Abstract
Calreticulin (CRT) is an endoplasmic reticulum luminal Ca2+-binding chaperone protein. By immunizing mice with recombinant fragment (rCRT/39-272), six clones of monoclonal antibodies (mAbs) were generated and characterized. Based on these mAbs, a microplate chemiluminescent enzyme immunoassay (CLEIA) system with a measured limit of detection of 0.09 ng/ml was developed. Using this CLEIA system, it was found that soluble CRT (sCRT) level in serum samples from 58 lung cancer patients was significantly higher than that from 40 healthy individuals (only 9 were detectable, P < 0.0001). Among them, serum sCRT in the small cell lung cancer was lower than that in adenocarcinoma (P = 0.0085), while both were lower than that in the squamous cell carcinoma (P = 0.013, P = 0.0012, respectively). Moreover, it was found that sCRT in sera from the patients after chemotherapy was higher than that from the patients without chemotherapy (P = 0.042). Further study by immunohistochemistry showed that CRT was also highly expressed in the cytoplasm and on the membrane of the lung cancer cells, while there was a trace amount of CRT expression in normal lung cells. Correspondingly, the expression level of CRT on lung cancer cell membrane was associated with the tumor pathological grade. This study demonstrates that sCRT concentration in sera of lung cancer patients is higher than that in sera of healthy individuals, and CRT expression level on lung cancer cell membrane is associated with tumor pathological classification and grade. These findings suggest that CRT may be used as a biomarker in lung cancer prediction and diagnosis.
Similar content being viewed by others
Abbreviations
- CRT:
-
Calreticulin
- CLEIA:
-
Chemiluminescent enzyme immunoassay
- IHC:
-
Immunohistochemistry
References
Jemal A, Center MM, DeSantis C, Ward EM (2010) Global patterns of cancer incidence and mortality rates and trends. Cancer Epidemiol Biomarkers Prev 19(8):1893–1907
Molina R, Auge JM, Bosch X, Escudero JM, Vinolas N, Marrades R, Ramirez J, Carcereny E, Filella X (2009) Usefulness of serum tumor markers, including progastrin-releasing peptide, in patients with lung cancer: correlation with histology. Tumour Biol 30(3):121–129
Ramalingam SS, Owonikoko TK, Khuri FR (2011) Lung cancer: new biological insights and recent therapeutic advances. CA Cancer J Clin 61(2):91–112
Johnson S, Michalak M, Opas M, Eggleton P (2001) The ins and outs of calreticulin: from the ER lumen to the extracellular space. Trends Cell Biol 11(3):122–129
Gardai SJ, McPhillips KA, Frasch SC, Janssen WJ, Starefeldt A, Murphy-Ullrich JE, Bratton DL, Oldenborg PA, Michalak M, Henson PM (2005) Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell 123(2):321–334
Tongu M, Harashima N, Yamada T, Harada T, Harada M (2010) Immunogenic chemotherapy with cyclophosphamide and doxorubicin against established murine carcinoma. Cancer Immunol Immunother 59(5):769–777
Perez CA, Fu A, Onishko H, Hallahan DE, Geng L (2009) Radiation induces an antitumour immune response to mouse melanoma. Int J Radiat Biol 85(12):1126–1136
Obeid M, Panaretakis T, Joza N, Tufi R, Tesniere A, van Endert P, Zitvogel L, Kroemer G (2007) Calreticulin exposure is required for the immunogenicity of gamma-irradiation and UVC light-induced apoptosis. Cell Death Differ 14(10):1848–1850
Hong C, Qiu X, Li Y, Huang Q, Zhong Z, Zhang Y, Liu X, Sun L, Lv P, Gao XM (2010) Functional analysis of recombinant calreticulin fragment 39–272: implications for immunobiological activities of calreticulin in health and disease. J Immunol 185(8):4561–4569
Chignard N, Shang S, Wang H, Marrero J, Brechot C, Hanash S, Beretta L (2006) Cleavage of endoplasmic reticulum proteins in hepatocellular carcinoma: detection of generated fragments in patient sera. Gastroenterology 130(7):2010–2022
Kageyama S, Isono T, Matsuda S, Ushio Y, Satomura S, Terai A, Arai Y, Kawakita M, Okada Y, Yoshiki T (2009) Urinary calreticulin in the diagnosis of bladder urothelial carcinoma. Int J Urol 16(5):481–486
Kageyama S, Isono T, Iwaki H, Wakabayashi Y, Okada Y, Kontani K, Yoshimura K, Terai A, Arai Y, Yoshiki T (2004) Identification by proteomic analysis of calreticulin as a marker for bladder cancer and evaluation of the diagnostic accuracy of its detection in urine. Clin Chem 50(5):857–866
Chen CN, Chang CC, Su TE, Hsu WM, Jeng YM, Ho MC, Hsieh FJ, Lee PH, Kuo ML, Lee H, Chang KJ (2009) Identification of calreticulin as a prognosis marker and angiogenic regulator in human gastric cancer. Ann Surg Oncol 16(2):524–533
Alfonso P, Nunez A, Madoz-Gurpide J, Lombardia L, Sanchez L, Casal JI (2005) Proteomic expression analysis of colorectal cancer by two-dimensional differential gel electrophoresis. Proteomics 5(10):2602–2611
Eric A, Juranic Z, Milovanovic Z, Markovic I, Inic M, Stanojevic-Bakic N, Vojinovic-Golubovic V (2009) Effects of humoral immunity and calreticulin overexpression on postoperative course in breast cancer. Pathol Oncol Res 15(1):89–90
Lwin ZM, Guo C, Salim A, Yip GW, Chew FT, Nan J, Thike AA, Tan PH, Bay BH (2010) Clinicopathological significance of calreticulin in breast invasive ductal carcinoma. Mod Pathol 23(12):1559–1566
Bergner A, Kellner J, Tufman A, Huber RM (2009) Endoplasmic reticulum Ca2+-homeostasis is altered in small and non-small cell lung cancer cell lines. J Exp Clin Cancer Res 28:25
Korbelik M, Zhang W, Merchant S (2011) Involvement of damage-associated molecular patterns in tumor response to photodynamic therapy: surface expression of calreticulin and high-mobility group box-1 release. Cancer Immunol Immunother 60(10):1431–1437
Gong J, Zhu C, Zhuang R, Song C, Li Q, Xu Z, Wei Y, Yang K, Yang A, Chen L, Jin B (2009) Establishment of an enzyme-linked immunosorbent assay system for determining soluble CD96 and its application in the measurement of sCD96 in patients with viral hepatitis B and hepatic cirrhosis. Clin Exp Immunol 155(2):207–215
Gold LI, Eggleton P, Sweetwyne MT, Van Duyn LB, Greives MR, Naylor SM, Michalak M, Murphy-Ullrich JE (2010) Calreticulin: non-endoplasmic reticulum functions in physiology and disease. FASEB J 24(3):665–683
Liu F, Li Y, Song C, Dong B, Liu Z, Zhang K, Li H, Sun Y, Wei Y, Yang A, Yang K, Jin B (2010) Highly sensitive microplate chemiluminescence enzyme immunoassay for the determination of staphylococcal enterotoxin B based on a pair of specific monoclonal antibodies and its application to various matrices. Anal Chem 82(18):7758–7765
Panaretakis T, Kepp O, Brockmeier U, Tesniere A, Bjorklund AC, Chapman DC, Durchschlag M, Joza N, Pierron G, van Endert P, Yuan J, Zitvogel L, Madeo F, Williams DB, Kroemer G (2009) Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death. EMBO J 28(5):578–590
Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, Castedo M, Mignot G, Panaretakis T, Casares N, Metivier D, Larochette N, van Endert P, Ciccosanti F, Piacentini M, Zitvogel L, Kroemer G (2007) Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med 13(1):54–61
Tesniere A, Apetoh L, Ghiringhelli F, Joza N, Panaretakis T, Kepp O, Schlemmer F, Zitvogel L, Kroemer G (2008) Immunogenic cancer cell death: a key-lock paradigm. Curr Opin Immunol 20(5):504–511
Coppolino MG, Woodside MJ, Demaurex N, Grinstein S, St-Arnaud R, Dedhar S (1997) Calreticulin is essential for integrin-mediated calcium signalling and cell adhesion. Nature 386(6627):843–847
Orr AW, Pallero MA, Xiong WC, Murphy-Ullrich JE (2004) Thrombospondin induces RhoA inactivation through FAK-dependent signaling to stimulate focal adhesion disassembly. J Biol Chem 279(47):48983–48992
Nanney LB, Woodrell CD, Greives MR, Cardwell NL, Pollins AC, Bancroft TA, Chesser A, Michalak M, Rahman M, Siebert JW, Gold LI (2008) Calreticulin enhances porcine wound repair by diverse biological effects. Am J Pathol 173(3):610–630
Chaput N, De Botton S, Obeid M, Apetoh L, Ghiringhelli F, Panaretakis T, Flament C, Zitvogel L, Kroemer G (2007) Molecular determinants of immunogenic cell death: surface exposure of calreticulin makes the difference. J Mol Med 85(10):1069–1076
Obeid M, Panaretakis T, Tesniere A, Joza N, Tufi R, Apetoh L, Ghiringhelli F, Zitvogel L, Kroemer G (2007) Leveraging the immune system during chemotherapy: moving calreticulin to the cell surface converts apoptotic death from “silent” to immunogenic. Cancer Res 67(17):7941–7944
Tufi R, Panaretakis T, Bianchi K, Criollo A, Fazi B, Di Sano F, Tesniere A, Kepp O, Paterlini-Brechot P, Zitvogel L, Piacentini M, Szabadkai G, Kroemer G (2008) Reduction of endoplasmic reticulum Ca2+ levels favors plasma membrane surface exposure of calreticulin. Cell Death Differ 15(2):274–282
Zeng G, Aldridge ME, Tian X, Seiler D, Zhang X, Jin Y, Rao J, Li W, Chen D, Langford MP, Duggan C, Belldegrun AS, Dubinett SM (2006) Dendritic cell surface calreticulin is a receptor for NY-ESO-1: direct interactions between tumor-associated antigen and the innate immune system. J Immunol 177(6):3582–3589
Chao MP, Jaiswal S, Weissman-Tsukamoto R, Alizadeh AA, Gentles AJ, Volkmer J, Weiskopf K, Willingham SB, Raveh T, Park CY, Majeti R, Weissman IL (2010) Calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by CD47. Sci Transl Med 2(63):63ra94
Liu M, Imam H, Oberg K, Zhou Y (2005) Gene transfer of vasostatin, a calreticulin fragment, into neuroendocrine tumor cells results in enhanced malignant behavior. Neuroendocrinology 82(1):1–10
Hsu WM, Hsieh FJ, Jeng YM, Kuo ML, Chen CN, Lai DM, Hsieh LJ, Wang BT, Tsao PN, Lee H, Lin MT, Lai HS, Chen WJ (2005) Calreticulin expression in neuroblastoma: a novel independent prognostic factor. Ann Oncol 16(2):314–321
Acknowledgments
We thank Institute of Biology and Medical Sciences Soochow University (Suzhou, China) for the antigen rCRT39/272. This work was supported by the National Natural Science Foundation of China (30972683 and 31070794).
Conflict of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding authors
Additional information
R. Liu, J. Gong and J. Chen contributed equally to the work.
Rights and permissions
About this article
Cite this article
Liu, R., Gong, J., Chen, J. et al. Calreticulin as a potential diagnostic biomarker for lung cancer. Cancer Immunol Immunother 61, 855–864 (2012). https://doi.org/10.1007/s00262-011-1146-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00262-011-1146-8