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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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MDDCs obtained from patients with early-staged CRC respond poorly to LPS, secreting low levels of IL-12p70. Peripheral blood monocytes were obtained from colorectal cancer patients before surgical removal of their tumour. Tumour explants of these patients were also obtained, cultured in vitro for 72 h and TCM collected. (stage I, n=1, stage II, n=11, stage III, n=1). MDDCs were pretreated for 4 h with TCM and then LPS (1 μg ml−1) added and cells cultured for a further 18 h. Levels of IL-12p70 and IL-10 in supernatants were determined by ELISA (A). Expression of maturation markers (CD54, CD80 and CD86) were measured by flow cytometry (B). Statistical differences were determined using Wilcoxon signed-rank test. *P<0.05, **P<0.01.
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fig3: MDDCs obtained from patients with early-staged CRC respond poorly to LPS, secreting low levels of IL-12p70. Peripheral blood monocytes were obtained from colorectal cancer patients before surgical removal of their tumour. Tumour explants of these patients were also obtained, cultured in vitro for 72 h and TCM collected. (stage I, n=1, stage II, n=11, stage III, n=1). MDDCs were pretreated for 4 h with TCM and then LPS (1 μg ml−1) added and cells cultured for a further 18 h. Levels of IL-12p70 and IL-10 in supernatants were determined by ELISA (A). Expression of maturation markers (CD54, CD80 and CD86) were measured by flow cytometry (B). Statistical differences were determined using Wilcoxon signed-rank test. *P<0.05, **P<0.01.

Mentions: Next, to determine if the local microenvironment of the tumour could modulate the function of autologous DCs, we cultured MDDC from presurgery blood samples in the presence of TCM generated from the patient's own tumour (n=13, stage I, n=1, stage II, n=11, stage III, n=1). Median age of the cohort was 60.8 years (range, 50–84). Nine patients were male. Further clinical details of this cohort are presented in Table 2. TCM used in these experiments suppressed the responsiveness of healthy control MDDCs to LPS as we have previously observed (Figure 3). However, as the response of the patient MDDCs to LPS was poor, TCM pretreatment of patient DC did not have an additional inhibitory effect. Of particular note, MDDCs obtained from this cohort of patients with early-staged CRC secreted very low levels of IL-12p70 in response to LPS. Although pretreatment of patient or control MDDCs with TCM increased IL-10 production in response to LPS, this did not reach statistical significance in these experiments.


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)

MDDCs obtained from patients with early-staged CRC respond poorly to LPS, secreting low levels of IL-12p70. Peripheral blood monocytes were obtained from colorectal cancer patients before surgical removal of their tumour. Tumour explants of these patients were also obtained, cultured in vitro for 72 h and TCM collected. (stage I, n=1, stage II, n=11, stage III, n=1). MDDCs were pretreated for 4 h with TCM and then LPS (1 μg ml−1) added and cells cultured for a further 18 h. Levels of IL-12p70 and IL-10 in supernatants were determined by ELISA (A). Expression of maturation markers (CD54, CD80 and CD86) were measured by flow cytometry (B). Statistical differences were determined using Wilcoxon signed-rank test. *P<0.05, **P<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: MDDCs obtained from patients with early-staged CRC respond poorly to LPS, secreting low levels of IL-12p70. Peripheral blood monocytes were obtained from colorectal cancer patients before surgical removal of their tumour. Tumour explants of these patients were also obtained, cultured in vitro for 72 h and TCM collected. (stage I, n=1, stage II, n=11, stage III, n=1). MDDCs were pretreated for 4 h with TCM and then LPS (1 μg ml−1) added and cells cultured for a further 18 h. Levels of IL-12p70 and IL-10 in supernatants were determined by ELISA (A). Expression of maturation markers (CD54, CD80 and CD86) were measured by flow cytometry (B). Statistical differences were determined using Wilcoxon signed-rank test. *P<0.05, **P<0.01.
Mentions: Next, to determine if the local microenvironment of the tumour could modulate the function of autologous DCs, we cultured MDDC from presurgery blood samples in the presence of TCM generated from the patient's own tumour (n=13, stage I, n=1, stage II, n=11, stage III, n=1). Median age of the cohort was 60.8 years (range, 50–84). Nine patients were male. Further clinical details of this cohort are presented in Table 2. TCM used in these experiments suppressed the responsiveness of healthy control MDDCs to LPS as we have previously observed (Figure 3). However, as the response of the patient MDDCs to LPS was poor, TCM pretreatment of patient DC did not have an additional inhibitory effect. Of particular note, MDDCs obtained from this cohort of patients with early-staged CRC secreted very low levels of IL-12p70 in response to LPS. Although pretreatment of patient or control MDDCs with TCM increased IL-10 production in response to LPS, this did not reach statistical significance in these experiments.

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