJECTIVE: To identify and compare tumor-infiltrating immune markers with those present in the adjacent normal thyroid tissue, and collate these immune infiltrates with tumor characteristics. METHODS: Twenty-nine adult tissue samples containing tumor and stromal regions were collected from patients with DTC. The samples were analyzed using multiplex immunofluorescence (MxIF) with antibodies against cell-surface molecules CD56, PD-1, PD-L1, FOXP3, CD3, CD8, CD4, CD45, CD68, CD163, INOS, HLA-DR, CD33, and CD19. 17 of the specimens were analyzed using HALO and a positive threshold was assigned based on review by a trained researcher. RESULTS: In evaluating the immune profiles, important differences in the immune infiltrates between different stages of the cancer were observed. Generally, PD-1 and PD-L1 were highly expressed within the tumor, despite variability in lymphocyte infiltration, indicating the importance of PD-1 and PD-L1 as potential predictive biomarkers for the aggressiveness of thyroid cancer. Tumor from patients with distant metastases demonstrated higher T cell infiltration, T regulatory cells, macrophages and PD-L1 positive cells as compared to localized tumor. CONCLUSION: Immune profiling demonstrated significant differences between tumor and adjacent healthy regions, particularly in terms of PD-1 and PD-L1 expression and lymphocyte infiltration, indicating that higher intratumor infiltration of T regulatory cells, macrophages and PD-1/PD-L1 positive cells may be associated with advanced thyroid cancer. Therefore, the data demonstrates the efficacy of MxIF in gathering valuable information regarding the tumor microenvironment, which will have major implications in guiding the selection of patients for immunotherapy.https://digitalcommons.unmc.edu/surp2021/1042/thumbnail.jp

MicroRNA-1 Attenuates the Growth and Metastasis of Small Cell Lung Cancer through CXCR4/FOXM1/RRM2 Axis

BACKGROUND: Small cell lung cancer (SCLC) is an aggressive lung cancer subtype that is associated with high recurrence and poor prognosis. Due to lack of potential drug targets, SCLC patients have few therapeutic options. MicroRNAs (miRNAs) provide an interesting repertoire of therapeutic molecules; however, the identification of miRNAs regulating SCLC growth and metastasis and their precise regulatory mechanisms are not well understood. METHODS: To identify novel miRNAs regulating SCLC, we performed miRNA-sequencing from donor/patient serum samples and analyzed the bulk RNA-sequencing data from the tumors of SCLC patients. Further, we developed a nanotechnology-based, highly sensitive method to detect microRNA-1 (miR-1, identified miRNA) in patient serum samples and SCLC cell lines. To assess the therapeutic potential of miR-1, we developed various in vitro models, including miR-1 sponge (miR-1Zip) and DOX-On-miR-1 (Tet-ON) inducible stable overexpression systems. Mouse models derived from intracardiac injection of SCLC cells (miR-1Zip and DOX-On-miR-1) were established to delineate the role of miR-1 in SCLC metastasis. In situ hybridization and immunohistochemistry were used to analyze the expression of miR-1 and target proteins (mouse and human tumor specimens), respectively. Dual-luciferase assay was used to validate the target of miR-1, and chromatin immunoprecipitation assay was used to investigate the protein-gene interactions. RESULTS: A consistent downregulation of miR-1 was observed in tumor tissues and serum samples of SCLC patients compared to their matched normal controls, and these results were recapitulated in SCLC cell lines. Gain of function studies of miR-1 in SCLC cell lines showed decreased cell growth and oncogenic signaling, whereas loss of function studies of miR-1 rescued this effect. Intracardiac injection of gain of function of miR-1 SCLC cell lines in the mouse models showed a decrease in distant organ metastasis, whereas loss of function of miR-1 potentiated growth and metastasis. Mechanistic studies revealed that CXCR4 is a direct target of miR-1 in SCLC. Using unbiased transcriptomic analysis, we identified CXCR4/FOXM1/RRM2 as a unique axis that regulates SCLC growth and metastasis. Our results further showed that FOXM1 directly binds to the RRM2 promoter and regulates its activity in SCLC. CONCLUSIONS: Our findings revealed that miR-1 is a critical regulator for decreasing SCLC growth and metastasis. It targets the CXCR4/FOXM1/RRM2 axis and has a high potential for the development of novel SCLC therapies. MicroRNA-1 (miR-1) downregulation in the tumor tissues and serum samples of SCLC patients is an important hallmark of tumor growth and metastasis. The introduction of miR-1 in SCLC cell lines decreases cell growth and metastasis. Mechanistically, miR-1 directly targets CXCR4, which further prevents FOXM1 binding to the RRM2 promoter and decreases SCLC growth and metastasis