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Prognostic impact of CXCL16 and CXCR6 in non-small cell lung cancer: combined high CXCL16 expression in tumor stroma and cancer cells yields improved survival.

Hald SM, Kiselev Y, Al-Saad S, Richardsen E, Johannessen C, Eilertsen M, Kilvaer TK, Al-Shibli K, Andersen S, Busund LT, Bremnes RM, Donnem T - BMC Cancer (2015)

Bottom Line: In univariate analysis, ↑ stromal cell CXCL16 expression was a significant positive prognostic factor (P = 0.016).CXCR6 was expressed in cancer cells, but did not show any prognostic impact.In the multivariate analysis, combined ↑cancer, and ↑stromal cell CXCL16 expression was an independent positive prognostic factor when compared to ↓stromal and ↓cancer cell expression (HR: 0.42; 95 % CI: 0.20-0.88; P = 0.022).

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Medicine, UiT The Arctic University of Norway, 9037, Tromso, Norway. sigurd.hald@uit.no.

ABSTRACT

Background: The chemokine CXCL16 and its receptor CXCR6 are expressed by a variety of immune cells and have been shown to influence angiogenesis. The expression of CXCR6 and CXCL16 has been examined in numerous human cancers; however no studies have yet investigated their influence on prognosis in non-small cell lung cancer (NSCLC). We aimed to explore their prognostic significance in NSCLC, in addition to examining associations with previously investigated markers.

Methods: Resected tumor tissue from 335 consecutive unselected stage I-IIIA NSCLC patients (1990-2005) were collected. Immunohistochemistry was used to evaluate the expression of CXCR6 and CXCL16 on tissue microarrays. In vitro, NSCLC cells (NCI-H460, A549 cells) were transfected with CXCL16 siRNA to examine effects on proliferation.

Results: In univariate analysis, ↑ stromal cell CXCL16 expression was a significant positive prognostic factor (P = 0.016). CXCR6 was expressed in cancer cells, but did not show any prognostic impact. In the multivariate analysis, combined ↑cancer, and ↑stromal cell CXCL16 expression was an independent positive prognostic factor when compared to ↓stromal and ↓cancer cell expression (HR: 0.42; 95 % CI: 0.20-0.88; P = 0.022). Knockdown of CXCL16 by siRNA resulted in accelerated proliferation of NSCLC cell lines.

Conclusion: We have shown that combined ↑cancer and ↑stromal cell CXCL16 expression is an independent positive prognostic factor in NSCLC. Further studies are warranted to elucidate the biological mechanism underlying this finding.

No MeSH data available.


Related in: MedlinePlus

Knockdown of CXCL16 with siRNA caused activation of proliferation compared to the negative scrambled control in NSCLC cell lines A549 and NCI-H460. Cells were trypsinized briefly until detached, resuspended in complete growth media and counted. According to the manufacturer’s instructions, cells were seeded in duplicates into E-plates 16 after baseline measurement. Plates were incubated for 1 hour at room temperature, and then placed into the RTCA DP instrument located in an incubator preserving same temperature and CO2 concentration as were used for routine cultivation of the cells. siRNA transfection mix was added to the cells 6 hours after seeding, and left there for 4 hours. Subsequently, the transfection mix was replaced with regular growth media. Cell index (arbitrary unit reflecting the cell-sensor impedance) was measured every 15 minutes during the first 4 hours for better resolution at attachment and spreading phase. Further measurements were taken every 30 minutes. Doubling times were calculated with RTCA software 1.2 (Roche). ± S.D of 4 technical replicates are shown. * P <0.001
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Fig4: Knockdown of CXCL16 with siRNA caused activation of proliferation compared to the negative scrambled control in NSCLC cell lines A549 and NCI-H460. Cells were trypsinized briefly until detached, resuspended in complete growth media and counted. According to the manufacturer’s instructions, cells were seeded in duplicates into E-plates 16 after baseline measurement. Plates were incubated for 1 hour at room temperature, and then placed into the RTCA DP instrument located in an incubator preserving same temperature and CO2 concentration as were used for routine cultivation of the cells. siRNA transfection mix was added to the cells 6 hours after seeding, and left there for 4 hours. Subsequently, the transfection mix was replaced with regular growth media. Cell index (arbitrary unit reflecting the cell-sensor impedance) was measured every 15 minutes during the first 4 hours for better resolution at attachment and spreading phase. Further measurements were taken every 30 minutes. Doubling times were calculated with RTCA software 1.2 (Roche). ± S.D of 4 technical replicates are shown. * P <0.001

Mentions: The increased survival seen in patients with combined high CXCL16 expression in cancer and stromal cells led us to investigate the effect of CXCL16 on cell proliferation. We employed the xCELLigence platform (Roche) which facilitates studying of cell proliferation in real time. This is a micro electric assay based on changing impedance of bottom electrodes in presence of the cells. Attachment and initial spreading of the cells typically took 3–6 hours, after which cells were transfected with siRNAs. We repeatedly observed that knockdown of CXCL16 with siRNA caused activation of proliferation compared to the negative scrambled control (P < 0.001, Fig. 4). This was evident both from the growth curves and from the doubling time calculations. The same effect was observed in two different NSCLC cell lines: A549 and NCI-H460.Fig. 4


Prognostic impact of CXCL16 and CXCR6 in non-small cell lung cancer: combined high CXCL16 expression in tumor stroma and cancer cells yields improved survival.

Hald SM, Kiselev Y, Al-Saad S, Richardsen E, Johannessen C, Eilertsen M, Kilvaer TK, Al-Shibli K, Andersen S, Busund LT, Bremnes RM, Donnem T - BMC Cancer (2015)

Knockdown of CXCL16 with siRNA caused activation of proliferation compared to the negative scrambled control in NSCLC cell lines A549 and NCI-H460. Cells were trypsinized briefly until detached, resuspended in complete growth media and counted. According to the manufacturer’s instructions, cells were seeded in duplicates into E-plates 16 after baseline measurement. Plates were incubated for 1 hour at room temperature, and then placed into the RTCA DP instrument located in an incubator preserving same temperature and CO2 concentration as were used for routine cultivation of the cells. siRNA transfection mix was added to the cells 6 hours after seeding, and left there for 4 hours. Subsequently, the transfection mix was replaced with regular growth media. Cell index (arbitrary unit reflecting the cell-sensor impedance) was measured every 15 minutes during the first 4 hours for better resolution at attachment and spreading phase. Further measurements were taken every 30 minutes. Doubling times were calculated with RTCA software 1.2 (Roche). ± S.D of 4 technical replicates are shown. * P <0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4447015&req=5

Fig4: Knockdown of CXCL16 with siRNA caused activation of proliferation compared to the negative scrambled control in NSCLC cell lines A549 and NCI-H460. Cells were trypsinized briefly until detached, resuspended in complete growth media and counted. According to the manufacturer’s instructions, cells were seeded in duplicates into E-plates 16 after baseline measurement. Plates were incubated for 1 hour at room temperature, and then placed into the RTCA DP instrument located in an incubator preserving same temperature and CO2 concentration as were used for routine cultivation of the cells. siRNA transfection mix was added to the cells 6 hours after seeding, and left there for 4 hours. Subsequently, the transfection mix was replaced with regular growth media. Cell index (arbitrary unit reflecting the cell-sensor impedance) was measured every 15 minutes during the first 4 hours for better resolution at attachment and spreading phase. Further measurements were taken every 30 minutes. Doubling times were calculated with RTCA software 1.2 (Roche). ± S.D of 4 technical replicates are shown. * P <0.001
Mentions: The increased survival seen in patients with combined high CXCL16 expression in cancer and stromal cells led us to investigate the effect of CXCL16 on cell proliferation. We employed the xCELLigence platform (Roche) which facilitates studying of cell proliferation in real time. This is a micro electric assay based on changing impedance of bottom electrodes in presence of the cells. Attachment and initial spreading of the cells typically took 3–6 hours, after which cells were transfected with siRNAs. We repeatedly observed that knockdown of CXCL16 with siRNA caused activation of proliferation compared to the negative scrambled control (P < 0.001, Fig. 4). This was evident both from the growth curves and from the doubling time calculations. The same effect was observed in two different NSCLC cell lines: A549 and NCI-H460.Fig. 4

Bottom Line: In univariate analysis, ↑ stromal cell CXCL16 expression was a significant positive prognostic factor (P = 0.016).CXCR6 was expressed in cancer cells, but did not show any prognostic impact.In the multivariate analysis, combined ↑cancer, and ↑stromal cell CXCL16 expression was an independent positive prognostic factor when compared to ↓stromal and ↓cancer cell expression (HR: 0.42; 95 % CI: 0.20-0.88; P = 0.022).

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Medicine, UiT The Arctic University of Norway, 9037, Tromso, Norway. sigurd.hald@uit.no.

ABSTRACT

Background: The chemokine CXCL16 and its receptor CXCR6 are expressed by a variety of immune cells and have been shown to influence angiogenesis. The expression of CXCR6 and CXCL16 has been examined in numerous human cancers; however no studies have yet investigated their influence on prognosis in non-small cell lung cancer (NSCLC). We aimed to explore their prognostic significance in NSCLC, in addition to examining associations with previously investigated markers.

Methods: Resected tumor tissue from 335 consecutive unselected stage I-IIIA NSCLC patients (1990-2005) were collected. Immunohistochemistry was used to evaluate the expression of CXCR6 and CXCL16 on tissue microarrays. In vitro, NSCLC cells (NCI-H460, A549 cells) were transfected with CXCL16 siRNA to examine effects on proliferation.

Results: In univariate analysis, ↑ stromal cell CXCL16 expression was a significant positive prognostic factor (P = 0.016). CXCR6 was expressed in cancer cells, but did not show any prognostic impact. In the multivariate analysis, combined ↑cancer, and ↑stromal cell CXCL16 expression was an independent positive prognostic factor when compared to ↓stromal and ↓cancer cell expression (HR: 0.42; 95 % CI: 0.20-0.88; P = 0.022). Knockdown of CXCL16 by siRNA resulted in accelerated proliferation of NSCLC cell lines.

Conclusion: We have shown that combined ↑cancer and ↑stromal cell CXCL16 expression is an independent positive prognostic factor in NSCLC. Further studies are warranted to elucidate the biological mechanism underlying this finding.

No MeSH data available.


Related in: MedlinePlus