Liquid Biopsy Transcriptomics of Extracellular Vesicles (EV) in Plasma of Pancreatic Ductal Adenocarcinoma (PDAC)

https://openworks.mdanderson.org/sumexp22/1089/thumbnail.jp

Determination and comparison rate of expression markers of osteoblast derived of Adipose derived stem cells markers in monolayer and pellet culture models

Abstract: Nowadays high accident rates, fractures leading to permanent bone disorders and the impossibility of bone transplant have made scientists to look for new methods of repairing injured bones. Considering the application of stem cells in bone tissue engineering, there exists the necessity to investigate various culture methods and suitable fields and scaffolds. Thus, we decided to induce adipose-derived stem cells into osteoblast cells in two systems of pellet culture and monolayer and compare osteogenic markers. Methods: Stem cells have been separated via mechanical and enzymatic methods and cultured in monolayer and pellet culture models with osteogenic medium. Then, RNA was separated from differentiated cells, complementary DNA (cDNA) was synthesized and amplified. Polymerase chain reaction (PCR) product was transferred to electrophoresis gel. The intensity of the bands was measured by Image-J software and analyzed by SPSS. Results: average osteopontin, osteocalcin and Runx2 genes in differentiated cells in the two culture systems showed a significant difference. The expression of osteocalcin, osteopontin and Runx2 gense in pellet system were more than monolayer systems in 21 days. Conclusion: This study indicated that pellet and monolayer culture systems are appropriate for bone engineering but osteocalcin, osteopontin and Runx2 genes expressions were different in the two culture system

Analyzing Extracellular Vesicles for Disease Monitoring in Metastatic Colorectal Cancer Patients

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. Metastases are the main cause of cancer-related death, and the most frequent metastatic sites in patients with CRCs are liver and lung. To confirm the diagnosis of metastatic CRC (mCRCs) and to classify mCRCs, tumor biopsy of a suspected metastasis is often required. The consensus molecular subtype (CMS) classification which is based on gene expression profiles of CRC tumor specimens, is a predictive factor for treatment outcomes of standard chemotherapies for mCRCs. The practicality of repeated tumor biopsies for disease monitoring in mCRC patients is often limited. Liquid biopsy utilizing extracellular vesicles (EVs) is an alternative approach for obtaining insights into molecular landscapes of mCRC. Cancer-derived EVs play a critical role in cancer cell-to-cell communication and carry surface proteins and cell-specific cargos of nucleic acids. For this reason, we aimed to characterize ribonucleic acid (RNA) cargoes and surface proteins on EVs disseminated from CRC cancer cells to longitudinally monitor mCRC patients and identify immune modulatory signaling molecules that potentially play a role in the progression and metastasis of CRC. Using novel bioinformatics deconvolution pipelines applied to RNA sequencing data of circulating EVs, we demonstrate that transcriptomic profiling of EVs can be used for the detection, classification, and longitudinal monitoring of mCRC. In the context of both conventional and targeted therapies, assessing the transcriptomic cargo of circulating EVs can provide pivotal insights into molecular pathways that are altered under treatment selection pressure, and which likely contribute to the emergence of resistance. Additionally, we were able to characterize cancer-related protein composition of EVs originating from colorectal cancer cells. We demonstrate the immunomodulatory protein CD276/B7H3 as a prominent surface protein on CRC-associated EVs. This study highlights the potential of EVs in both detection and therapeutic monitoring mCRC, offering valuable insights into cancer-derived protein and RNA cargo, and emphasizing the role of longitudinal sampling via liquid biopsy in the clinical care of patients with advanced cancer

Transcriptomic comparison of osteopontin, osteocalcin and core binding factor 1 genes between human adipose derived differentiated osteoblasts and native osteoblasts

Background: There are significant limitations in repair of irrecoverable bone defects. Stem-cell therapy is a promising approach for the construction of bone tissue. Mesenchymal stem cells (MSCs) have been introduced as basic tools for bone tissue generation. Through MSCs, adipose-derived stem cells (ADSCs) are more interesting. Since the similarity of native osteoblasts and differentiated osteoblasts from ADSCs in terms of gene expression pattern is unknown, this study was designed to compare gene expression patterns of some genes involved in osteogenesis between human native osteoblasts and adipose-derived differentiated osteoblasts. Materials and Methods: Realtime qRT-PCR was used for studying the gene expression of osteocalcin, osteopontin, and core binding factor alpha 1 (Cbfa1) in human native osteoblasts and adipose derived osteogenic osteoblasts at days 7, 14, 21, and 28 of differentiation. Results: This study demonstrated that native osteoblasts and differentiated osteoblasts, cultured in common osteogenic medium, have significant differences in gene expression levels for osteocalcin and osteopontin. Compared to native osteoblasts, these genes are expressed lower in all four groups of differentiated osteoblastic cells. We also found, there is a progressive increase in cbfa1 expression over the differentiation period of ADSCs from day 7 to day 28. Conclusions: Our findings help for better assessment of adipose-derived differentiated cells as a source for cell-based therapy

The effect of Kisspeptin-10 on mesenchymal stem cells migration in vitro and in vivo

Background: Kisspeptins (kp) activate a receptor coupled to a Gαq subunit (GPR54 or KiSS-1R) receptor to perform a variety of functions, including inhibition of cell motility, chemotaxis, and metastasis. In this study we have investigated whether kp-10, the most potent member of the kisspeptin family, can modulate CXCR4 (C-X-C chemokine receptor type 4) expression and mesenchymal stem cells (MSCs) migration that may influence the development of tumors. Materials and Methods: We compared the directional migration of MSCs treated with 10-100 or 500 nM kp-10 for 24 hours and no treated cells using an in vitro transmembrane migration assay. In addition, Chloromethylbenzamido Dialkylacarbocyanine (CM-Dil) labeled adipose-derived mesenchymal stem cells treated with 10-100 or 500 nM kp-10 and no treated cells were transfused via the tail vein to the melanoma tumor bearing C57BL/6 mice. After 24 hours, the mice were scarified, the tumors were dissected, and the tumor cell suspensions were analyzed by flow cytometry for detection of CM-Dil + MSCs. Results: We have found that kp-10 increased the MSCs migration at 100 nM, while it decreased the MSCs migration at 500 nM, both in vitro and in vivo, with a significant increase of CXCR4 expression at 100 nM kp-10 compared to the no treated cells, but it had no significant difference between the various concentrations of kp-10. Conclusion: Thus, our data showed that kp-10 can differently affect MSCs migration in various concentrations, probably through different effects on CXCR4 expression in various concentrations

Phenotypic expansion illuminates multilocus pathogenic variation

Purpose: Multilocus variation-pathogenic variants in two or more disease genes-can potentially explain the underlying genetic basis for apparent phenotypic expansion in cases for which the observed clinical features extend beyond those reported in association with a "known" disease gene. Methods: Analyses focused on 106 patients, 19 for whom apparent phenotypic expansion was previously attributed to variation at known disease genes. We performed a retrospective computational reanalysis of whole-exome sequencing data using stringent Variant Call File filtering criteria to determine whether molecular diagnoses involving additional disease loci might explain the observed expanded phenotypes. Results: Multilocus variation was identified in 31.6% (6/19) of families with phenotypic expansion and 2.3% (2/87) without phenotypic expansion. Intrafamilial clinical variability within two families was explained by multilocus variation identified in the more severely affected sibling. Conclusion: Our findings underscore the role of multiple rare variants at different loci in the etiology of genetically and clinically heterogeneous cohorts. Intrafamilial phenotypic and genotypic variability allowed a dissection of genotype-phenotype relationships in two families. Our data emphasize the critical role of the clinician in diagnostic genomic analyses and demonstrate that apparent phenotypic expansion may represent blended phenotypes resulting from pathogenic variation at more than one locus

WNT Signaling Perturbations Underlie the Genetic Heterogeneity of Robinow Syndrome.

Locus heterogeneity characterizes a variety of skeletal dysplasias often due to interacting or overlapping signaling pathways. Robinow syndrome is a skeletal disorder historically refractory to molecular diagnosis, potentially stemming from substantial genetic heterogeneity. All current known pathogenic variants reside in genes within the noncanonical Wnt signaling pathway including ROR2, WNT5A, and more recently, DVL1 and DVL3. However, ∼70% of autosomal-dominant Robinow syndrome cases remain molecularly unsolved. To investigate this missing heritability, we recruited 21 families with at least one family member clinically diagnosed with Robinow or Robinow-like phenotypes and performed genetic and genomic studies. In total, four families with variants in FZD2 were identified as well as three individuals from two families with biallelic variants in NXN that co-segregate with the phenotype. Importantly, both FZD2 and NXN are relevant protein partners in the WNT5A interactome, supporting their role in skeletal development. In addition to confirming that clustered -1 frameshifting variants in DVL1 and DVL3 are the main contributors to dominant Robinow syndrome, we also found likely pathogenic variants in candidate genes GPC4 and RAC3, both linked to the Wnt signaling pathway. These data support an initial hypothesis that Robinow syndrome results from perturbation of the Wnt/PCP pathway, suggest specific relevant domains of the proteins involved, and reveal key contributors in this signaling cascade during human embryonic development. Contrary to the view that non-allelic genetic heterogeneity hampers gene discovery, this study demonstrates the utility of rare disease genomic studies to parse gene function in human developmental pathways