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Fibroblast-Derived Exosomes Contribute to Chemoresistance through Priming Cancer Stem Cells in Colorectal Cancer.

Hu Y, Yan C, Mu L, Huang K, Li X, Tao D, Wu Y, Qin J - PLoS ONE (2015)

Bottom Line: In addition, fibroblast-derived conditioned medium (CM) promoted percentage, clonogenicity and tumor growth of CSCs (i.e., CD133+ and TOP-GFP+) upon treatment with 5-fluorouracil (5-Fu) or oxaliplatin (OXA).Further investigations exhibited that exosomes, isolated from CM, similarly took the above effects.Inhibition of exosome secretion decreased the percentage, clonogenicity and tumor growth of CSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Molecular Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

ABSTRACT
Chemotherapy resistance observed in patients with colorectal cancer (CRC) may be related to the presence of cancer stem cells (CSCs), but the underlying mechanism(s) remain unclear. Carcinoma-associated fibroblasts (CAFs) are intimately involved in tumor recurrence, and targeting them increases chemo-sensitivity. We investigated whether fibroblasts might increase CSCs thus mediating chemotherapy resistance. CSCs were isolated from either patient-derived xenografts or CRC cell lines based on expression of CD133. First, CSCs were found to be inherently resistant to cell death induced by chemotherapy. In addition, fibroblast-derived conditioned medium (CM) promoted percentage, clonogenicity and tumor growth of CSCs (i.e., CD133+ and TOP-GFP+) upon treatment with 5-fluorouracil (5-Fu) or oxaliplatin (OXA). Further investigations exhibited that exosomes, isolated from CM, similarly took the above effects. Inhibition of exosome secretion decreased the percentage, clonogenicity and tumor growth of CSCs. Altogether, our findings suggest that, besides targeting CSCs, new therapeutic strategies blocking CAFs secretion even before chemotherapy shall be developed to gain better clinical benefits in advanced CRCs.

No MeSH data available.


Related in: MedlinePlus

Fibroblast-derived exosomes prime CSCs to be more chemoresistance.(A) Transmission electron microscopic image of the exosomes derived from 18Co cells and CAFs. Scale bars, 100nm. (B) Western blotting of CD81 in exosomes. (C) Representative microscopy of SW620 cells exposed to DiI-labeled exosomes for 12h. (D, E) Sphere-forming capacity of SW620 or XhCRC CSCs treated with 18Co/CAF-derived exosomes during chemotherapy (5-Fu or OXA). (F) Sphere-forming capacity of SW620 CSCs treated with ultracentrifugation-purified 18Co-derived exosomes during chemotherapy (5-Fu or OXA). (G, H)Fibroblasts were treated with 10mM GW4869 (dissolves in DMSO) for 24h. The CMs derived from GW4869/DMSO-pretreated fibroblasts were added to CSCs. (I-L) CAF-derived exosomes affected on tumor growth of XhCRC CSCs in female NOD/SCID mice treated with 5-Fu or OXA. Tumor growth curves are shown in I(5-Fu) and K(OXA), and tumor weights and images at the end of experiments are shown in L(5-Fu) and J(OXA). Data are presented as mean ± SD; *P< 0.1; **P< 0.05.
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pone.0125625.g005: Fibroblast-derived exosomes prime CSCs to be more chemoresistance.(A) Transmission electron microscopic image of the exosomes derived from 18Co cells and CAFs. Scale bars, 100nm. (B) Western blotting of CD81 in exosomes. (C) Representative microscopy of SW620 cells exposed to DiI-labeled exosomes for 12h. (D, E) Sphere-forming capacity of SW620 or XhCRC CSCs treated with 18Co/CAF-derived exosomes during chemotherapy (5-Fu or OXA). (F) Sphere-forming capacity of SW620 CSCs treated with ultracentrifugation-purified 18Co-derived exosomes during chemotherapy (5-Fu or OXA). (G, H)Fibroblasts were treated with 10mM GW4869 (dissolves in DMSO) for 24h. The CMs derived from GW4869/DMSO-pretreated fibroblasts were added to CSCs. (I-L) CAF-derived exosomes affected on tumor growth of XhCRC CSCs in female NOD/SCID mice treated with 5-Fu or OXA. Tumor growth curves are shown in I(5-Fu) and K(OXA), and tumor weights and images at the end of experiments are shown in L(5-Fu) and J(OXA). Data are presented as mean ± SD; *P< 0.1; **P< 0.05.

Mentions: Recent evidence suggests that exosomes, soluble factors which are secreted by a variety of cells, have been implicated in metastasis and drug resistance [8, 27, 28]. We hypothesized that exosomes might also contribute to drug resistance in our experimental system. Therefore, we first purified exosomes from 18Co-CM and CAF-CM as described in Materials and Methods, and then confirmed their structural nature under phase-contrast electron microscopy (Fig 5A) and by immunoblotting of exosome marker protein CD81 (Fig 5B). To investigate whether fibroblast-secreted exosomes can transfer to CRC cells, we labeled exosomes with Dil, a lipophilic fluorescentcarbocyanine dye. We observed that Dil-labeled exosomes derived from 18Co cells were taken up by SW620 cells after 12 hours co-incubation (Fig 5C). We then treated CSCs with purified exosomes instead of CM, and found that both SW620 and XhCRC CSCs treated with exosomes generated more spheres (P<0.01) (Fig 5D and 5E), suggesting that exosomes, which are derived from fibroblasts, may prime CSCs to become more chemoresistance. To further confirm that fibroblast-secreted exosomes rather than other soluble factors take the above effects, we adopted standard differential ultracentrifugation, instead of Total Exosome Isolation Kit, to isolate exosomes. Similar to kit-purified exosomes, CM-pellet-treated SW620 CSCs formed more spheres when compared with control pellets (P<0.001 in 5-Fu group, P<0.01 in OXA group), and the exosome-depleted supernatant from 18Co-CM did not take this effect (NS control pellet vs supernatant) (Fig 5F). To further confirm whether fibroblast-derived exosomes mediate in drug resistance, we treated fibroblasts (18Co and CAFs) with GW4869, a specific neutral sphingomyelinase (nSMase) inhibitor which blocking exosomes release, and then obtained the CM (i.e., exosome-depleted CM). Consistent with the previous findings, exosome-depleted CM remarkably decreased chemoresistance of CSCs (Fig 5G, P<0.001, 5H, in 5-Fu group, P<0.001 in OXA group). In addition, in vivo experiments also showed that XhCRC CSCs, while treated with CAF-derived exosomes, generated faster-growing (Fig 5I and 5K) and larger tumors (P<0.05) (Fig 5J and 5L) during chemotherapy (5-Fu or OXA). These data clearly showed that fibroblasts-derived exosomes primed CSCs to be more drug resistance.


Fibroblast-Derived Exosomes Contribute to Chemoresistance through Priming Cancer Stem Cells in Colorectal Cancer.

Hu Y, Yan C, Mu L, Huang K, Li X, Tao D, Wu Y, Qin J - PLoS ONE (2015)

Fibroblast-derived exosomes prime CSCs to be more chemoresistance.(A) Transmission electron microscopic image of the exosomes derived from 18Co cells and CAFs. Scale bars, 100nm. (B) Western blotting of CD81 in exosomes. (C) Representative microscopy of SW620 cells exposed to DiI-labeled exosomes for 12h. (D, E) Sphere-forming capacity of SW620 or XhCRC CSCs treated with 18Co/CAF-derived exosomes during chemotherapy (5-Fu or OXA). (F) Sphere-forming capacity of SW620 CSCs treated with ultracentrifugation-purified 18Co-derived exosomes during chemotherapy (5-Fu or OXA). (G, H)Fibroblasts were treated with 10mM GW4869 (dissolves in DMSO) for 24h. The CMs derived from GW4869/DMSO-pretreated fibroblasts were added to CSCs. (I-L) CAF-derived exosomes affected on tumor growth of XhCRC CSCs in female NOD/SCID mice treated with 5-Fu or OXA. Tumor growth curves are shown in I(5-Fu) and K(OXA), and tumor weights and images at the end of experiments are shown in L(5-Fu) and J(OXA). Data are presented as mean ± SD; *P< 0.1; **P< 0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4418721&req=5

pone.0125625.g005: Fibroblast-derived exosomes prime CSCs to be more chemoresistance.(A) Transmission electron microscopic image of the exosomes derived from 18Co cells and CAFs. Scale bars, 100nm. (B) Western blotting of CD81 in exosomes. (C) Representative microscopy of SW620 cells exposed to DiI-labeled exosomes for 12h. (D, E) Sphere-forming capacity of SW620 or XhCRC CSCs treated with 18Co/CAF-derived exosomes during chemotherapy (5-Fu or OXA). (F) Sphere-forming capacity of SW620 CSCs treated with ultracentrifugation-purified 18Co-derived exosomes during chemotherapy (5-Fu or OXA). (G, H)Fibroblasts were treated with 10mM GW4869 (dissolves in DMSO) for 24h. The CMs derived from GW4869/DMSO-pretreated fibroblasts were added to CSCs. (I-L) CAF-derived exosomes affected on tumor growth of XhCRC CSCs in female NOD/SCID mice treated with 5-Fu or OXA. Tumor growth curves are shown in I(5-Fu) and K(OXA), and tumor weights and images at the end of experiments are shown in L(5-Fu) and J(OXA). Data are presented as mean ± SD; *P< 0.1; **P< 0.05.
Mentions: Recent evidence suggests that exosomes, soluble factors which are secreted by a variety of cells, have been implicated in metastasis and drug resistance [8, 27, 28]. We hypothesized that exosomes might also contribute to drug resistance in our experimental system. Therefore, we first purified exosomes from 18Co-CM and CAF-CM as described in Materials and Methods, and then confirmed their structural nature under phase-contrast electron microscopy (Fig 5A) and by immunoblotting of exosome marker protein CD81 (Fig 5B). To investigate whether fibroblast-secreted exosomes can transfer to CRC cells, we labeled exosomes with Dil, a lipophilic fluorescentcarbocyanine dye. We observed that Dil-labeled exosomes derived from 18Co cells were taken up by SW620 cells after 12 hours co-incubation (Fig 5C). We then treated CSCs with purified exosomes instead of CM, and found that both SW620 and XhCRC CSCs treated with exosomes generated more spheres (P<0.01) (Fig 5D and 5E), suggesting that exosomes, which are derived from fibroblasts, may prime CSCs to become more chemoresistance. To further confirm that fibroblast-secreted exosomes rather than other soluble factors take the above effects, we adopted standard differential ultracentrifugation, instead of Total Exosome Isolation Kit, to isolate exosomes. Similar to kit-purified exosomes, CM-pellet-treated SW620 CSCs formed more spheres when compared with control pellets (P<0.001 in 5-Fu group, P<0.01 in OXA group), and the exosome-depleted supernatant from 18Co-CM did not take this effect (NS control pellet vs supernatant) (Fig 5F). To further confirm whether fibroblast-derived exosomes mediate in drug resistance, we treated fibroblasts (18Co and CAFs) with GW4869, a specific neutral sphingomyelinase (nSMase) inhibitor which blocking exosomes release, and then obtained the CM (i.e., exosome-depleted CM). Consistent with the previous findings, exosome-depleted CM remarkably decreased chemoresistance of CSCs (Fig 5G, P<0.001, 5H, in 5-Fu group, P<0.001 in OXA group). In addition, in vivo experiments also showed that XhCRC CSCs, while treated with CAF-derived exosomes, generated faster-growing (Fig 5I and 5K) and larger tumors (P<0.05) (Fig 5J and 5L) during chemotherapy (5-Fu or OXA). These data clearly showed that fibroblasts-derived exosomes primed CSCs to be more drug resistance.

Bottom Line: In addition, fibroblast-derived conditioned medium (CM) promoted percentage, clonogenicity and tumor growth of CSCs (i.e., CD133+ and TOP-GFP+) upon treatment with 5-fluorouracil (5-Fu) or oxaliplatin (OXA).Further investigations exhibited that exosomes, isolated from CM, similarly took the above effects.Inhibition of exosome secretion decreased the percentage, clonogenicity and tumor growth of CSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Molecular Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

ABSTRACT
Chemotherapy resistance observed in patients with colorectal cancer (CRC) may be related to the presence of cancer stem cells (CSCs), but the underlying mechanism(s) remain unclear. Carcinoma-associated fibroblasts (CAFs) are intimately involved in tumor recurrence, and targeting them increases chemo-sensitivity. We investigated whether fibroblasts might increase CSCs thus mediating chemotherapy resistance. CSCs were isolated from either patient-derived xenografts or CRC cell lines based on expression of CD133. First, CSCs were found to be inherently resistant to cell death induced by chemotherapy. In addition, fibroblast-derived conditioned medium (CM) promoted percentage, clonogenicity and tumor growth of CSCs (i.e., CD133+ and TOP-GFP+) upon treatment with 5-fluorouracil (5-Fu) or oxaliplatin (OXA). Further investigations exhibited that exosomes, isolated from CM, similarly took the above effects. Inhibition of exosome secretion decreased the percentage, clonogenicity and tumor growth of CSCs. Altogether, our findings suggest that, besides targeting CSCs, new therapeutic strategies blocking CAFs secretion even before chemotherapy shall be developed to gain better clinical benefits in advanced CRCs.

No MeSH data available.


Related in: MedlinePlus