Extracellular Vesicle Measurements with Nanoparticle Tracking Analysis: A Different Appreciation of Up and Down Secretion

Abstract

As is the case with most eucaryotic cells, cancer cells are able to secrete extracellular vesicles (EVs) as a communication means towards their environment and surrounding cells. EVs are represented by microvesicles and smaller vesicles called exosomes, which are known for their involvement in cancer aggressiveness. The release of such EVs requires the intervention of trafficking-associated proteins, mostly represented by the RAB-GTPases family. In particular, RAB27A is known for its role in addressing EVs-to-be secreted towards the the plasma membrane. In this study, shRNAs targeting RAB27A were used in colorectal (CRC) and glioblastoma (GB) cell lines in order to alter EVs secretion. To study and monitor EVs secretion in cell lines' supernatants, nanoparticle tracking analysis (NTA) was used through the NanoSight NS300 device. Since it appeared that NanoSight failed to detect the decrease in the EVs secretion, we performed another approach to drop EVs secretion (RAB27A-siRNA, indomethacin, Nexihnib20). Similar results were obtained i.e., no variation in EVs concentration. Conversely, NTA allowed us to monitor EVs up-secretion following rotenone treatment or hypoxia conditions. Therefore, our data seemed to point out the insufficiency of using only this technique for the assessment of EVs secretion decrease.

Keywords: RAB27A; colorectal cancer; exosomes; extracellular vesicles; glioblastoma; nanoparticle tracking analysis (NTA).

Figure 1

Validation of the shRAB27A models in both HCT-116 and U87-MG cell lines. (A)—Downregulation of RAB27A expression was evaluated by RT-qPCR in both pLKO (control) and shRAB27A cell lines. Whatever the cell line, sh1 and sh3 showed the strongest impairment in RAB27A expression compared to pLKO. The Mann–Whitney test was used for evaluation of the significance; * p < 0.05; *** p < 0.001 n = 6. (B)—Downregulation of RAB27A expression was evaluated by Western blot in HCT-116 and U87-MG models (left panels). The quantification of RAB27A expression (right panel) was consistent with RT-qPCR analysis. The Mann–Whitney test was used for evaluation of the significance; * p < 0.05; ** p < 0.01; *** p < 0.001; n = 6. (C)—Western blot analysis showed a decreased expression of HSC-70 in purified exosomes from both cell lines when RAB27A was down regulated; n = 3.

Figure 1

Validation of the shRAB27A models in both HCT-116 and U87-MG cell lines. (A)—Downregulation of RAB27A expression was evaluated by RT-qPCR in both pLKO (control) and shRAB27A cell lines. Whatever the cell line, sh1 and sh3 showed the strongest impairment in RAB27A expression compared to pLKO. The Mann–Whitney test was used for evaluation of the significance; * p < 0.05; *** p < 0.001 n = 6. (B)—Downregulation of RAB27A expression was evaluated by Western blot in HCT-116 and U87-MG models (left panels). The quantification of RAB27A expression (right panel) was consistent with RT-qPCR analysis. The Mann–Whitney test was used for evaluation of the significance; * p < 0.05; ** p < 0.01; *** p < 0.001; n = 6. (C)—Western blot analysis showed a decreased expression of HSC-70 in purified exosomes from both cell lines when RAB27A was down regulated; n = 3.

Figure 2

NTA analysis of extracellular vesicles from HCT-116 and U87-MG pLKO vs. shRAB27A. (A)—Evaluation of the size repartition of the EVs secreted by the HCT-116 (upper panel) and U87-MG (lower panel) pLKO and shRAB27A cell lines. The cells were cultured as mentioned in the material and methods, and the supernatants were directly analyzed using the Nanosight300. Most of the EVs population is comprised of between 50 and 180 nm, which is the size range commonly given to exosomes (n = 3). (B)—Concomitantly with the supernatant collection, cells were counted, and the EVs/cell ratio was calculated for each cell line. There is no difference concerning this ratio between pLKO and shRAB27A cell lines. (C)—EVs mean size (nm) from each cell line after 48 h of culture in EFM. (D)—EVs size mode (nm) from each cell line after 48 h of culture in EFM. There is no difference between the control and shRAB27A cell lines in both U87-MG and HCT-116, n = 3. (E–H)—The same analysis of EVs characteristics was performed at the same time (48 h) after the ultracentrifugation of the supernatant, according to Théry’s protocol (n = 3).

Figure 2

NTA analysis of extracellular vesicles from HCT-116 and U87-MG pLKO vs. shRAB27A. (A)—Evaluation of the size repartition of the EVs secreted by the HCT-116 (upper panel) and U87-MG (lower panel) pLKO and shRAB27A cell lines. The cells were cultured as mentioned in the material and methods, and the supernatants were directly analyzed using the Nanosight300. Most of the EVs population is comprised of between 50 and 180 nm, which is the size range commonly given to exosomes (n = 3). (B)—Concomitantly with the supernatant collection, cells were counted, and the EVs/cell ratio was calculated for each cell line. There is no difference concerning this ratio between pLKO and shRAB27A cell lines. (C)—EVs mean size (nm) from each cell line after 48 h of culture in EFM. (D)—EVs size mode (nm) from each cell line after 48 h of culture in EFM. There is no difference between the control and shRAB27A cell lines in both U87-MG and HCT-116, n = 3. (E–H)—The same analysis of EVs characteristics was performed at the same time (48 h) after the ultracentrifugation of the supernatant, according to Théry’s protocol (n = 3).

Figure 3

NTA analysis following different attempts for EVs secretion alteration in HCT-116 and U87-MG cell lines. (A)—Downregulation of RAB27A expression using siRAB27A was evaluated by Western lot (left panels). RAB27A expression quantification (right panels) in siCTL (control) and siRAB27A conditions in both HCT-116 and U87-MG cell lines (n = 3, n = 4, respectively). The Mann–Whitney test was performed in at least three independent experiments; * p < 0.05; ** p < 0.01. (B)—Evaluation of the size repartition of the EVs secreted by the HCT-116 (upper panel) and U87-MG (lower panel) pLKO transfected with siCTL or siRAB27A. (C)—EVs/cell ratio was calculated for each cell line. There is no difference concerning this ratio between pLKO siCTL and siRAB27A cell lines in both HCT-116 and U87-MG models (n = 3, n = 4, respectively). (D)—EVs mean size (nm) from each cell line after 48 h of culture in EFM. (E)—EVs size mode (nm) from each cell line after 48 h of culture in EFM. (F–I)—HCT-116 and U87-MG cell lines were treated in EFM with either indomethacin (n = 3, n = 4, respectively) or Nexinhib20 (n = 3, n = 6, respectively). (G)—EVs/cell ratio was calculated for each condition. There is no difference concerning this ratio between pLKO alone and pLKO treated with either indomethacin or Nx20 in both the HCT-116 and U87-MG models (n = 3 each). (H)—EVs mean size (nm) from each cell line after 48 h of culture in EFM. (I)—EVs size mode (nm) from each cell line after 48 h of culture in EFM.

Figure 3

NTA analysis following different attempts for EVs secretion alteration in HCT-116 and U87-MG cell lines. (A)—Downregulation of RAB27A expression using siRAB27A was evaluated by Western lot (left panels). RAB27A expression quantification (right panels) in siCTL (control) and siRAB27A conditions in both HCT-116 and U87-MG cell lines (n = 3, n = 4, respectively). The Mann–Whitney test was performed in at least three independent experiments; * p < 0.05; ** p < 0.01. (B)—Evaluation of the size repartition of the EVs secreted by the HCT-116 (upper panel) and U87-MG (lower panel) pLKO transfected with siCTL or siRAB27A. (C)—EVs/cell ratio was calculated for each cell line. There is no difference concerning this ratio between pLKO siCTL and siRAB27A cell lines in both HCT-116 and U87-MG models (n = 3, n = 4, respectively). (D)—EVs mean size (nm) from each cell line after 48 h of culture in EFM. (E)—EVs size mode (nm) from each cell line after 48 h of culture in EFM. (F–I)—HCT-116 and U87-MG cell lines were treated in EFM with either indomethacin (n = 3, n = 4, respectively) or Nexinhib20 (n = 3, n = 6, respectively). (G)—EVs/cell ratio was calculated for each condition. There is no difference concerning this ratio between pLKO alone and pLKO treated with either indomethacin or Nx20 in both the HCT-116 and U87-MG models (n = 3 each). (H)—EVs mean size (nm) from each cell line after 48 h of culture in EFM. (I)—EVs size mode (nm) from each cell line after 48 h of culture in EFM.

Figure 4

NTA analysis following EVs secretion increase attempts in HCT-116 and U87-MG cell lines. (A)—Evaluation of the size repartition of the EVs secreted by the HCT-116 (upper panel) and U87-MG (lower panel) untreated pLKO (CTL) or treated with 0.5µM of rotenone for 48 h. (B)—EVs/cell ratio was calculated for each condition in both HCT-116 and U87-MG cells (n = 3 each). The Mann–Whitney test was performed in three independent experiments; p < 0.05 was considered significant. (C)—EVs size range after 48 h under normoxia (N) or hypoxia (H) conditions was determined for both HCT-116 and U87-MG cells. (D)—EVs/cell ratio was calculated for each cell line in hypoxia compared to normoxia conditions. The Mann–Whitney test was performed in three independent experiments; *** p < 0.001.

Figure 5

Analysis of the expression of other RAB-GTPases. (A)—RAB27B and RAB35 expressions were analyzed by RT-qPCR assays in both HCT-116 and U87-MG cell lines. RAB35 was not detected in the U87-MG cell line. The Mann–Whitney test was performed on three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001. (B)—RAB27B and RAB35 expressions were assessed by Western blot in both HCT-116 and U87-MG cell lines. RAB35 failed to be detected in U87-MG. The Mann–Whitney test was performed in three independent experiments; ** p < 0.01; *** p < 0.001. (C)—RAB11 expression was analyzed by Western blot in both HCT-116 and U87-MG cell lines. The Mann–Whitney test was performed on three independent experiments.

Figure 5

Analysis of the expression of other RAB-GTPases. (A)—RAB27B and RAB35 expressions were analyzed by RT-qPCR assays in both HCT-116 and U87-MG cell lines. RAB35 was not detected in the U87-MG cell line. The Mann–Whitney test was performed on three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001. (B)—RAB27B and RAB35 expressions were assessed by Western blot in both HCT-116 and U87-MG cell lines. RAB35 failed to be detected in U87-MG. The Mann–Whitney test was performed in three independent experiments; ** p < 0.01; *** p < 0.001. (C)—RAB11 expression was analyzed by Western blot in both HCT-116 and U87-MG cell lines. The Mann–Whitney test was performed on three independent experiments.