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Effect of cancer-associated fibroblast exosomes on murine T cells

ABSTRACT: We profiled how cancer associated fibroblast exosomes alter T cells in mouse breast tumor setting using mass spectrometry. Briefly, exosomes were isolated from breast cancer associated fibroblasts from the MCa-P1362 cell line and then applied to primary CD8+ T cells from BALB/c mice. After exposure for 24 hours, T cells stimulated with anti-CD3/CD28 antibodies for indicated times and were harvested and subjected to quantitative proteomic and phosphoproteomic analysis by mass spectrometry to capture changes in protein expression and signaling. Unstimulated cells and stimulated cells not exposed to exosomes were used as comparators.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): T Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Carl White

LAB HEAD: Dennis Jones

PROVIDER: PXD074262 | Pride | 2026-03-30

REPOSITORIES: Pride

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			Action	DRS
	20210412_ML_Jones_TCell_Prot_F10_Split_1_164327.raw	Raw
	20210412_ML_Jones_TCell_Prot_F10_Split_2_164610.raw	Raw
	20210412_ML_Jones_TCell_Prot_F10_Split_3_164809.raw	Raw
	20210412_ML_Jones_TCell_Prot_F11_Split_1_164957.raw	Raw
	20210412_ML_Jones_TCell_Prot_F11_Split_2_165238.raw	Raw

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Project description:The innate immune system is vital to rapidly responding to pathogens and Toll-like receptors (TLRs) are a critical component of this response. Nanovesicular exosomes play a role in immunity, but to date their exact contribution to the dissemination of the TLR response is unknown. To understand the effect of exosomal cargo released from locally stimulated cells on distal cell expression, we collected exosomes from local ovarian adenocarcinoma (HEY) cells that were either unstimulated (control-exosomes), stimulated with pIC (pIC-exosomes), or lipopolysaccharide (LPS-exosomes) for 48 hours. The three groups of exosomes were added to naÃ¯ve (distal) cells and the gene expression profiles were compared between local TLR stimulation (for 6 hours) and distal stimulation mediated by exosomes at the 48-hour time point The goal of the study was to delineate the differential effector function of TLR-exosomes based on the innate immune activation state (control-, LPS-, pIC-stimulation) of the cell of origin in vitro. We used microarrays to understand the changes in gene expression in 2 samples of local hey cells either unstimulated(control) or stimulated with poly(I:C) or LPS each. We also profiled 2 samples of distal naÃ¯ve cells that were exposed to either control, LPS or poly(I:C) exosomes from local cells. Here we show that exosomes from TLR stimulated cells (TLR-exosomes) can largely recapitulate TLR activation in distal cells in vitro. We can abrogate the action-at-a-distance signaling of exosomes by UV irradiation, demonstrating that RNA is crucial for their effector function. This work definitively establishes the differential effector function for TLR-exosomes in communicating the activation state of the cell of origin. HEY cells were either unstimulated (Local control) or stimulated with LPS or poly (I:C) for 6 hours and the RNA from these cells was profiled using a microarray. Exosomes were collected from local cells that were unstimulated or stimulated for 48 hours and added to distal (naive) HEY cells that were exposed to control exosomes, LPS exosomes or pIC exosomes and then the RNA was collected for analysis by microarray. mRNA expression was captured on Affymetrix U133 Plus 2 chips. mRNA microarray data were analyzed using the Expression Console software (Affymetrix) and Bioconductor tools written in the R statistical programming language. Pre-processing of raw signal intensities and normalization was performed using GCRMA (R). Linear modelling of the transformed data was determined by using Limma in R with the Benjamini and Hochberg correction. Differentially expressed probesets were identified using a threshold 5% FDR correction and a fold change â¥ 1.4 was applied

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Effect of cancer-associated fibroblast exosomes on murine T cells

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