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Physical Intimacy of Breast Cancer Cells with Mesenchymal Stem Cells Elicits Trastuzumab Resistance through Src Activation.

Daverey A, Drain AP, Kidambi S - Sci Rep (2015)

Bottom Line: The development of resistance to trastuzumab is a major obstacle for lasting effective treatment of patients with ErbB2-overexpressing tumors.To our knowledge, this is the first report that showed PTEN loss without the use of chemical inhibitors, matrix stiffness, or silencing RNAs.Our findings show that MSCs are potent mediators of resistance to trastuzumab and might reveal targets to enhance trastuzumab efficacy in patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, NE, 68588.

ABSTRACT
The development of resistance to trastuzumab is a major obstacle for lasting effective treatment of patients with ErbB2-overexpressing tumors. Here, we demonstrate that the physical contact of breast cancer cells with mesenchymal stem cells (MSCs) is a potential modulator of trastuzumab response by activation of nonreceptor tyrosine kinase c-Src and down regulation of phosphatase and tensin homolog (PTEN). Using an in vitro patterned breast cancer/MSC co-culture model, we find that the presence of MSCs results in Src activation that is missing in cancer cells monoculture, transwell co-culture, and cells treated with MSCs conditioned media. Interestingly, the co-culture model also results in PTEN loss and activation of PI3K/AKT pathway that has been demonstrated as fundamental proliferative and survival pathways in clinical settings. To our knowledge, this is the first report that showed PTEN loss without the use of chemical inhibitors, matrix stiffness, or silencing RNAs. In addition, breast cancer cells in co-culture with MSCs conferred trastuzumab resistance in vitro as observed in the lack of inhibition of proliferative and migrative properties of the cancer cells. Our findings show that MSCs are potent mediators of resistance to trastuzumab and might reveal targets to enhance trastuzumab efficacy in patients.

No MeSH data available.


Related in: MedlinePlus

Breast cancer cells in co-culture with MSCs contribute to trastuzumab resistance.(a) Migration assay data detailing the migration of cells out from a confluent monolayer onto a featureless scratch or wound. Left, representative fluorescent images of breast cancer cells (CFDSE, green) and breast cancer cells co-cultured with MSCs (PKH26, red) before and after treatment with trastuzumab (Ttzm) at time = 0 h and 48 h. Scale bar 500 μm. Right, quantification of wound closure. The percentage of wound closure was determined by using following equation: % wound closure  =  [(Area @ t = 0–Area @ t = 48)/Area @ t = 0h]× 100%. Data are mean ± SD; n = 3 independent experiments; **p < 0.01 compared with untreated monoculture; ***p < 0.001 compared untreated monoculture; ^^p < 0.05 compared with random co-culture; ^p < 0.01 compared with random co-culture. (b) MSCs in co-culture with breast cancer cells confer trastuzumab resistance. Left, western blot analysis of Ki67 in breast cancer cells sorted by FACS after co-culture with MSCs, and breast cancer cells exposed to MSCs-CM in the presence or absence of Ttzm. Right, respective densitometry of bands normalized with respective untreated group after loading control (GAPDH) correction. Mean ± SD; n = 3 independent experiments; *p < 0.05 compared with untreated monoculture. (c) Suggested model of trastuzumab resistance mediated by MSCs via activation of Src. Figure drawn by Dr. Amita Daverey.
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f5: Breast cancer cells in co-culture with MSCs contribute to trastuzumab resistance.(a) Migration assay data detailing the migration of cells out from a confluent monolayer onto a featureless scratch or wound. Left, representative fluorescent images of breast cancer cells (CFDSE, green) and breast cancer cells co-cultured with MSCs (PKH26, red) before and after treatment with trastuzumab (Ttzm) at time = 0 h and 48 h. Scale bar 500 μm. Right, quantification of wound closure. The percentage of wound closure was determined by using following equation: % wound closure  =  [(Area @ t = 0–Area @ t = 48)/Area @ t = 0h]× 100%. Data are mean ± SD; n = 3 independent experiments; **p < 0.01 compared with untreated monoculture; ***p < 0.001 compared untreated monoculture; ^^p < 0.05 compared with random co-culture; ^p < 0.01 compared with random co-culture. (b) MSCs in co-culture with breast cancer cells confer trastuzumab resistance. Left, western blot analysis of Ki67 in breast cancer cells sorted by FACS after co-culture with MSCs, and breast cancer cells exposed to MSCs-CM in the presence or absence of Ttzm. Right, respective densitometry of bands normalized with respective untreated group after loading control (GAPDH) correction. Mean ± SD; n = 3 independent experiments; *p < 0.05 compared with untreated monoculture. (c) Suggested model of trastuzumab resistance mediated by MSCs via activation of Src. Figure drawn by Dr. Amita Daverey.

Mentions: To investigate if the physical intimacy of MSCs may confer resistance to trastuzumab in breast cancer cells, we assessed the migratory potential of cancer cells in co-culture when exposed to the drug. Trastuzumab has been shown to inhibit cell migration in both in vitro and in vivo studies, however the drug loses this ability in drug resistant tumors3. In order to evaluate the effect of direct contact of MSC on breast cancer cells migration, we probed the migration capacity of BT-474 and 21MT-1 in mono-cultures, exposed with MSCs-CM, and co-cultures with MSCs in the presence of trastuzumab (Fig. 5A). A scratch was made in a sub-confluent cell monolayer and cells were allowed to migrate into the cell-free area. The distance moved by the cells in control, MSC-CM treated cells, and co-cultured plates, respectively, was compared when exposed to the drug. To distinguish the cell populations, breast cancer cells and MSCs were stained with green dye, CFDSE and red dye, PKH26 respectively. In the absence of trastuzumab, the co-culture of BT-474 and 21MT-1 cells with MSCs showed significantly (p < 0.001) higher migration after 48 h of co-culture as compared to the respective monocultures. On the other hand, only 21MT-1 cells treated with MSCs-CM demonstrated significant migration as compared to monoculture (p < 0.05). When BT-474 mono-culture cells were exposed to Ttzm, there was no significant change in the migratory property in mono-culture and MSCs-CM conditions but BT-474 in co-culture showed significantly (p < 0.01) higher migration when compared to untreated co-culture samples. In contrast, when 21MT-1 mono-culture and MSC-CMs cells were exposed to Ttzm, there was no significant no signficiant inhibition in the migratory potential while 21MT-1 in co-culture showed significantly (p < 0.05) even after exposed to Ttzm. This data suggest that direct interaction of MSCs with breast cancer cells promotes migration of breast cancer cells and potentially regulates the Ttzm responsiveness in breast tumor cells when in physical contact.


Physical Intimacy of Breast Cancer Cells with Mesenchymal Stem Cells Elicits Trastuzumab Resistance through Src Activation.

Daverey A, Drain AP, Kidambi S - Sci Rep (2015)

Breast cancer cells in co-culture with MSCs contribute to trastuzumab resistance.(a) Migration assay data detailing the migration of cells out from a confluent monolayer onto a featureless scratch or wound. Left, representative fluorescent images of breast cancer cells (CFDSE, green) and breast cancer cells co-cultured with MSCs (PKH26, red) before and after treatment with trastuzumab (Ttzm) at time = 0 h and 48 h. Scale bar 500 μm. Right, quantification of wound closure. The percentage of wound closure was determined by using following equation: % wound closure  =  [(Area @ t = 0–Area @ t = 48)/Area @ t = 0h]× 100%. Data are mean ± SD; n = 3 independent experiments; **p < 0.01 compared with untreated monoculture; ***p < 0.001 compared untreated monoculture; ^^p < 0.05 compared with random co-culture; ^p < 0.01 compared with random co-culture. (b) MSCs in co-culture with breast cancer cells confer trastuzumab resistance. Left, western blot analysis of Ki67 in breast cancer cells sorted by FACS after co-culture with MSCs, and breast cancer cells exposed to MSCs-CM in the presence or absence of Ttzm. Right, respective densitometry of bands normalized with respective untreated group after loading control (GAPDH) correction. Mean ± SD; n = 3 independent experiments; *p < 0.05 compared with untreated monoculture. (c) Suggested model of trastuzumab resistance mediated by MSCs via activation of Src. Figure drawn by Dr. Amita Daverey.
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f5: Breast cancer cells in co-culture with MSCs contribute to trastuzumab resistance.(a) Migration assay data detailing the migration of cells out from a confluent monolayer onto a featureless scratch or wound. Left, representative fluorescent images of breast cancer cells (CFDSE, green) and breast cancer cells co-cultured with MSCs (PKH26, red) before and after treatment with trastuzumab (Ttzm) at time = 0 h and 48 h. Scale bar 500 μm. Right, quantification of wound closure. The percentage of wound closure was determined by using following equation: % wound closure  =  [(Area @ t = 0–Area @ t = 48)/Area @ t = 0h]× 100%. Data are mean ± SD; n = 3 independent experiments; **p < 0.01 compared with untreated monoculture; ***p < 0.001 compared untreated monoculture; ^^p < 0.05 compared with random co-culture; ^p < 0.01 compared with random co-culture. (b) MSCs in co-culture with breast cancer cells confer trastuzumab resistance. Left, western blot analysis of Ki67 in breast cancer cells sorted by FACS after co-culture with MSCs, and breast cancer cells exposed to MSCs-CM in the presence or absence of Ttzm. Right, respective densitometry of bands normalized with respective untreated group after loading control (GAPDH) correction. Mean ± SD; n = 3 independent experiments; *p < 0.05 compared with untreated monoculture. (c) Suggested model of trastuzumab resistance mediated by MSCs via activation of Src. Figure drawn by Dr. Amita Daverey.
Mentions: To investigate if the physical intimacy of MSCs may confer resistance to trastuzumab in breast cancer cells, we assessed the migratory potential of cancer cells in co-culture when exposed to the drug. Trastuzumab has been shown to inhibit cell migration in both in vitro and in vivo studies, however the drug loses this ability in drug resistant tumors3. In order to evaluate the effect of direct contact of MSC on breast cancer cells migration, we probed the migration capacity of BT-474 and 21MT-1 in mono-cultures, exposed with MSCs-CM, and co-cultures with MSCs in the presence of trastuzumab (Fig. 5A). A scratch was made in a sub-confluent cell monolayer and cells were allowed to migrate into the cell-free area. The distance moved by the cells in control, MSC-CM treated cells, and co-cultured plates, respectively, was compared when exposed to the drug. To distinguish the cell populations, breast cancer cells and MSCs were stained with green dye, CFDSE and red dye, PKH26 respectively. In the absence of trastuzumab, the co-culture of BT-474 and 21MT-1 cells with MSCs showed significantly (p < 0.001) higher migration after 48 h of co-culture as compared to the respective monocultures. On the other hand, only 21MT-1 cells treated with MSCs-CM demonstrated significant migration as compared to monoculture (p < 0.05). When BT-474 mono-culture cells were exposed to Ttzm, there was no significant change in the migratory property in mono-culture and MSCs-CM conditions but BT-474 in co-culture showed significantly (p < 0.01) higher migration when compared to untreated co-culture samples. In contrast, when 21MT-1 mono-culture and MSC-CMs cells were exposed to Ttzm, there was no significant no signficiant inhibition in the migratory potential while 21MT-1 in co-culture showed significantly (p < 0.05) even after exposed to Ttzm. This data suggest that direct interaction of MSCs with breast cancer cells promotes migration of breast cancer cells and potentially regulates the Ttzm responsiveness in breast tumor cells when in physical contact.

Bottom Line: The development of resistance to trastuzumab is a major obstacle for lasting effective treatment of patients with ErbB2-overexpressing tumors.To our knowledge, this is the first report that showed PTEN loss without the use of chemical inhibitors, matrix stiffness, or silencing RNAs.Our findings show that MSCs are potent mediators of resistance to trastuzumab and might reveal targets to enhance trastuzumab efficacy in patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, NE, 68588.

ABSTRACT
The development of resistance to trastuzumab is a major obstacle for lasting effective treatment of patients with ErbB2-overexpressing tumors. Here, we demonstrate that the physical contact of breast cancer cells with mesenchymal stem cells (MSCs) is a potential modulator of trastuzumab response by activation of nonreceptor tyrosine kinase c-Src and down regulation of phosphatase and tensin homolog (PTEN). Using an in vitro patterned breast cancer/MSC co-culture model, we find that the presence of MSCs results in Src activation that is missing in cancer cells monoculture, transwell co-culture, and cells treated with MSCs conditioned media. Interestingly, the co-culture model also results in PTEN loss and activation of PI3K/AKT pathway that has been demonstrated as fundamental proliferative and survival pathways in clinical settings. To our knowledge, this is the first report that showed PTEN loss without the use of chemical inhibitors, matrix stiffness, or silencing RNAs. In addition, breast cancer cells in co-culture with MSCs conferred trastuzumab resistance in vitro as observed in the lack of inhibition of proliferative and migrative properties of the cancer cells. Our findings show that MSCs are potent mediators of resistance to trastuzumab and might reveal targets to enhance trastuzumab efficacy in patients.

No MeSH data available.


Related in: MedlinePlus