Three-dimensional culture system identifies a new mode of cetuximab resistance and disease-relevant genes in colorectal cancer.
We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor15-PGDH/HPGD, and the most up-regulated gene in SC wasversican(VCAN) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232.|Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232.|Jiaen Genetics Laboratory, Beijing Jiaen Hospital, Beijing 100191.|Molecular Pathology, Cancer Research Center, Medical College of Xiamen University, Xiamen, Fujian 361102.
Cunxi
C
Li
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232.|Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232.
Bhuminder
B
Singh
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232.|Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232.
Ramona
R
Graves-Deal
Whitehead Institute for Biomedical Research, Cambridge, MA 02142.
Haiting
H
Ma
Interdisciplinary Graduate Program, Vanderbilt University School of Medicine, Nashville, TN 37232.
Alina
A
Starchenko
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232.|Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232.
William H
WH
Fry
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232.|Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232.
Yuanyuan
Y
Lu
Department of Human Genetics, Emory University, Atlanta, GA 30084.
Yang
Y
Wang
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232.|Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232.
Galina
G
Bogatcheva
Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232.
Mohseen P
MP
Khan
Division of Clinical Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232.
Ginger L
GL
Milne
Center for Quantitative Sciences, Vanderbilt University School of Medicine, Nashville, TN 37232.
Shilin
S
Zhao
Division of Cancer Biostatistics, Department of Biostatistics, Vanderbilt University School of Medicine, Nashville, TN 37232.
Gregory Daniel
GD
Ayers
Jiaen Genetics Laboratory, Beijing Jiaen Hospital, Beijing 100191.
Nenggan
N
Li
Jiaen Genetics Laboratory, Beijing Jiaen Hospital, Beijing 100191.
Huaying
H
Hu
Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232.
Mary Kay
MK
Washington
Gibbs Cancer Center & Research Institute, Spartanburg, SC 29303.
Timothy J
TJ
Yeatman
Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232.
Oliver G
OG
McDonald
Center for Quantitative Sciences, Vanderbilt University School of Medicine, Nashville, TN 37232.|Department of Biomedical Informatics, Vanderbilt University School of Medicine, Nashville, TN 37232.
Qi
Q
Liu
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232; robert.coffey@vanderbilt.edu.|Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232.|Veterans Affairs Medical Center, Nashville, TN 37212.
Robert J
0000-0002-2180-3844
RJ
Coffey
D000818:Animals; D000074322:Antineoplastic Agents, Immunological / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D000068818:Cetuximab / Q000494:pharmacology*; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology*; D000077547:Crizotinib; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000235:genetics; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics; D046888:Tissue Array Analysis; D053675:Versicans / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
D000074322:Antineoplastic Agents, Immunological; D011720:Pyrazoles; D011725:Pyridines; D018125:Receptors, Transforming Growth Factor beta; C508322:VCAN protein, human; D053675:Versicans; D000077547:Crizotinib; D006911:Hydroxyprostaglandin Dehydrogenases; C035859:15-hydroxyprostaglandin dehydrogenase; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D000077294:Receptor, Transforming Growth Factor-beta Type II; D000068818:Cetuximab
3D culture; HPGD; cetuximab resistance; colorectal cancer; versican