A novel antibody against cancer stem cell biomarker, DCLK1-S, is potentially useful for assessing colon cancer risk after screening colonoscopy

Shubhashish Sarkar, Vsevolod Popov, Malaney R. O'Connell, Heather Stevenson-Lerner, Brian S. Lee, Robert A. Obeid, Gurinder Luthra, Pomila Singh

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Abstract

DCLK1 expression is critically required for maintaining growth of human colon cancer cells (hCCCs). Human colorectal tumors (CRCs) and hCCCs express a novel short isoform of DCLK1 (DCLK1-S; isoform 2) from β-promoter of hDCLK1 gene, while normal colons express long isoform (DCLK1-L; isoform 1) from 5'(α)-promoter, suggesting that DCLK1-S, and not DCLK1-L, marks cancer stem cells (CSCs). Even though DCLK1-S differs from DCLK1-L by only six amino acids, we succeeded in generating a monospecific DCLK1-S-Antibody (PS41014), which does not cross-react with DCLK1-L, and specifically detects CSCs. Subcellular localization of S/L-isoforms was examined by immune-electron-microscopy (IEM). Surprisingly, besides plasma membrane and cytosolic fractions, S/L also localized to nuclear/mitochondrial fractions, with pronounced localization of S-isoform in the nuclei and mitochondria. Sporadic CRCs develop from adenomas. Screening colonoscopy is used for detection/resection of growths, and morphological/pathological criteria are used for risk assessment and recommendations for follow-up colonoscopy. But, these features are not precise and majority of the patients will never develop cancer. We hypothesized that antibody-based assay(s), which identify CSCs, will significantly improve prognostic value of morphological/pathological criteria. We conducted a pilot retrospective study with PS41014-Ab, by staining archived adenoma specimens from patients who developed (high-risk), or did not develop (low-risk) adenocarcinomas within 10-15 years. PS41014-Ab stained adenomas from initial and follow-up colonoscopies of high-risk patients, at significantly higher levels (three to fivefold) than adenomas from low-risk patients, suggesting that PS41014-Ab could be used as an additional tool for assessing CRC risk. CRC patients, with high DCLK1-S-expressing tumors (by qRT-PCR), were reported to have worse overall survival than low expressers. We now report that DCLK1-S-specific Ab may help to identify high-risk patients at the time of index/screening colonoscopy.

Original languageEnglish (US)
Pages (from-to)1245-1261
Number of pages17
JournalLaboratory investigation; a journal of technical methods and pathology
Volume97
Issue number10
DOIs
StatePublished - Oct 1 2017

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Neoplastic Stem Cells
Colonoscopy
Colonic Neoplasms
Protein Isoforms
Biomarkers
Adenoma
Antibodies
Growth
Colorectal Neoplasms
Neoplasms
Electron Microscopy
Mitochondria
Colon
Adenocarcinoma
Retrospective Studies
Cell Membrane
Staining and Labeling
Amino Acids
Polymerase Chain Reaction
Survival

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Biology
  • Cell Biology

Cite this

@article{48156df63e974994bc71e805795b523c,
title = "A novel antibody against cancer stem cell biomarker, DCLK1-S, is potentially useful for assessing colon cancer risk after screening colonoscopy",
abstract = "DCLK1 expression is critically required for maintaining growth of human colon cancer cells (hCCCs). Human colorectal tumors (CRCs) and hCCCs express a novel short isoform of DCLK1 (DCLK1-S; isoform 2) from β-promoter of hDCLK1 gene, while normal colons express long isoform (DCLK1-L; isoform 1) from 5'(α)-promoter, suggesting that DCLK1-S, and not DCLK1-L, marks cancer stem cells (CSCs). Even though DCLK1-S differs from DCLK1-L by only six amino acids, we succeeded in generating a monospecific DCLK1-S-Antibody (PS41014), which does not cross-react with DCLK1-L, and specifically detects CSCs. Subcellular localization of S/L-isoforms was examined by immune-electron-microscopy (IEM). Surprisingly, besides plasma membrane and cytosolic fractions, S/L also localized to nuclear/mitochondrial fractions, with pronounced localization of S-isoform in the nuclei and mitochondria. Sporadic CRCs develop from adenomas. Screening colonoscopy is used for detection/resection of growths, and morphological/pathological criteria are used for risk assessment and recommendations for follow-up colonoscopy. But, these features are not precise and majority of the patients will never develop cancer. We hypothesized that antibody-based assay(s), which identify CSCs, will significantly improve prognostic value of morphological/pathological criteria. We conducted a pilot retrospective study with PS41014-Ab, by staining archived adenoma specimens from patients who developed (high-risk), or did not develop (low-risk) adenocarcinomas within 10-15 years. PS41014-Ab stained adenomas from initial and follow-up colonoscopies of high-risk patients, at significantly higher levels (three to fivefold) than adenomas from low-risk patients, suggesting that PS41014-Ab could be used as an additional tool for assessing CRC risk. CRC patients, with high DCLK1-S-expressing tumors (by qRT-PCR), were reported to have worse overall survival than low expressers. We now report that DCLK1-S-specific Ab may help to identify high-risk patients at the time of index/screening colonoscopy.",
author = "Shubhashish Sarkar and Vsevolod Popov and O'Connell, {Malaney R.} and Heather Stevenson-Lerner and Lee, {Brian S.} and Obeid, {Robert A.} and Gurinder Luthra and Pomila Singh",
year = "2017",
month = "10",
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language = "English (US)",
volume = "97",
pages = "1245--1261",
journal = "Laboratory Investigation",
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T1 - A novel antibody against cancer stem cell biomarker, DCLK1-S, is potentially useful for assessing colon cancer risk after screening colonoscopy

AU - Sarkar, Shubhashish

AU - Popov, Vsevolod

AU - O'Connell, Malaney R.

AU - Stevenson-Lerner, Heather

AU - Lee, Brian S.

AU - Obeid, Robert A.

AU - Luthra, Gurinder

AU - Singh, Pomila

PY - 2017/10/1

Y1 - 2017/10/1

N2 - DCLK1 expression is critically required for maintaining growth of human colon cancer cells (hCCCs). Human colorectal tumors (CRCs) and hCCCs express a novel short isoform of DCLK1 (DCLK1-S; isoform 2) from β-promoter of hDCLK1 gene, while normal colons express long isoform (DCLK1-L; isoform 1) from 5'(α)-promoter, suggesting that DCLK1-S, and not DCLK1-L, marks cancer stem cells (CSCs). Even though DCLK1-S differs from DCLK1-L by only six amino acids, we succeeded in generating a monospecific DCLK1-S-Antibody (PS41014), which does not cross-react with DCLK1-L, and specifically detects CSCs. Subcellular localization of S/L-isoforms was examined by immune-electron-microscopy (IEM). Surprisingly, besides plasma membrane and cytosolic fractions, S/L also localized to nuclear/mitochondrial fractions, with pronounced localization of S-isoform in the nuclei and mitochondria. Sporadic CRCs develop from adenomas. Screening colonoscopy is used for detection/resection of growths, and morphological/pathological criteria are used for risk assessment and recommendations for follow-up colonoscopy. But, these features are not precise and majority of the patients will never develop cancer. We hypothesized that antibody-based assay(s), which identify CSCs, will significantly improve prognostic value of morphological/pathological criteria. We conducted a pilot retrospective study with PS41014-Ab, by staining archived adenoma specimens from patients who developed (high-risk), or did not develop (low-risk) adenocarcinomas within 10-15 years. PS41014-Ab stained adenomas from initial and follow-up colonoscopies of high-risk patients, at significantly higher levels (three to fivefold) than adenomas from low-risk patients, suggesting that PS41014-Ab could be used as an additional tool for assessing CRC risk. CRC patients, with high DCLK1-S-expressing tumors (by qRT-PCR), were reported to have worse overall survival than low expressers. We now report that DCLK1-S-specific Ab may help to identify high-risk patients at the time of index/screening colonoscopy.

AB - DCLK1 expression is critically required for maintaining growth of human colon cancer cells (hCCCs). Human colorectal tumors (CRCs) and hCCCs express a novel short isoform of DCLK1 (DCLK1-S; isoform 2) from β-promoter of hDCLK1 gene, while normal colons express long isoform (DCLK1-L; isoform 1) from 5'(α)-promoter, suggesting that DCLK1-S, and not DCLK1-L, marks cancer stem cells (CSCs). Even though DCLK1-S differs from DCLK1-L by only six amino acids, we succeeded in generating a monospecific DCLK1-S-Antibody (PS41014), which does not cross-react with DCLK1-L, and specifically detects CSCs. Subcellular localization of S/L-isoforms was examined by immune-electron-microscopy (IEM). Surprisingly, besides plasma membrane and cytosolic fractions, S/L also localized to nuclear/mitochondrial fractions, with pronounced localization of S-isoform in the nuclei and mitochondria. Sporadic CRCs develop from adenomas. Screening colonoscopy is used for detection/resection of growths, and morphological/pathological criteria are used for risk assessment and recommendations for follow-up colonoscopy. But, these features are not precise and majority of the patients will never develop cancer. We hypothesized that antibody-based assay(s), which identify CSCs, will significantly improve prognostic value of morphological/pathological criteria. We conducted a pilot retrospective study with PS41014-Ab, by staining archived adenoma specimens from patients who developed (high-risk), or did not develop (low-risk) adenocarcinomas within 10-15 years. PS41014-Ab stained adenomas from initial and follow-up colonoscopies of high-risk patients, at significantly higher levels (three to fivefold) than adenomas from low-risk patients, suggesting that PS41014-Ab could be used as an additional tool for assessing CRC risk. CRC patients, with high DCLK1-S-expressing tumors (by qRT-PCR), were reported to have worse overall survival than low expressers. We now report that DCLK1-S-specific Ab may help to identify high-risk patients at the time of index/screening colonoscopy.

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