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Adipose cells promote resistance of breast cancer cells to trastuzumab-mediated antibody-dependent cellular cytotoxicity.

Duong MN, Cleret A, Matera EL, Chettab K, Mathé D, Valsesia-Wittmann S, Clémenceau B, Dumontet C - Breast Cancer Res. (2015)

Bottom Line: The results were validated in vivo in a mouse xenograft model.Using a transcriptomic approach, we found that cancer cells undergo major modifications when exposed to adipocyte-conditioned medium.Collectively, our findings underline the importance of adipose tissue in the resistance to trastuzumab and suggest that approaches targeting the adipocyte-cancer cell crosstalk may help sensitize cancer cells to trastuzumab-based therapy.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM UMR 1052, CNRS 5286, 8 Avenue Rockefeller, 69008, Lyon, France. mn.duong@hotmail.com.

ABSTRACT

Introduction: Trastuzumab has been used in the treatment of human epidermal growth factor receptor 2 (HER2)-expressing breast cancer, but its efficacy is limited by de novo or acquired resistance. Although many mechanisms have been proposed to explain resistance to trastuzumab, little is known concerning the role of the tumor microenvironment. Given the importance of antibody-dependent cellular cytotoxicity (ADCC) in the antitumor effect of trastuzumab and the abundance of adipose tissue in the breast, we investigated the impact of adipocytes on ADCC.

Methods: We set up a coculture system to study the effect of adipocytes on ADCC in vitro. The results were validated in vivo in a mouse xenograft model.

Results: We found that adipocytes, as well as preadipocytes, inhibited trastuzumab-mediated ADCC in HER2-expressing breast cancer cells via the secretion of soluble factors. The inhibition of ADCC was not due to titration or degradation of the antibody. We found that adipose cells decreased the secretion of interferon-γ by natural killer cells, but did not alter natural killer cells' cytotoxicity. Preincubation of breast cancer cells with the conditioned medium derived from adipocytes reduced the sensitivity of cancer cells to ADCC. Using a transcriptomic approach, we found that cancer cells undergo major modifications when exposed to adipocyte-conditioned medium. Importantly, breast tumors grafted next to lipomas displayed resistance to trastuzumab in mouse xenograft models.

Conclusions: Collectively, our findings underline the importance of adipose tissue in the resistance to trastuzumab and suggest that approaches targeting the adipocyte-cancer cell crosstalk may help sensitize cancer cells to trastuzumab-based therapy.

No MeSH data available.


Related in: MedlinePlus

#hMADS-CM and hMADS-CM do not alter natural killer cell cytotoxicity. (A) Expression of markers and receptors on NK-92-CD16 cells from antibody-dependent cellular cytotoxicity (ADCC) assays in the presence of conditioned media of differentiated human multipotent adipose-derived stem cells (#hMADS-CM) or human multipotent adipose-derived stem cells (hMADS-CM) or in the control medium. Dotted red lines indicate unstained NK-92-CD16 cells, dotted green lines indicate control medium, solid coral lines indicate #hMADS-CM and dashed blue line indicate hMADS-CM conditions, respectively. (B) Enzyme-linked immunosorbent assay of the supernatants from NK-92-CD16 cells after ADCC assay. IFN, Interferon. (C) ADCC assays using NK-92-CD16 cells preincubated overnight with #hMADS-CM or hMADS-CM or the control media. (D) Cytotoxicity assay using K-562 cells as target cells at an effector to target ratio of 5:1 in the presence of #hMADS-CM, hMADS-CM or their control media. Results representative of three independent experiments are shown in (A). Mean ± SD values of three independent experiments, each performed in duplicate (B) or triplicate (C and D), are shown. *P < 0.05; **P < 0.01; ns, Not significant.
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Fig4: #hMADS-CM and hMADS-CM do not alter natural killer cell cytotoxicity. (A) Expression of markers and receptors on NK-92-CD16 cells from antibody-dependent cellular cytotoxicity (ADCC) assays in the presence of conditioned media of differentiated human multipotent adipose-derived stem cells (#hMADS-CM) or human multipotent adipose-derived stem cells (hMADS-CM) or in the control medium. Dotted red lines indicate unstained NK-92-CD16 cells, dotted green lines indicate control medium, solid coral lines indicate #hMADS-CM and dashed blue line indicate hMADS-CM conditions, respectively. (B) Enzyme-linked immunosorbent assay of the supernatants from NK-92-CD16 cells after ADCC assay. IFN, Interferon. (C) ADCC assays using NK-92-CD16 cells preincubated overnight with #hMADS-CM or hMADS-CM or the control media. (D) Cytotoxicity assay using K-562 cells as target cells at an effector to target ratio of 5:1 in the presence of #hMADS-CM, hMADS-CM or their control media. Results representative of three independent experiments are shown in (A). Mean ± SD values of three independent experiments, each performed in duplicate (B) or triplicate (C and D), are shown. *P < 0.05; **P < 0.01; ns, Not significant.

Mentions: To investigate whether hMADS- and #hMADS-secreted factors alter NK cell phenotype or functions, we analyzed NK cell markers at the end of the 4-hour ADCC assay. As shown in Figure 4A, no modification of CD16 or CD107a expression levels was observed in the presence of hMADS-CM or #hMADS-CM. Additionally, the expression levels of the activation markers CD25 and CD69—as well as of the other NK receptors, NKG2D, NKp30, NKp44 and NKG2A—were unchanged (Figure 4A). Interestingly, the presence of adipocyte-conditioned media decreased the secretion of interferon (IFN)-γ by NK cells as compared with the control media (Figure 4B). However, when NK-92-CD16 cells were preincubated overnight with hMADS-CM or #hMADS-CM, we did not observe any modification of NK cell viability or of NK cells’ ability to exert their cytotoxicity in trastuzumab-mediated ADCC (Figure 4C and Additional file 7: Figure S5). Furthermore, the adipocyte-conditioned media did not inhibit the spontaneous lysis of K-562 cells by NK-92 cells (Figure 4D). Overall, these data suggest that NK-92-CD16 cells may be altered by hMADS-CM or #hMADS-CM for their secretion of cytokines, but not for their cytotoxicity.Figure 4


Adipose cells promote resistance of breast cancer cells to trastuzumab-mediated antibody-dependent cellular cytotoxicity.

Duong MN, Cleret A, Matera EL, Chettab K, Mathé D, Valsesia-Wittmann S, Clémenceau B, Dumontet C - Breast Cancer Res. (2015)

#hMADS-CM and hMADS-CM do not alter natural killer cell cytotoxicity. (A) Expression of markers and receptors on NK-92-CD16 cells from antibody-dependent cellular cytotoxicity (ADCC) assays in the presence of conditioned media of differentiated human multipotent adipose-derived stem cells (#hMADS-CM) or human multipotent adipose-derived stem cells (hMADS-CM) or in the control medium. Dotted red lines indicate unstained NK-92-CD16 cells, dotted green lines indicate control medium, solid coral lines indicate #hMADS-CM and dashed blue line indicate hMADS-CM conditions, respectively. (B) Enzyme-linked immunosorbent assay of the supernatants from NK-92-CD16 cells after ADCC assay. IFN, Interferon. (C) ADCC assays using NK-92-CD16 cells preincubated overnight with #hMADS-CM or hMADS-CM or the control media. (D) Cytotoxicity assay using K-562 cells as target cells at an effector to target ratio of 5:1 in the presence of #hMADS-CM, hMADS-CM or their control media. Results representative of three independent experiments are shown in (A). Mean ± SD values of three independent experiments, each performed in duplicate (B) or triplicate (C and D), are shown. *P < 0.05; **P < 0.01; ns, Not significant.
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Fig4: #hMADS-CM and hMADS-CM do not alter natural killer cell cytotoxicity. (A) Expression of markers and receptors on NK-92-CD16 cells from antibody-dependent cellular cytotoxicity (ADCC) assays in the presence of conditioned media of differentiated human multipotent adipose-derived stem cells (#hMADS-CM) or human multipotent adipose-derived stem cells (hMADS-CM) or in the control medium. Dotted red lines indicate unstained NK-92-CD16 cells, dotted green lines indicate control medium, solid coral lines indicate #hMADS-CM and dashed blue line indicate hMADS-CM conditions, respectively. (B) Enzyme-linked immunosorbent assay of the supernatants from NK-92-CD16 cells after ADCC assay. IFN, Interferon. (C) ADCC assays using NK-92-CD16 cells preincubated overnight with #hMADS-CM or hMADS-CM or the control media. (D) Cytotoxicity assay using K-562 cells as target cells at an effector to target ratio of 5:1 in the presence of #hMADS-CM, hMADS-CM or their control media. Results representative of three independent experiments are shown in (A). Mean ± SD values of three independent experiments, each performed in duplicate (B) or triplicate (C and D), are shown. *P < 0.05; **P < 0.01; ns, Not significant.
Mentions: To investigate whether hMADS- and #hMADS-secreted factors alter NK cell phenotype or functions, we analyzed NK cell markers at the end of the 4-hour ADCC assay. As shown in Figure 4A, no modification of CD16 or CD107a expression levels was observed in the presence of hMADS-CM or #hMADS-CM. Additionally, the expression levels of the activation markers CD25 and CD69—as well as of the other NK receptors, NKG2D, NKp30, NKp44 and NKG2A—were unchanged (Figure 4A). Interestingly, the presence of adipocyte-conditioned media decreased the secretion of interferon (IFN)-γ by NK cells as compared with the control media (Figure 4B). However, when NK-92-CD16 cells were preincubated overnight with hMADS-CM or #hMADS-CM, we did not observe any modification of NK cell viability or of NK cells’ ability to exert their cytotoxicity in trastuzumab-mediated ADCC (Figure 4C and Additional file 7: Figure S5). Furthermore, the adipocyte-conditioned media did not inhibit the spontaneous lysis of K-562 cells by NK-92 cells (Figure 4D). Overall, these data suggest that NK-92-CD16 cells may be altered by hMADS-CM or #hMADS-CM for their secretion of cytokines, but not for their cytotoxicity.Figure 4

Bottom Line: The results were validated in vivo in a mouse xenograft model.Using a transcriptomic approach, we found that cancer cells undergo major modifications when exposed to adipocyte-conditioned medium.Collectively, our findings underline the importance of adipose tissue in the resistance to trastuzumab and suggest that approaches targeting the adipocyte-cancer cell crosstalk may help sensitize cancer cells to trastuzumab-based therapy.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM UMR 1052, CNRS 5286, 8 Avenue Rockefeller, 69008, Lyon, France. mn.duong@hotmail.com.

ABSTRACT

Introduction: Trastuzumab has been used in the treatment of human epidermal growth factor receptor 2 (HER2)-expressing breast cancer, but its efficacy is limited by de novo or acquired resistance. Although many mechanisms have been proposed to explain resistance to trastuzumab, little is known concerning the role of the tumor microenvironment. Given the importance of antibody-dependent cellular cytotoxicity (ADCC) in the antitumor effect of trastuzumab and the abundance of adipose tissue in the breast, we investigated the impact of adipocytes on ADCC.

Methods: We set up a coculture system to study the effect of adipocytes on ADCC in vitro. The results were validated in vivo in a mouse xenograft model.

Results: We found that adipocytes, as well as preadipocytes, inhibited trastuzumab-mediated ADCC in HER2-expressing breast cancer cells via the secretion of soluble factors. The inhibition of ADCC was not due to titration or degradation of the antibody. We found that adipose cells decreased the secretion of interferon-γ by natural killer cells, but did not alter natural killer cells' cytotoxicity. Preincubation of breast cancer cells with the conditioned medium derived from adipocytes reduced the sensitivity of cancer cells to ADCC. Using a transcriptomic approach, we found that cancer cells undergo major modifications when exposed to adipocyte-conditioned medium. Importantly, breast tumors grafted next to lipomas displayed resistance to trastuzumab in mouse xenograft models.

Conclusions: Collectively, our findings underline the importance of adipose tissue in the resistance to trastuzumab and suggest that approaches targeting the adipocyte-cancer cell crosstalk may help sensitize cancer cells to trastuzumab-based therapy.

No MeSH data available.


Related in: MedlinePlus