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Tumour microenvironment of both early- and late-stage colorectal cancer is equally immunosuppressive.

O'Toole A, Michielsen AJ, Nolan B, Tosetto M, Sheahan K, Mulcahy HE, Winter DC, Hyland JM, O'Connell PR, Fennelly D, O'Donoghue D, O'Sullivan J, Doherty GA, Ryan EJ - Br. J. Cancer (2014)

Bottom Line: The profile of inflammatory mediators in TCM was similar across stages, and all increased pSTAT3 expression by DCs.CRC patient DCs (n=31) secreted low levels of IL-12p70 and failed to upregulate expression of maturation markers in response to LPS.Furthermore, in vitro culture of autologous DCs with TCM did not change the hypo-responsiveness of patient DCs.Measures to reverse the negative influence of the TME on DCs will optimise cancer vaccines in both early- and late-stage CRC.

View Article: PubMed Central - PubMed

Affiliation: 1] Centre for Colorectal Disease, St Vincent's University Hospital, Dublin 4, Ireland [2] School of Medicine and Medical Sciences, University College Dublin, Dublin 4, Ireland.

ABSTRACT

Background: Tumour microenvironment (TME) of advanced colorectal cancer (CRC) suppresses dendritic cell (DC) maturation. Here, our aim was to determine how the microenvironment of early-stage tumours influences DCs.

Methods: Tumour-conditioned media (TCM) was generated by culturing explant tumour tissue in vitro (n=50). Monocyte-derived DCs (MDDCs) of healthy donors or cancer patients were pretreated with TCM and stimulated with lipopolysaccharide (LPS). DC maturation was assessed by flow cytometry and cytokine production measured by ELISA.

Results: TCM from both early- and late-staged tumours abrogated LPS-induction of IL-12p70 secretion, while increasing IL-10. The profile of inflammatory mediators in TCM was similar across stages, and all increased pSTAT3 expression by DCs.CRC patient DCs (n=31) secreted low levels of IL-12p70 and failed to upregulate expression of maturation markers in response to LPS. Furthermore, in vitro culture of autologous DCs with TCM did not change the hypo-responsiveness of patient DCs.

Conclusion: Our data demonstrates that the TME of all stages of CRC contains inflammatory mediators capable of suppressing local DCs. MDDCs obtained from CRC patients are hyporesponsive to stimuli such as LPS. Measures to reverse the negative influence of the TME on DCs will optimise cancer vaccines in both early- and late-stage CRC.

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TME of all colorectal cancer stages suppresses DC maturation. Immature monocyte-derived DCs were treated for 4 h with tumour-conditioned media (TCM). LPS (1 μg ml−1) was added and the cells cultured for a further 18 h. Dendritic cells were cultured in media alone as a negative control and LPS stimulated dendritic cells with no TCM pretreatment were included as a positive control. (A) Supernatants were collected and the levels of IL-12p70 and IL-10 were measured by ELISA. Error bars presented are s.e.m. Statistical differences were determined by ANOVA. *P<0.05, **P<0.01 and ***P<0.001. (B) Expression of DC maturation markers were assessed by flow cytometry. Percentage inhibition was calculated relative to cells stimulated with LPS alone. Representative flow cytometry histograms from one experiment showing CD54 expression are shown. Statistical significance was calculated using the Wilcoxon signed-rank test. *P<0.05.
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fig1: TME of all colorectal cancer stages suppresses DC maturation. Immature monocyte-derived DCs were treated for 4 h with tumour-conditioned media (TCM). LPS (1 μg ml−1) was added and the cells cultured for a further 18 h. Dendritic cells were cultured in media alone as a negative control and LPS stimulated dendritic cells with no TCM pretreatment were included as a positive control. (A) Supernatants were collected and the levels of IL-12p70 and IL-10 were measured by ELISA. Error bars presented are s.e.m. Statistical differences were determined by ANOVA. *P<0.05, **P<0.01 and ***P<0.001. (B) Expression of DC maturation markers were assessed by flow cytometry. Percentage inhibition was calculated relative to cells stimulated with LPS alone. Representative flow cytometry histograms from one experiment showing CD54 expression are shown. Statistical significance was calculated using the Wilcoxon signed-rank test. *P<0.05.

Mentions: Tumour-conditioned media (TCM) was generated from CRC tumours with different stages of disease progression (stage I, n=8; stage II, n=21; stage III, n=14, stage IV, n=7). Tumours were assigned as stage I–IV according to the American Joint Committee on Cancer (AJCC) classification system (Edge and Compton, 2010). We found that TCM from all stages of CRC significantly suppressed IL-12p70, whereas levels of IL-10 were increased in response to LPS (Figure 1A). TCM pretreatment of MDDCs also suppressed the upregulation of maturation markers in response to LPS (Figure 1B and C). Immature MDDCs were cultured with TCM without the addition of LPS as a control, however this had no effect on cytokine secretion or maturation marker expression when compared with immature DCs cultured in media alone (data not shown). These data clearly illustrate that the microenvironment of both early and advanced colorectal cancer has the potential to potently suppress cell-mediated immunity.


Tumour microenvironment of both early- and late-stage colorectal cancer is equally immunosuppressive.

O'Toole A, Michielsen AJ, Nolan B, Tosetto M, Sheahan K, Mulcahy HE, Winter DC, Hyland JM, O'Connell PR, Fennelly D, O'Donoghue D, O'Sullivan J, Doherty GA, Ryan EJ - Br. J. Cancer (2014)

TME of all colorectal cancer stages suppresses DC maturation. Immature monocyte-derived DCs were treated for 4 h with tumour-conditioned media (TCM). LPS (1 μg ml−1) was added and the cells cultured for a further 18 h. Dendritic cells were cultured in media alone as a negative control and LPS stimulated dendritic cells with no TCM pretreatment were included as a positive control. (A) Supernatants were collected and the levels of IL-12p70 and IL-10 were measured by ELISA. Error bars presented are s.e.m. Statistical differences were determined by ANOVA. *P<0.05, **P<0.01 and ***P<0.001. (B) Expression of DC maturation markers were assessed by flow cytometry. Percentage inhibition was calculated relative to cells stimulated with LPS alone. Representative flow cytometry histograms from one experiment showing CD54 expression are shown. Statistical significance was calculated using the Wilcoxon signed-rank test. *P<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150274&req=5

fig1: TME of all colorectal cancer stages suppresses DC maturation. Immature monocyte-derived DCs were treated for 4 h with tumour-conditioned media (TCM). LPS (1 μg ml−1) was added and the cells cultured for a further 18 h. Dendritic cells were cultured in media alone as a negative control and LPS stimulated dendritic cells with no TCM pretreatment were included as a positive control. (A) Supernatants were collected and the levels of IL-12p70 and IL-10 were measured by ELISA. Error bars presented are s.e.m. Statistical differences were determined by ANOVA. *P<0.05, **P<0.01 and ***P<0.001. (B) Expression of DC maturation markers were assessed by flow cytometry. Percentage inhibition was calculated relative to cells stimulated with LPS alone. Representative flow cytometry histograms from one experiment showing CD54 expression are shown. Statistical significance was calculated using the Wilcoxon signed-rank test. *P<0.05.
Mentions: Tumour-conditioned media (TCM) was generated from CRC tumours with different stages of disease progression (stage I, n=8; stage II, n=21; stage III, n=14, stage IV, n=7). Tumours were assigned as stage I–IV according to the American Joint Committee on Cancer (AJCC) classification system (Edge and Compton, 2010). We found that TCM from all stages of CRC significantly suppressed IL-12p70, whereas levels of IL-10 were increased in response to LPS (Figure 1A). TCM pretreatment of MDDCs also suppressed the upregulation of maturation markers in response to LPS (Figure 1B and C). Immature MDDCs were cultured with TCM without the addition of LPS as a control, however this had no effect on cytokine secretion or maturation marker expression when compared with immature DCs cultured in media alone (data not shown). These data clearly illustrate that the microenvironment of both early and advanced colorectal cancer has the potential to potently suppress cell-mediated immunity.

Bottom Line: The profile of inflammatory mediators in TCM was similar across stages, and all increased pSTAT3 expression by DCs.CRC patient DCs (n=31) secreted low levels of IL-12p70 and failed to upregulate expression of maturation markers in response to LPS.Furthermore, in vitro culture of autologous DCs with TCM did not change the hypo-responsiveness of patient DCs.Measures to reverse the negative influence of the TME on DCs will optimise cancer vaccines in both early- and late-stage CRC.

View Article: PubMed Central - PubMed

Affiliation: 1] Centre for Colorectal Disease, St Vincent's University Hospital, Dublin 4, Ireland [2] School of Medicine and Medical Sciences, University College Dublin, Dublin 4, Ireland.

ABSTRACT

Background: Tumour microenvironment (TME) of advanced colorectal cancer (CRC) suppresses dendritic cell (DC) maturation. Here, our aim was to determine how the microenvironment of early-stage tumours influences DCs.

Methods: Tumour-conditioned media (TCM) was generated by culturing explant tumour tissue in vitro (n=50). Monocyte-derived DCs (MDDCs) of healthy donors or cancer patients were pretreated with TCM and stimulated with lipopolysaccharide (LPS). DC maturation was assessed by flow cytometry and cytokine production measured by ELISA.

Results: TCM from both early- and late-staged tumours abrogated LPS-induction of IL-12p70 secretion, while increasing IL-10. The profile of inflammatory mediators in TCM was similar across stages, and all increased pSTAT3 expression by DCs.CRC patient DCs (n=31) secreted low levels of IL-12p70 and failed to upregulate expression of maturation markers in response to LPS. Furthermore, in vitro culture of autologous DCs with TCM did not change the hypo-responsiveness of patient DCs.

Conclusion: Our data demonstrates that the TME of all stages of CRC contains inflammatory mediators capable of suppressing local DCs. MDDCs obtained from CRC patients are hyporesponsive to stimuli such as LPS. Measures to reverse the negative influence of the TME on DCs will optimise cancer vaccines in both early- and late-stage CRC.

Show MeSH
Related in: MedlinePlus