Stromal Extracellular Vesicles Research Articles

Extracellular vesicles secreted by human uterine stromal cells regulate decidualization, angiogenesis, and trophoblast differentiation

In humans, the uterus undergoes a dramatic transformation to form an endometrial stroma-derived secretory tissue, termed decidua, during early pregnancy. The decidua secretes various factors that act in an autocrine/paracrine manner to promote stromal differentiation, facilitate maternal angiogenesis, and influence trophoblast differentiation and development, which are critical for the formation of a functional placenta. Here, we investigated the mechanisms by which decidual cells communicate with each other and with other cell types within the uterine milieu. We discovered that primary human endometrial stromal cells (HESCs) secrete extracellular vesicles (EVs) during decidualization and that this process is controlled by a conserved HIF2α-RAB27B pathway. Mass spectrometry revealed that the decidual EVs harbor a variety of protein cargo, including cell signaling molecules, growth modulators, metabolic regulators, and factors controlling endothelial cell expansion and remodeling. We tested the hypothesis that EVs secreted by the decidual cells mediate functional communications between various cell types within the uterus. We demonstrated that the internalization of EVs, specifically those carrying the glucose transporter 1 (GLUT1), promotes glucose uptake in recipient HESCs, supporting and advancing the decidualization program. Additionally, delivery of HESC-derived EVs into human endothelial cells stimulated their proliferation and led to enhanced vascular network formation. Strikingly, stromal EVs also promoted the differentiation of trophoblast stem cells into the extravillous trophoblast lineage. Collectively, these findings provide a deeper understanding of the pleiotropic roles played by EVs secreted by the decidual cells to ensure coordination of endometrial differentiation and angiogenesis with trophoblast function during the progressive phases of decidualization and placentation.

Abstract 1487: Role of micro-RNA199a-3p in lymph node stromal extracellular vesicles on tumorigenesis in colorectal cancer

Abstract Introduction: MicroRNAs (miRNAs) are short non-coding RNAs implicated in post-transcriptional regulation of gene expression. Abnormal expression of miRNAs in cancer is associated with enhanced tumorigenesis and metastatic potential. Our previous data has shown that extracellular vesicles (EV) secreted by human lymph node stromal cells (LNSC) enhance tumorigenesis in colorectal cancer (CRC). By systemic searching and modifying miRNAs carried by EV through overexpression and inhibition on LNSC, we aim to demonstrate their effect on CRC progression in vitro and orthotopic mouse model. Methods: Total microRNA sequencing was analyzed using in-silico computer prediction models. Four overexpressed miRNAs in the LNSC-EV were selected (miR-155-5p, miR-199a-3p, mi-143-3p, and mi-214-3p). Tumor cell proliferation assay was performed using a CRC SW620 cell line transfected with selected miRNA mimics or inhibitors or controls. In our mouse model, miRNA transfected SW620 cells tagged with luciferase/RFP, were injected into the rectal submucosa of NOD/SCID mice. Tumor growth and metastases were observed by weekly bioluminescent imaging (BLI). At week 7, tumors were weighed and BLI of the tumors and organs were recorded. Immunohistochemistry staining was performed on the paraffin sections of tumors, livers and lung collected from these mice. Results: In vitro proliferation assays, cells transfected with the mimic of miRNA 199a-3p significantly increased CRC SW620 cell proliferation (p<0.01) while cells treated with the inhibitor showed decreased proliferation (p<0.05). In our orthotopic mouse model, mice with SW620 cells transfected with the mimic had increased frequencies of tumorigenesis (94% vs. 14%) and liver and lung metastasis comparing to control group (56% vs 9.5%, 50% vs 0%). Mice injected with SW620 cells transfected with the miRNA inhibitor had decreased tumor growth compared to the negative control (p<0.05). Conclusion: Our study shows that miRNAs carried in LNSC-EV are crucial for CRC progression. Specifically, inhibition of miRNA-199a-3p may serve as a therapeutic target in the treatment of CRC. Understanding the role and mechanisms of this miRNA in CRC could lead to the improved management of patients with CRC. Citation Format: Hallie Baer, Jessica Simon, Lara McKean-Baste, Alicia N. Ray, Heather Green, Grace Maresh, Xin Zhang, David Marjolin, Jennifer Paruch, Li Li. Role of micro-RNA199a-3p in lymph node stromal extracellular vesicles on tumorigenesis in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1487.

Bone Marrow Stromal Cell Mediated Drug Resistance in AML Cells: Role of Extracellular Vesicles and AKT Signaling

Accumulating evidence suggests that bone marrow stromal micro-environment act as sanctuary site for acute myeloid leukemia (AML) cells and affords protection from conventional chemotherapy agents. Recently, there has been considerable interest in extracellular vesicles (EVs) and their role in intercellular communication. We had previously reported that there is significant stromal cell mediated drug resistance in acute promyelocytic leukemia (APL) which was predominantly driven by up-regulation of the NF-kB pathway in the malignant cells (Ganesan S et al, Leukemia 2016). Though there are reports of similar stromal cell mediated protection against chemotherapy in non-M3 AML cells, there is limited data on the role EVs play, if any, in inducing such chemotherapy resistance. We undertook a preliminary study to evaluate the role of EVs in inducing resistance to chemotherapeutic agents in AML.We demonstrated that direct stromal co-culture experiments with AML cell lines as well as primary AML cells showed a significant stromal mediated protective effect against both cytosine arabinoside (AraC) and daunorubicin (DNR) [U937: AraC p < 0.01, DNR p < 0.001 (n=5); primary AML cells: AraC p < 0.01, DNR p < 0.001 (n=26)]. Gene expression profiling (GEP) analysis of AML cell line U937 co-cultured with HS-5 revealed a significant enrichment of genes involved in PI3K-AKT signaling pathway (p < 0.0001) followed by NF-kB pathway genes. It has been previously reported that on direct co-culture with stromal cells there is activation of the PI3K-AKT pathway in AML cells leading to drug resistance (Tabe Y et al. Cancer Res 2007). It is also well recognized that PI3K-AKT pathway can drive NF-kB signaling, which is consistent with our earlier reported observation in APL. We hypothesize that this effect of PI3K-AKT activation could be mediated by EVs without direct malignant and stromal cell interaction as well. Towards this, we isolated stromal EVs as previously described (Suzanne M et al, Blood 2016). The size and concentration of EVs was characterized by NanoSight and flow cytometry (data not shown).We demonstrated an activation of PI3K-AKT signaling by showing phosphorylation of AKT (P-AKTser473) in U937 cells when treated with stromal EVs (figure 1A). We further demonstrated that stromal EVs were able to mediate a protective effect against AraC and DNR similar to that seen on direct co-culture with HS-5 cells [AraC p < 0.01, DNR p < 0.01 (n=3)]. This was further confirmed by looking at the pathway genes using Q-PCR, where an increased expression of PI3K-AKT signaling was observed in AML cells upon treatment with stromal EVs. The activation PI3K signaling was also confirmed by showing down regulation of PTEN [p < 0.01 (n=5)] which is a negative regulator of PI3K-AKT signaling. Further, to demonstrate the role of stromal EVs induced PI3K-AKT signaling in drug resistance, we treated the leukemic cells with a small molecule inhibitor of PI3 kinase (LY294002-10uM) along with AraC and DNR in the presence of stromal EVs. We noted that inhibiting PI3K-AKT signaling along with AraC or DNR was able to overcome the resistance mediated by stromal EVs (figure 1B). The mechanism of inducing apoptosis by LY294002 along with DNR or AraC (in the presence of stroma or stromal EVs) probably involves a TP53 dependent apoptosis pathway as evidenced through upregulation of TP53 and its target apoptotic genes such as BAD, CASP9 and PUMA (AraC- figure 1C, DNR - data not shown).Our study thus illustrates the complexity of cellular crosstalk between leukemic cells and its microenvironment in inducing drug resistance. This resistance can be mediated by non-contact setting via stromal EVs through up regulation of PI3K-AKT signaling in myeloid leukemic cells. Our data highlights the need for additional studies that are required to explore and characterize in detail the cargo of these EVs and the mechanisms by which they mediate drug resistance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.