Detection of circulating tumor cells in blood of pancreatic ductal adenocarcinoma patients

Abstract

Aim: Previous studies suggest that circulating tumor cells (CTC) are present at very low frequencies in blood of pancreatic cancer (PC) patients. However, no technique has proven efficient for their detection, in part due to the lack of accurate tumor markers. Here, we evaluated the potential utility of two marker candidates - Mucin 16 (MUC16) and Tetraspanin 1 (TSPAN1) - identified through a detailed review of the literature.

Methods: To evaluate the pattern of expression of both markers in pancreatic tumor cells vs. normal blood, we used cell lines derived from pancreatic cancer patients and blood from healthy adults.

Results: Antibodies against both MUC16 and TSPAN1 showed expression in three pancreatic cancer (PC) cell lines while they were absent in blood cells. To evaluate the efficiency of isolating tumor cells from blood, PC cell lines were spiked at different frequencies in blood, sequentially stained with biotin-conjugated TSPAN1 and MUC16 antibodies and a streptavidin ferrofluids, followed by immunomagnetic enrichment. The recovery of spiked TSPAN1⁺ tumor cells was high with limited contamination by leukocytes. In contrast, no PC cells were isolated when the biotin MUC16 reagent was used because the biotin-conjugated clone did not recognize PC cells.

Conclusion: The combination of MUC16, TSPAN1, and epithelial cell adhesion molecule (EpCAM) antibodies will likely increase the efficiency of capturing circulating tumor cell in blood of pancreatic ductal adenocarcinoma. To further develop a protocol for isolation of circulating tumor cell in blood of PC patients, high amounts of antibodies (5-10 mg) against EpCAM, MUC16, and TSPAN1 will be needed.

Keywords

Pancreatic ductal adenocarcinoma / circulating biomarkers / circulating tumor cells

References

[1] Riva F,Khomenko DI,Louvet C.Clinical applications of circulating tumor DNA and circulating tumor cells in pancreatic cancer..Mol Oncol2016;10:481-93 PMCID:PMC5528974

[2] Bunt SK,Bailey JM,Hollingworth MA.Rosiglitazone and Gemcitabine in combination reduces immune suppression and modulates T cell populations in pancreatic cancer..Cancer Immunol Immunother2013;62:225-36 PMCID:PMC3873637

[3] Tanemura M,Nagano H,Matsunami K.Cancer immunotherapy for pancreatic cancer utilizing alpha-gal epitope/natural anti-Gal antibody reaction..World J Gastroenterol2015;21:11396-410 PMCID:PMC4616216

[4] Amedei A,Prisco D.Pancreatic cancer: role of the immune system in cancer progression and vaccine-based immunotherapy..Hum Vaccines Immunother2014;10:3354-68 PMCID:PMC4514060

[5] Baines AT,Rorje CJ.Current and emerging targeting strategies for treatment of pancreatic cancer..Prog Mol Biol Transl Sci2016;144:277-320

[6] Zhang Q,Zeng L,Lian G.New developments in the early diagnosis of pancreatic cancer..Expert Rev Gastroenterol Hepatol2017;11:149-56

[7] Chan A,Dimitromanolakis A,Serra S.Validation of biomarkers that complement CA19.9 in detecting early pancreatic cancer..Clin Cancer Res2014;20:5787-95 PMCID:PMC4233184

[8] Imamura T,Ichikawa D,Miyamae M.Liquid biopsy in patients with pancreatic cancer: Circulating tumor cells and cell-free nucleic acids..World J Gastroenterol2016;22:5627-41 PMCID:PMC4932201

[9] Hou FQ,Yao JL,Zhang W.Tetraspanin 1 is involved in survival, proliferation and carcinogenesis of pancreatic cancer..Oncol Rep2015;34:3069-76

[10] Shukla SK,Abrego J,Mishra A.MUC16-mediated activation of mTOR and c-Myc reprograms pancreatic cancer metabolism..Oncotarget2015;6:19118-31 PMCID:PMC4662479

[11] Haridas D,Ponnusamy MP,Rachagani S.Pathological implications of MUC16 expression in pancreatic cancer..PLoS One2011;6:e26839 PMCID:PMC3204976

[12] Hölters S,Jansen L,Dürst M.Tetraspanin 1 promotes invasiveness of cervical cancer cells..Int J Oncol2013;43:503-12

[13] Chen Y,Lu Y,Zhu YY.miR-200a enhances the migrations of A549 and SK-MES-1 cells by regulating the expression of TSPAN1..J Biosci2013;38:523-32

[14] Chen L,Zhao R,Zhu J.Suppression of TSPAN1 by RNA interference inhibits proliferation and invasion of colon cancer cells in vitro..Tumori2010;96:744-50

[15] Desouki MM,Huang H,Nowak NJ.Identification of metastasis-associated breast cáncer genes using a high-resolution whole genome profiling approach..J Cancer Res Clin Oncol2011;137:795-809

[16] Chen L,Li H,Wu YY.Knockdown of TSPAN1 by RNA silencing and antisense technique inhibits proliferation and infiltration of human skin squamous carcinoma cells..Tumori2010;95:289-95

[17] Gutierrez ML,Teodosio C,del Mar Abad.Identification and characterization of the gene expression profiles for protein coding and non-coding RNAs of pancreatic ductal adenocarcinomas..Oncotarget2015;6:19070-86 PMCID:PMC4662476

[18] Zhang X,Gao F,Wang H.TSPAN1 upregulates MMP2 to promote pancreatic cancer cell migration and invasion via PLCγ..Oncol Rep2019;41:2117-25 PMCID:PMC6412570

[19] Scholz CJ,Koretz K,Kreienberg R.Tspan-1 is a tetraspanin preferentially expressed by mucinous and endometrioid subtypes of human ovarian carcinomas..Cancer Lett2009;275:198-203

[20] Kalina T,van der Velden VH,Bottcher S.EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols..Leukemia2012;26:1986-2010 PMCID:PMC3437409

[21] Millner LM,Valdes Jr R.Circulating tumor cells: a review of present methods and the need to identify heterogeneous phenotypes..Ann Clin Lab Sci2013;34:295-304 PMCID:PMC5060940

[22] Bidard FC,Louvet C,Bouché O.Circulating tumor cells in locally advanced pancreatic adenocarcinoma: the ancillary CirCe 07 study to the LAP 07 trial..Ann Oncol2013;24:2057-61

[23] Cristofanilli M,Ellis MJ,Matera J.Circulating tumor cells, disease progression, and survival in metastatic breast cancer..N Engl J Med2004;351:781-91

[24] Alix-Panabieres C.Technologies for detection of circulating tumor cells: facts and vision..Lab Chip2014;14:57-62

[25] Went PT,Meier S,Mirlacher M.Frequent EpCAM protein expression in human carcinomas..Human Pathol2004;35:122-8

[26] Akita H,Takeda Y,Kobayashi S.EpCAM is a significant prognostic factor in pancreatic cancer patients by suppressing cell activity..Oncogene2011;30:3468-76

[27] Fong D,Obrist P,Margreiter R.EpCAM expression in pancreatic and ampullary carcinomas: frequency and prognostic relenvance..J Clin Pathol2008;61:31-5

[28] Adams DL,Alpaugh RK,Tsai S.Circulating giant macrophages as a potential biomarker of solid tumors..Proc Natl Acad Sci U S A2014;111:3514-9 PMCID:PMC3948254

[29] Armstrong EA,Chakedis J,Moris D.Exosomes in pancreatic cancer: from early detection to treatment..J Gastrointest Surg2018;4:737-50

[30] Giampieri R,Occhipinti G,Righetti A.Clinical impact of different exosomes’ protein expression in pancreatic ductal carcinoma patients treated with standard first line palliative chemotherapy..PLoS One2019;14:e0215990 PMCID:PMC6497273

[31] Kahlert C,Protopopov A,Seth S.Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer..J Biol Chem2014;289:3869-75 PMCID:PMC3924256