論文の公開元へGlycan profile of signet ring cell gastric cancer and potential applicability of lectin drug conjugate therapy
概要
筑
波 大
学
博士（医学）学位論文
Glycan profile of signet ring cell gastric
cancer and potential applicability of
lectin drug conjugate therapy
（シグネットリングセル胃癌細胞表面糖鎖の発現
解析と糖鎖を標的する治療法の開発）
2022
筑波大学大学院博士課程人間総合科学研究科
YANG
1
YU
Abbreviations:
BPL
Bauhinia Purpurea Lectin
BSA
bovine serum albumin
Con A
Concanavalin A lectin
FBS
fetal bovine serum
GC
gastric cancer
HER2
human epidermal growth factor receptor 2
IC50
50% inhibitory concentration
IMDM
Iscove's Modified Dulbecco's Medium
LDC
lectin-drug conjugate
NSRC
non-signet ring cell
PBS
phosphate-buffered saline
rBC2LCN
recombinant N-terminal domain of BC2L-N lectin
RPMI 1640
Roswell Park Memorial Institute medium
SRC
signet ring cell carcinoma
2
Table of contents
Abbreviations
2
Chapter 1 : Glycan expression profile of signet ring cell (SRC) gastric cancer
1-1.
Background
8
1-1-1.
Present situation of signet ring cell gastric cancer
1-1-2.
Glycosylation and lectins
1-2.
Aim
10
1-3.
Material and methods
11
1-4.
1-3-1.
Cell lines
1-3-2.
Clinical tissue samples
1-3-3.
Protein extraction
1-3-4.
Lectin microarray
1-3-5.
Statistical analysis
Results
13
1-4-1. High affinity lectins and its binding glycan structure in SRC cell lines
1-4-2. Low affinity lectins and its binding glycan structure in SRC cell lines
3
1-4-3.
Glycan expression in clinical tissue samples
1-5.
Discussion
15
1-6.
Conclusion
18
Chapter 2 : Glycan targeting therapy for signet ring cell gastric cancer using lectin
drug conjugate
2-1.
Background
19
2-2.
Aim
19
2-3.
Material and methods
20
2-3-1.
Lectin staining
2-3-1-1.
Immunohistochemistry
2-3-1-2.
Live cell staining
2-3-2.
Protein blotting assay
2-3-2-1. Lectin blotting
2-3-2-2. Silver blotting
2-3-3.
Flow cytometry
4
2-3-4.
Cell viability assay
2-3-5.
Hemagglutination assay
2-3-6.
Animal models
2-3-7.
in vivo treatment assay using lectin drug conjugate
2-3-8.
Statistical analysis
2-4. Results
25
2-4-1.
Lectin selection for glycan targeting therapy
2-4-2.
BPL lectin
2-4-2-1. Lectin affinity assay
2-4-2-2. Cytotoxicity of lectin drug conjugate
2-4-2-3. Hemagglutination assay
2-4-3.
rBC2LCN lectin
2-4-3-1. Lectin affinity assay
2-4-3-2. Cytotoxicity of lectin drug conjugate
2-4-3-3. in vivo treatment assay using lectin drug conjugate
5
2-5.
Discussion
29
2-6.
Conclusion
33
Chapter 3 : Summary and plan of future
34
Figure and Tables
36
Table 1: Differential glycan analysis between SRC and NSRC cell line array of 96
lectins that showed significant difference
Figure 1: Location of 96 lectins in lectin micro-array
Figure 2: Glycan expression file of GC cell lines
Figure 3: Glycan expression file of GC clinical samples
Figure 4: Lectin affinity confirming for BPL
Figure 5: Evaluation of BPL-staining to GC clinical samples
Figure 6: Hemagglutination assay
Figure 7: Lectin affinity confirming for rBC2LCN
Figure 8: Evaluation of rBC2LCN-staining to GC clinical samples
6
Figure 9: Therapeutic efficacy evaluation of rBC2LCN-PE38 conjugate in vivo
Supplementary
48
References
53
Acknowledgements
62
Source
64
7
Chapter 1. Glycan expression profile of signet ring cell gastric cancer
1-1.
Background
1-1-1. Present situation of signet ring cell gastric cancer
Gastric cancer (GC) is the 2nd cancer leading death cause worldwide, even the
incidence has decreased in last decades, it remains the 4th common malignancy in the
world, especially in Eastern Asia [1][2][3]. There are several subtypes of GC, in which,
signet ring cell carcinoma (SRC) is a typical histological subtype, accounts for 16–32%
of all GC [4]. In Japan, 10% of all gastric cancers are SRC, even the rates of mortality
have declined but the incidence rates are continuing to be high [5] [6].
According to Bormann classification, most of SRC is categorized as Bormann type 4,
also known as linitis plastica (LP), but not all LPs are SRC, 10% ~ 20% of LP patients
are not diagnosed as SRC [7] [8]. The WHO system defines 5 main types of GC, SRC
is included in one of them, poorly cohesive gastric carcinoma [9]. Compare with other
subtypes, SRC is more aggressive, infiltrating, and more metastatic. The age
distribution of SRC patients is younger, also, SRC occurs more often in female [10].
Detection of gastric cancer usually relies on examination through endoscope [11], as
for SRC, histologic appearance of SRC is a solid diagnosis. Whereas there are some
limitations. First, endoscopic examination may ignore tiny cancer foci leads to the
inaccurate detection [12] [13]. Second, an important characteristic of SRC, aggressive
behavior, also results in detection is usually at an advanced stage [14] [15].
8
Benefit from the development of novel cytotoxic medicaments and surgical techniques,
the prognosis for GC has been improved. However, the majority of patients with SRC
who are diagnosed during late stages are more likely resistant to chemotherapy [16][17].
1-1-2. Glycosylation and lectins
Glycans are covering the most outside layer of cell membrane, and glycosylation is a
common modification of proteins in human bodies, aberrant glycosylation plays a role
of tumor invasiveness, metastasis and prognosis by formatting sialylation or other
formations which will lead to glycans associated to tumor [18][19][20].
Several studies were reported for glycosylation in GC and some lectins, which were
bind to specific structure of glycosylation, were associated with GC and its prognosis.
In the comparison between GC and normal tissue or gastric ulcer tissues, GalNAc
glycans were highly expressed in GC and its binding lectin (VVA) was associated with
lymph node metastases [21] [22].
However, it remains unclear that the difference of glycan expression and glycosylation
of GC cells between each histological subtypes including SRC. I therefore investigated
that the glycan profile of SRC by the comparison with NSRC using lectin microarray.
9
1-2.
Aim of research
To explore the glycan expression of SRC and find out the specific lectin with intensive
affinity.
glycoprotein
drug
lectin
host carbohydrate
lectin
LDC
sm
pla
o
t
cy
glycoprotein
Man
Sia
Glc
GalNAc
Fuc
GlcNAc
cell membrane
Gal
10
1-3.
1-3-1.
Materials and methods
Cell lines
Human gastric adenocarcinoma cell lines, KATO-III, SNU-1, and NCI-N87 were
purchased from ATCC (Manassas, VA, USA), NUGC-4 was purchased from RIKEN
BioResource Research Center (Tsukuba, Ibaraki, Japan), and MKN-45 was purchased
from the Japanese Collection of Research Bioresources cell bank (Sennan, Osaka,
Japan). KATO-III and NUGC-4 are signet ring cell GC cell lines, which I defined as
SRC cell lines. NCI-N87 is a well differentiated, and SNU-1 and MKN-45 are poorly
differentiated gastric adenocarcinomas without signet ring cells, which I defined as
NSRC. The attributes of the aforementioned cell lines were confirmed using the Cancer
Cell Line Encyclopedia. SNU-1 and NCI-N87 cells were cultured in RPMI 1640
(ATCC), KATO-III cells in IMDM (ATCC), and MKN-45 and NUGC-4 cells in RPMI
1640 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The media were
supplemented with 10% or 20% FBS (GIBCO, Paisley, RF, UK) and 1% penicillinstreptomycin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). All cells
were incubated at 37 °C in a 5% CO2 atmosphere and were passaged directly or via
trypsinization.
1-3-2.
Clinical tissue samples
Thirty-two pairs of formalin-fixed and paraffin-embedded (FFPE) human GC tissue
sections and frozen adjacent normal tissues were obtained from the Department of
11
Pathology, University of Tsukuba Hospital, Tsukuba, Japan. All clinical samples were
obtained with written informed consent from the patients. All the protocols followed
the regulations relating to ethics of human subject research at the University of Tsukuba,
Tsukuba, Japan. This study was approved by the Tsukuba Clinical Research and
Development Organization (T-CReDO), Tsukuba, Japan.
1-3-3.
Protein extraction
A total of 5×104–107 live cells, or frozen tissue sections were lysed with CelLytic MEM
Protein extraction kit (Sigma-Aldrich, St. Louis, Missouri, USA) the protein
concentrations were determined using the Micro BCATM Protein Assay kit (Thermo
Fisher Scientific, Waltham, Massachusetts, USA), and γ-globulin was used as the
standard (Bio-Rad Laboratories, Hercules, California, USA). The procedures were
performed according to the manufacturer’s instructions.
1-3-4.
Lectin micro-array
A lectin microarray was constructed and analyzed as previously described [23][24][25].
In brief, 96 lectins were immobilized (Fig. 1). Protein samples were labeled with
fluorescent dye Cy3 (GE Healthcare, Cat#:PA13104), then, the Cy3-labelled lysates
were applied to each well of the microarray slides, after incubating at 20°C overnight,
the chips were scanned with an evanescent field activated fluorescence scanner (BioREX Scan 200, Rexxam Co., Ltd, Osaka, Japan). Automatic intensity analysis was
performed after high–speed scan, the quantified signal was analyzed using Cluster 3.0
12
(yellow: high; blue: low; black: intermediate), mean-normalized, log-transformed and
analyzed via the average linkage were performed.
1-3-5.
Statistical analysis
Cluster 3.0 and Java tree view were used to perform cluster analysis, and the results
were exported as heat maps. Student’s t-test in SPSS Statistics 21.0 (SPSS Inc.) was
used to evaluate the differences in lectin array signals between the two data sets.
Differences were considered statistically significant at p<0.05.
1-4.
1-4-1.
Results
High affinity lectins and its binding glycan structure in SRC cell lines
Glycan expression in SRC cell lines was compared with that in NSRC cell lines using
high-density lectin microarrays. ...
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