Hypoxic enhancement of exosome release by breast cancer cells

Abstract

Background: Exosomes are nanovesicles secreted by tumour cells which have roles in paracrine signalling during tumour progression, including tumour-stromal interactions, activation of proliferative pathways and bestowing immunosuppression. Hypoxia is an important feature of solid tumours which promotes tumour progression, angiogenesis and metastasis, potentially through exosome-mediated signalling.

Methods: Breast cancer cell lines were cultured under either moderate (1% O2) or severe (0.1% O2) hypoxia. Exosomes were isolated from conditioned media and quantitated by nanoparticle tracking analysis (NTA) and immunoblotting for the exosomal protein CD63 in order to assess the impact of hypoxia on exosome release. Hypoxic exosome fractions were assayed for miR-210 by real-time reverse transcription polymerase chain reaction and normalised to exogenous and endogenous control genes. Statistical significance was determined using the Student T test with a P value of < 0.05 considered significant.

Results: Exposure of three different breast cancer cell lines to moderate (1% O2) and severe (0.1% O2) hypoxia resulted in significant increases in the number of exosomes present in the conditioned media as determined by NTA and CD63 immunoblotting. Activation of hypoxic signalling by dimethyloxalylglycine, a hypoxia-inducible factor (HIF) hydroxylase inhibitor, resulted in significant increase in exosome release. Transfection of cells with HIF-1α siRNA prior to hypoxic exposure prevented the enhancement of exosome release by hypoxia. The hypoxically regulated miR-210 was identified to be present at elevated levels in hypoxic exosome fractions.

Conclusions: These data provide evidence that hypoxia promotes the release of exosomes by breast cancer cells, and that this hypoxic response may be mediated by HIF-1α. Given an emerging role for tumour cell-derived exosomes in tumour progression, this has significant implications for understanding the hypoxic tumour phenotype, whereby hypoxic cancer cells may release more exosomes into their microenvironment to promote their own survival and invasion.

Figure 1

Exosome isolation by ultracentrifugation and Exoquick.^TMprecipitation from MCF7 conditioned media. (A, B) Transmission electron microscopy and CD63 immunolabelling of MCF7 exosomes isolated by ultracentrifugation (A) and Exoquick^TM precipitation (B). (C) Nanoparticle tracking analysis (NTA) of MCF7 exosomes isolated by ultracentrifugation and Exoquick^TM precipitation. Data represent the average size distribution profile of n = 4 for each purification method normalised to the total nanoparticle concentrations. Data for each individual sample derive from four different videos and analyses. (D) Immunoblotting of ultracentrifugation pellet for exosomal proteins CD63, TSG101, cofilin and flotillin-1.

Figure 2

Comparison of exosome isolation efficiency by ultracentrifugation and Exoquick.^TMprecipitation. (A) Exosome fractions from ultracentrifugation or Exoquick^TM precipitation were analysed by NTA. Nanoparticle concentrations per mL of original volume of media conditioned by 48 hour MCF7 culture (10.5 mL and 0.2 mL for ultracentrifugation and Exoquick^TM respectively) were determined, and are expressed here relative to the ultracentrifugation results (n = 8; ± SEM). (B) CD63 immunoblot, performed under non-reducing conditions, of exosomes isolated by ultracentrifugation or Exoquick^TM precipitation from 24 mL or 0.5 mL MCF7 conditioned media respectively. Exosome pellets were resuspended in equal volumes of PBS for both methods, and loading was controlled by volume. *** corresponds with P value < 0.001.

Figure 3

Hypoxic enhancement of exosome release by breast cancer cells. (A) MCF7, SKBR3 and MDA-MB 231 cells were cultured at 1% O₂ for 48 hours. Exosomes were isolated from conditioned media by Exoquick^TM precipitation. Nanoparticle concentrations were determined by NTA and expressed relative to the normoxic control. Data for each sample were derived from four different videos and analyses (n ≥ 3; ± SEM). (B) MCF7, SKBR3 and MDA-MB 231 cells were cultured at 0.1% O₂ for 24 hours and exosomes were isolated and analysed as described in (A). (C) CD63 immunoblot of MCF7 Exoquick^TM precipitants from a 48 hour culture under normoxia or 1% O₂, including band intensity quantitation.(D) CD63 immunoblot of MDA-MB 231 Exoquick^TM precipitants from a 24 hour culture under normoxia or 0.1% O₂, including band intensity quantitation_. (E, F) Nanoparticle size distribution profiles obtained by NTA for hypoxic (0.1% O₂) MCF7 Exoquick^TM precipitants were normalised to final cell counts for normoxia and hypoxia (E) and relative nanoparticle size distribution profiles were obtained by normalising to total nanoparticle concentration (F). All CD63 immunoblots were performed under non-reducing conditions as described previously [26]. Annotations *, **, and *** correspond with P values <0.05, <0.01 and <0.001 respectively.

Figure 4

Exosome release is modulated by induction of hypoxia inducible factor. (A) MDA-MB 231 breast cancer cells were treated with 1 mM DMOG for 24 hours. Exosomes were isolated and quantitated by NTA as previously described (n = 4; ± SEM). (B) MDA-MB 231 cells were transfected with negative control (NC) siRNA or siRNA targeting HIF-1α prior to exposure to 0.1% O₂ for 24 hours. Exosomes were isolated by Exoquick^TM and quantitated by NTA (n = 4; ± SEM). Annotations *, **, and *** correspond with P values <0.05, <0.01 and <0.001 respectively.

Figure 5

Cellular and exosomal microRNA expression levels in response to hypoxia. (A) Mean normalised expression levels of MCF7 cellular miRNAs as determined by miRNA-specific Taqman real-time RT-PCR assays in response to 1% O₂ for 48 hours and normalised to RNU6B. Expression values are presented relative to normoxic control. (n = 3; ± SEM). (B, C) Exoquick^TM precipitants isolated from MCF7 cell culture after 48 hours at 1% O₂ were spiked with cel miR-54 and used for RNA extractions. This exosomal RNA was then assayed for miR-16, let7a, miR-210 and cel miR-54 by real-time RT-PCR and normalised to exogenous cel miR-54 (B) or endogenous miR-16 (C) (± SEM). Normoxia n = 4; Hypoxia n = 5. ** corresponds with P value < 0.01.