Goto et al. Diagnostic Pathology(2022) 17:16
https://doi.org/10.1186/s13000-021-01176-2

SHORT REPORTOpen Access

Establishment of an antibody specific for
AMIGO2 improves immunohistochemicalevaluation of liver metastases and clinicaloutcomes in patients with colorectal cancer
Keisuke Goto1,2, Mitsuhiko Osaki1,3, Runa Izutsu1, Hiroshi Tanaka4, Ryo Sasaki1, Akimitsu Tanio2, Hiroyuki Satofuka3,
Yasuhiro Kazuki3,5, Manabu Yamamoto2, Hiroyuki Kugoh3,5, Hisao Ito1, Mitsuo Oshimura3,4, Yoshiyuki Fujiwara2 andFutoshi Okada1,3*

Abstract
Instruction: The human amphoterin-induced gene and open reading frame (AMIGO) was identified as a novel celladhesion molecule of type I transmembrane protein. AMIGO2 is one of three members of the AMIGO family(AMIGO1, 2, and 3), and the similarity between them is approximately 40% at the amino acid level. We havepreviously shown that AMIGO2 functions as a driver of liver metastasis. Immunohistochemical analysis of AMIGO2
expression in colorectal cancer (CRC) using a commercially available anti-AMIGO2 mouse monoclonal antibody
clone sc-373699 (sc mAb) correlated with liver metastasis and poor prognosis. However, the sc mAb was found tobe cross-reactive with all three molecules in the AMIGO family.
Methods: We generated a rat monoclonal antibody clone rTNK1A0012 (rTNK mAb) for human AMIGO2. The rTNK
mAb was used to re-evaluate the association between AMIGO2 expression and liver metastases/clinical outcomesusing the same CRC tissue samples previously reported with sc mAb.
Results: Western blot analysis revealed that a rTNK mAb was identified as being specific for AMIGO2 protein anddid not cross-react with AMIGO1 and AMIGO3. The rTNK mAb and sc mAb showed higher AMIGO2 expression,
which correlates with a high frequency of liver metastases (65.3% and 47.5%, respectively), while multivariate
analysis showed that AMIGO2 expression was an independent prognostic factor for liver metastases (p = 7.930E-10
and p = 1.707E-5). The Kaplan-Meier analyses showed that the rTNK mAb (p = 0.004), but not sc mAb (p = 0.107),
predicted worse overall survival in patients with high AMIGO2 expression. The relationship between AMIGO2
expression and poor disease-specific survival showed a higher level of significance for rTNK mAb (p = 0.00004)
compared to sc mAb (p = 0.001).

* Correspondence: fuokada@tottori-u.ac.jp
1
Division of Experimental Pathology, Tottori University Faculty of Medicine,
86 Nishicho, 683-8503 Yonago, Japan
3
Chromosome Engineering Research Center (CERC), Tottori University,
683-8503 Yonago, Japan
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which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
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Goto et al. Diagnostic Pathology(2022) 17:16

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Conclusions: These results indicate that the developed rTNK1A0012 mAb is an antibody that specifically recognizes
AMIGO2 by immunohistochemistry and can be a more reliable and applicable method for the diagnostic detectionof liver metastases and worse prognosis in patients with high AMIGO2-expressing CRC.
Keywords: AMIGO2, Monoclonal antibody, Colorectal cancer, Liver metastasis, Prognosis

Introduction
Cancer survival has improved globally over the last two
decades, with an increase in the survival of patients withdeadly cancers, such as liver, pancreatic, and lung, by up
to 5% [1]. The main cause of reduced survival in cancer
patients and survivors is distant metastasis rather thangrowth of the primary tumor, accounting for approximately 90% of cancer-related deaths [2–4]. Of the common organs that cause distant metastases, the liver is themost frequent site (59%), except for regional lymph
nodes [5]. The precise mechanism of metastasis to distant organs, including the liver, that fundamentally undermines cancer therapeutic strategies, is not wellunderstood [6]. Therefore, finding diagnostic biomarkers
and therapeutic targets that reliably determine liver metastases is an urgent task.
We have identified that AMIGO2, a member of the
AMIGO family (AMIGO1, 2, and 3), functions as adriver gene for liver metastasis in a mouse model [7].
The AMIGO protein was originally identified as a novel
cell adhesion molecule that accompanies type I transmembrane proteins containing six leucine-rich repeats
and one immunoglobulin-like domain in the extracellular region that are preferentially expressed on fiber tractsof neuronal tissues which participate in axon tract development [8]. The three AMIGO family molecules are
structurally similar and have shown homophilic and heterophilic binding mechanisms among their molecules;
however, the organ expression pattern and biological
functions of each protein are independent [9]. We foundthat knocking down AMIGO2 expression, which ishighly expressed in liver metastatic tumor cells, reduces
the adhesion of tumor cells to hepatic endothelial cells
and suppresses liver metastasis. Conversely, it was verified that forced expression of the AMIGO2 gene in nonliver metastatic tumor cells increases the adhesion oftumor cells to hepatic endothelial cells and thereafter
forms liver metastases [10]. However, AMIGO2expressing tumor cells did not show increased adhesion
to lung endothelial cells and did not affect lung metastases or metastases to other organs. Thus, it was clarified
that tumor cells highly expressing AMIGO2 selectively
adhere to hepatic endothelial cells expressing AMIGO
family molecules by homophilic/heterophilic adhesion
patterns to cause liver metastasis [10].
Extrapolation of this phenomenon in human cancers
has been reported by immunohistochemical staining of

AMIGO2 expression in human colorectal cancer (CRC)
tissue, which is closely associated with AMIGO2 expression and liver metastasis, but not with lung metastasis
and peritoneal dissemination [11]. Multivariate analysis
showed that AMIGO2 expression in patients with CRC
was an independent predictive factor for liver metastasis[11]. Furthermore, transcriptionally high levels of
AMIGO2 are associated with shortened survival of patients with CRC [11], breast cancer [12], and gastric cancer [13]. AMIGO2 has been reported as a novel
pathogenesis-related gene in gastric cancer [14], melanoma [15, 16], ovarian cancer [17], and pituitary neuroendocrine tumors [18]. In addition, bioinformatics analysis
using the transcriptome database selected AMIGO2 as acancer-related gene candidate for CRC [19], pancreaticcancer [20], and endometrial cancer [21].
Most studies have been conducted on the expression of
AMIGO2 mRNA in tumor tissues. However, a high expression of AMIGO2 mRNA has been detected in normal
tissues, and is most prominent in the cerebellum, retina,
liver, and lung [9]. Lower but steady AMIGO2 expressionis also found in the cerebrum, kidneys, small intestine,spleen, and testis [9]. These physiological conditions indicate that as long as AMIGO2 expression is assessed as
tissue-wide mRNA expression, its association with carcinogenesis in each organ cannot be determined. Therefore, immunohistochemical staining is the most reliable
method for accurately evaluating AMIGO2 expression incancerous tissues. For this purpose, we have analyzed
AMIGO2 expression by using commercially available
anti-AMIGO2 mouse monoclonal antibody (sc-373699);
however, we noticed that the antibody recognizes
AMIGO1 and AMIGO3 as well as AMIGO2. We also investigated the specificity of three commercially available
affinity purified polyclonal antibodies. These antibodies
were also recognized as AMIGO family molecules.
In this study, we developed a new AMIGO2-specificantibody that does not react with AMIGO1 and AMIGO3,
and compared the efficacy of the new antibody using thesame tissue samples that previously reported a significant
correlation between AMIGO2 expression in primary CRC
tissue and liver metastasis [11].

Materials and methods
Cell culture and transfection

P3X63Ag8.653 myeloma cells (CRL-1580) and HepG2
hepatocellular carcinoma cells (HB-8065) wereGoto et al. Diagnostic Pathology(2022) 17:16

purchased from the American Type Culture Collection(ATCC, Manassas, VA). P3X63Ag8.653 cells and HepG2
cells were respectively maintained in RPMI-1640
medium (05911; Nissui Pharmaceutical, Tokyo, Japan)
and Dulbecco’s modified Eagle’s medium (05919; Nissui
Pharmaceutical) containing 10% heat-inactivated fetal
bovine serum (FBS, F7524, Sigma-Aldrich, St. Louis,MO) and L-glutamine. The cell lines were maintained at
37 °C in a humidified 5% CO2/95% air mixture.
Expression plasmids for human AMIGO1 (pEZ-M02/
AMIGO1),AMIGO2(pEZ-M02/AMIGO2),andAMIGO3 (pEZ-M02/AMIGO3) were purchased from
GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EXE1133-M02, Rockville, MD). The pEZ-M02 vector wasused as the control plasmid. HepG2 cells were transfected with these plasmids using Lipofectamine 2000 reagent (12566014, Thermo Fisher Scientific, Waltham,MA) according to the manufacturer’s instructions.
Transfectants stably expressing the introduced vector
plasmid were selected by continuous neomycin treatment at 450 µg/mL (10131035, Thermo Fisher Scientific). Neomycin-resistant cells were cloned by thelimiting dilution method and maintained in a medium
containing neomycin.
Antigens

To produce an antigen for immunization, a DNA fragment of AMIGO2 extracellular domain (NM_001143668) was amplified by PCR using primers (forward: 5′-GCG AAG CTT GTG TGC CCC ACC GCTTGC AT-3′ and reverse: 5′-GCG CTC GAG TGT GTTAAA TGC CTC ATG AGC ATG GG-3′), and was subcloned into pET-32b(+) (69016, Sigma-Aldrich) using
HindIII and XhoI restriction enzymes (resulting in
pET32b-AMIGO2-EX). AMIGO2-EX protein was difficult to express in Escherichia coli Rosseta-gami B (DE3)pLysS (71137, Merck KGaA, Darmstadt, Germany).
pET32b-AMIGO2-EX was digested with EcoRI (anEcoRI site is located near the upstream end of theleucine-rich repeat sequence), blunted using Blunting
high, and then digested with EcoRV (at a site upstreamof AMIGO2-EX) to eliminate its leucine-rich repeat sequence. Therefore, this vector consisted of the Ig-likedomain of AMIGO2 (named pET32b-AMIGO2-Ig) andproduces the Trx-AMIGO2-Ig recombinant protein. To
obtain the GST-AMIGO2-Ig recombinant fusion protein, pGEX-6P-1 (28954648, GE Healthcare, Chicago, IL)
was modified by inserting the synthesized DNA (5′ACG AGA TCT GCC ATG GAC AAG CTT GTC GAC
ACG AGC TCG AAT TCG GAT CCC CCG GGG CTC
GAG CAC CAC CAC CAC CAC CAC TGA GCT GAG
CGG CCG CTC A-3′) using BglII and NotI restriction
enzymes (resulting in pGEX-MCS-His). The amplified
AMIGO2-Ig fragment was also cloned into pGEX-MCS-

Page 3 of 12

His. Restriction enzymes and DNA-modifying enzymes
were purchased from New England Biolabs (Ipswich,MA) and Toyobo (Tokyo, Japan), respectively. Primers
were ordered from Eurofins (Huntsville, AL). E. coli
strain (DH5α) competent cells were obtained fromTakara Bio (9057, Shiga, Japan).
After transformation of E. coli gami B pLysS (DE3)with each vector, the recombinant proteins wereexpressed by induction with 1.0 mM isopropyl-β-D(-)-thiogalactopyranoside (094-05144, Fujifilm Wako
Pure Chemical, Osaka, Japan) in a LB medium. Transformation using the empty vector, pET-32b(+) was also
carried out to produce the Tag protein for use in hybridoma screening as a negative control. After harvesting thecells and sonication, the recombinant proteins were obtained as inclusion bodies. Following solubilization with
6 M guanidine hydrochloride (078-05003, Fujifilm Wako
Pure Chemical) in PBS with 0.1 mM glutathione (oxideform, 078-03333, Fujifilm Wako Pure Chemical) and 1
mM glutathione (redox form, 077-02011, Fujifilm Wako
Pure Chemical), recombinant protein was purified using
Ni-NTA columns (30410, Qiagen, Hulsterweg, Venlo,
Netherlands) with elution using 100 mM imidazole(091-00012, Fujifilm Wako Pure Chemical) containing
6 M guanidine hydrochloride. After dialyzing the eluted
fraction against PBS containing 0.4 M arginine (PBS-A,
091-04611, Fujifilm Wako Pure Chemical), samples werediluted to about 1 mg/mL and stored at -30 °C.

Immunization

Protein antigens were prepared in PBS or in PBS-A
at 1 mg/mL, and the volume corresponding to thedesired amount of protein was increased to an injectable volume with PBS or PBS-A. This volume
was then mixed 1:1 (v/v) with either Freund’s adjuvant, complete (F5881, Sigma-Aldrich), or Sigma adjuvant system (S6322, Sigma-Aldrich). For viscousadjuvants, the solution was mixed by repeated passage through a syringe until a smooth emulsion wasformed (over approximately 30 min on ice). Injections were performed on 6-week-old male and female Jcl:Wistar rats (Clea, Tokyo, Japan) using a 1
mL glass syringe and a 27-gauge needle. Prime andboost injections of 250 µg protein were injected intraperitoneally every two weeks. Final boosts of
250 µg of protein were delivered intravenously without adjuvant via the tail vein. Volumes varied depending on the injection route and experimental
requirements and were in accordance with the relevant JP Home Office animal license for the procedure. The experimental protocol was approved by theCommittee of the Institute for Animal Experimentation of Tottori University (17-Y-28).

Goto et al. Diagnostic Pathology(2022) 17:16

Hybridoma generation

After confirming induction of serum antibodies against
AMIGO2-Ig protein, spleens and lymph nodes from immunized rats were harvested from euthanized rats, homogenized to single-cell suspensions, and fused withmyeloma P3X63Ag8.653 cells using an electro-cellfusion generator (ECFG21; Nepagene, Chiba, Japan).
Fused hybridoma cells were plated in 96-well plates.
After approximately 14 days of culture, a primary screenof supernatants was performed by ELISA. Hybridoma
clones producing AMIGO2-specific Abs were identified
by ELISA using GST-AMIGO2-Ig following HAT selection. Positive wells were picked and passaged in 96-wellplates. Each supernatant was reanalyzed by ELISA using
Tag, Trx-AMIGO2-Ig, and GST-AMIGO2-Ig. Hybridoma clones that reacted with Trx-AMIGO2-Ig and
GST-AMIGO2-Ig, but not Tag, were established using
two or more limited dilutions.
Hybridoma screening

Hybridoma cells producing AMIGO2-specific mAb wereidentified using ELISA. In brief, a 96-well immunoassay
plate (44-2404, Thermo Fisher Scientific) were coatedwith 50 µg/well antigen, washed three times with PBS-T(0.05% v/v Tween-20: 160-21211, Fujifilm Wako Pure
Chemical), and blocked with PBS containing 5% skim
milk (232100, Difco, Detroit, MI) for 30 min. After incubation with 100 µL of serially diluted serum samples orsupernatant for 1 h, plates were again washed and incubated with 100 µL of anti-rat IgG (H+L)-HRP conjugate(A110-105P, Bethyl Laboratories, Montgomery, TX) diluted 50,000-fold in TBS-T (50 mM Tris-HCl, pH 8.0)
(T1503, Sigma-Aldrich), 150 mM NaCl (195-01663, Fujifilm Wako Pure Chemical), 0.05% v/v Tween 20) for
30 min. Plates were washed again as described above, developed using 100 µL of o-phenylenediamine (160-11022,
Fujifilm Wako Pure Chemical) for 30 min, and stopped
using 25 µL of 1 M H2SO4 (95626-06, Nacalai Tesque,
Kyoto, Japan). Absorbance was measured at 492 nm by
microplate reader Epoch2 (Bio Tek, Winooski, VT).
Western blotting

Cells were washed in cold PBS and lysed in lysis buffer
containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl,
0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 1% Triton X-100, 1 µg/mL aprotinin, and 1 µg/
mL leupeptin. Lysates were centrifuged at 15,000 rpm
for 5 min. Protein concentrations were estimated using
the Bradford protein assay (5000006, Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as thestandard. The cell lysate was incubated with or without
PNGase F (P0704S, New England Biolabs, UK) at 37 °C
for 1 h.

Page 4 of 12

Proteins were resolved by SDS-polyacrylamide gelelectrophoresis using 10% gels, followed by electrotransfer to polyvinylidene difluoride membranes (ISEQ00010,
Millipore, Bedford, MA). The membranes were thenblotted using primary antibodies, washed, then incubated with secondary antibodies. The membranes were
washed, and the bound antibodies were detected using
an enhanced chemiluminescence detection system(GERPN2209, Amersham, Buckinghamshire, UK). The
anti-AMIGO2 primary antibodies used in this study
were rTNK1A0012; sc-373699, Santa Cruz Biotechnology, Dallas, TX; LS-C404504, LifeSpan BioSciences, Seattle, WA; #36094, Signalway Antibody, College Park,
MD; HPA054004, Sigma-Aldrich), and anti-β-actin (1:2000; A1978, Sigma-Aldrich). The secondary antibodies
used in this study were as follows: anti-mouse IgG-HRP(PM009-7, MBL, Nagoya, Japan), anti-rabbit IgG-HRP(458, MBL, Nagoya, Japan), and goat anti-rat IgG-HRP
pre-adsorbed (ab98425, Abcam, Cambridge, UK).
Patient samples

Immunohistochemical analysis was performed using
paraffin-embedded colorectal cancer samples from 173 patients who underwent proctocolectomies at Tottori University Hospital between January 2007 and December 2015. For
173 cases, excluding the samples that were no longer available, the same samples reported in the previously were used[11]. Clinicopathological findings were determined using theJapanese classification of colorectal carcinoma [16]. None ofthe patients had received radiotherapy, chemotherapy, orother medical interventions before surgery.
Immunohistochemistry

Tissue samples were fixed in formalin and embedded in paraffin. Serial sections were cut at 4 μm, deparaffinized in xylene, and rehydrated using a graded alcohol series. For
retrieval of AMIGO2, the sections were heated at 121℃ for
20 min in an autoclave in 10 mM citrate buffer (pH 6.0).
The samples were incubated in 0.1% hydrogen peroxidase
for 15 min to block endogenous peroxidases and in 10% normal goat serum (424041, Nichirei Biosciences, Tokyo, Japan)
for 15 min to prevent non-specific antigen binding. The
slides were subsequently incubated with primary antibodies(rTNK1A0012a) overnight at 4 °C, then incubated with goatanti-rat IgG-HRP for 20 min. Staining was visualized with diaminobenzidine (SK-4105, Vector Laboratories, Burlingame,CA), and the sections were counterstained with hematoxylin.
The expression of AMIGO2 in CRC cells was evaluated in ablinded manner. In brief, five fields were chosen at random
and examined at x 400 magnification. The intensity of immunoreactivity was bisected according to a previous report[11], and the staining intensity of the primary CRC was defined as low (< 30% staining intensity) and high (≥ 30% staining intensity).

Goto et al. Diagnostic Pathology(2022) 17:16

Statistical analysis

All statistical analyses were performed using SPSS
version 25 software (IBM Japan, Tokyo, Japan). The
χ2 tests were used to compare the clinicopathologicalcharacteristics of tumors with high and low AMIGO2
expression. Univariate and multivariate analyses forthe identification of prognostic factors for overall survival were carried out using the Cox proportional
hazard regression model, and identification of prognostic factors for liver metastases were carried out
using the logistic regression model. Kaplan-Meier survival curves were plotted and compared using a generalized log-rank test. Statistical significance was set
at p < 0.05. Where appropriate, data are expressed asmean ± SD.

Results
Evaluation of reactivity of monoclonal antibody(rTNK1A0012) against human AMIGO2 protein

We developed rat monoclonal antibodies against theextracellular domain of human AMIGO2 for use in

Page 5 of 12

clinical diagnosis (Additional file 1 shows flowchart ofthe experimental method for establishing the monoclonal antibody [see Additional file 1]). The monoclonal
antibodies with high immunoglobulin titers were selected by enzyme-linked immunosorbent assay (ELISA)(An additional file shows this in more detail [see Additional file 2]). Of the 19 candidate antibodies, the monoclonal antibody clone rTNK1A0012 (rTNK mAb) wasestablished to detect human AMIGO2 for further study.
To evaluate whether the rTNK mAb reacted specifically with human AMIGO2, HepG2 cells were transfectedwith a plasmid containing AMIGO1, AMIGO2, or
AMIGO3 gene, and established stably expressing celllines. Western blot analysis showed that rTNK mAb wasreactive with AMIGO2 (HepG2-A2), while the rTNK
mAb did not react with the other AMIGO family molecules; that is, AMIGO1 (HepG2-A1), AMIGO3 (HepG2A3), or empty vector-transfected cells (HepG2-E)(Fig. 1 A). The rTNK mAb was found to be highly specific and sensitive to human AMIGO2. The same protein lysates obtained from the transfectants of theFig. 1 Specific detection of AMIGO2 by the monoclonal antibody rTNK1A0012. The large membrane was reacted with anti-AMIGO2 antibody,while the small membrane was reacted with an anti-β-actin antibody. A Cell lysates were prepared from HepG2 cells transfected with human
AMIGO1 (A1), AMIGO2 (A2), AMIGO3 (A3), and an empty vector (E). Immunoblotting with rTNK1A0012 (rTNK mAb). B Immunoblotting with sc373699 (sc mAb). C Cell lysates were treated with or without PNGase F and immunoblotted with rTNK mAb. D Cell lysates were treated with orwithout alkaline β-elimination and immunoblotted with rTNK mAb

Goto et al. Diagnostic Pathology(2022) 17:16

Page 6 of 12

Fig. 2 Detection of three types of AMIGO family molecules by commercially available antibodies. A The same cell lysates as shown in Fig. 1 wasused. Lysates were prepared from HepG2 cells transfected with human AMIGO1 (A1), AMIGO2 (A2), AMIGO3 (A3), and an empty vector (E).
Immunoblotting with LS-C404504 polyclonal antibody. B Immunoblotting with #36094 polyclonal antibody. C Immunoblotting with HPA054004
polyclonal antibody

AMIGO family molecules were used to examine the specificity of the commercially available mouse anti-human
AMIGO2 monoclonal antibody (sc-373699, sc mAb). On
the other hands, the sc mAb cross-reacted with all typesof AMIGO family molecules (Fig. 1B). Next, we investigated the specificity of three commercially available
polyclonal antibodies (LS-C404504, #36094, andHPA054004). However, these antibodies were recognized as all AMIGO family molecules (Fig. 2).

In addition to the predicted molecular weight of human AMIGO2 based on the amino acid sequence of
58 kDa [22], multiple/diffuse bands suggest that the

AMIGO2 protein may undergo a series of posttranslational modifications such as glycosylation [23].
Since there are two main types of protein glycosylation,
N-glycosylation and O-glycosylation [24], the peptide-Nglycosidase F (PNGase F), which cleaves N-linked oligosaccharides [25], was used first. Treatment of HepG2-A2cell lysates with PNGase F caused a shift in electrophoretic mobility and resulted in conversion of the multiple/
diffuse band to a single band (Fig. 1 C). In contrast,
treatment of HepG2-A2 cell lysates with alkaline βelimination, which releases O-linked glycans from N-glycosylated proteins [24], did not cause any shift in electrophoretic mobility (Fig. 1D). This indicates that the
AMIGO2 protein is not O-glycosylated. It was clarified

Fig. 3 Immunohistochemical staining for AMIGO2 expression by the monoclonal antibody rTNK1A0012 in CRC tissues. AMIGO2 negative
expression (A and C) and AMIGO2 high expression (B and D) are shown. The lower row are shown at higher magnification. Scale bars = 400 μm(A and B) and 100 μm (C and D)

Goto et al. Diagnostic Pathology(2022) 17:16

that the human AMIGO2 protein may undergo N-glycosylation as a post-translational modification but may notundergo O-glycosylation.
Immunohistochemical detection of AMIGO2 expression
by rTNK mAb and clinicopathologic risk factors

The efficacy of the rTNK mAb against AMIGO2 was reevaluated using the same CRC tissue as previously
reported by immunohistochemical staining with thecommercially available sc mA [11]. We performed
immunohistochemistry of AMIGO2 in 173 CRC tissue
samples. AMIGO2 was mainly expressed in tumor cells
and was rarely expressed in the stroma (Fig. 3).
AMIGO2 was located in both the cytoplasm and nucleus

Page 7 of 12of the tumor cells (Fig. 3B and 3D). The expression of
AMIGO2 was quantified using a visual grading system
based on the extent of staining. The cut-off value for
AMIGO2 expression was determined as the staining intensity of the primary CRC at 30%, as in a previous report [11], and the cases were divided into two groups:
high and low expression. High expression was defined as
an AMIGO2-positive tumor cell proportion of 30% or
higher (≥ 30%, Fig. 3B and 3D). On the other hand, low
expression was defined as less than 30% of positive
tumor cells (< 30%, Fig. 3A and 3C).
Of the 173 tumor specimens evaluated, 28.3% (49/173)
had AMIGO2 high expression, while 71.7% (124/173)

Table 1 AMIGO2 expression and clinicopathological factors affecting overall survival rate in 173 CRC patients
Variables

Total (n)p value

AMIGO2 expression
Low (n = 124)High (n = 49)

Age (years)
< 70

87

64 (74%)23 (26%)≥ 70

86

60 (70%)26 (30%)male

95

66 (70%)29 (30%)female78

58 (74%)20 (26%)

colon

122

89 (73%)33 (27%)

rectum51

35 (69%)16 (31%)< 4.0 cm

68

47 (69%)21 (31%)≥ 4.0 cm

105

77 (73%)28 (27%)differentiated typea

157

110 (70%)

47 (30%)undifferentiated typeb

16

14 (88%)2 (12%)T1/2

11

7 (64%)

4 (36%)

T3/4

162

117 (72%)45 (28%)absent90

70 (78%)20 (22%)

present

83

54 (65%)29 (35%)

ly0/1

65

48 (74%)17 (26%)

ly2/3

108

76 (70%)32 (30%)v0/1

97

75 (77%)22 (23%)v2/3

76

49 (65%)27 (35%)

0.580

Sex
0.478

Tumor location
0.565

Tumor size
0.548

Histological grade
0.115

Depth of invasionc
0.380

Lymph node metastasis
0.064

Lymphatic invasiond
0.623

Vascular invasione

a

0.050

Well- and moderately differentiated adenocarcinoma
Poorly differentiated adenocarcinoma, signet ring cell carcinoma, and mucinous carcinoma
T1, lamina propria or submucosa invasion; T2, muscularis propria invasion; T3, subserosa invasion or within adventitia; T4, serosa penetration or adjacent organ
invasiondly0, no invasion; ly1, minimal invasion; ly2, moderate invasion; ly3, severe invasionev0, no invasion; v1, minimal invasion; v2, moderate invasion; v3, severe invasionbc

Goto et al. Diagnostic Pathology(2022) 17:16

Page 8 of 12

Fig. 4 Relationship between AMIGO2 expression in primary colorectal cancer and metastatic site. A high expression of AMIGO2 was significantly
associated with liver metastases (A) and lung metastases (B), but not with peritoneal dissemination (C), as calculated using the X2 test

had AMIGO2 low expression (Table 1). To evaluate
the prognostic value of AMIGO2, we analyzed thecorrelations between clinicopathological variables andthe expression of AMIGO2 in tumor tissues (Table 1;
Fig. 4). A high expression of AMIGO2 (32/49; 65.3%)
was more closely associated with liver metastasis thanlow AMIGO2 expression (17/124; 13.7%) (p = 1.15E11; Fig. 4 A). Patients with high AMIGO2-expression
tumors were found to be more likely to develop lungmetastases (5/49; 10.2%) than patients with low expression tumors (3/124; 2.4%) (p = 0.042; Fig. 4B),
whereas AMIGO2 expression was found to be independent of peritoneal dissemination (high 2/49, 4.1%
vs. low 4/124, 3.2%: p = 0.546; Fig. 4 C). There wasno significant correlation between AMIGO2 expression and age, sex, tumor location, tumor size, histological grade, depth of invasion, lymph nodemetastasis, lymphatic invasion, and vascular invasion(Table 1).

Improved correlation between AMIGO2 expression
detected by rTNK1 mAb and liver metastasis and poor
prognosis

In an univariate Cox regression analysis for different
established risk factors for CRC, in addition to AMIGO2
expression (p = 0.005), age (p = 0.006) and vascular invasion (p = 0.019) were also associated with poor overall
survival (Table 2). To compare the independent predictive value of AMIGO2 status for overall survival, a multivariate analysis with Cox’s proportional hazard
regression model was performed. This analysis revealed
that AMIGO2 status (p = 0.022), age (p = 0.007), andvascular invasion (p = 0.029) had an independent prognostic impact (Table 2). Of particular interest waswhether AMIGO2 expression added any predictive value
as a widely used prognostic factor for liver metastasis in
CRC. Multivariate logistic regression analysis showedthat the risk factors for liver metastases were significantly higher in patients with AMIGO2 high-expressing

Table 2 Univariate and multivariate analysis for overall survival in CRC patients (Cox proportional hazard regression model)
Variablesa

Univariate analysis

Multivariate analysis
p value

Hazard ratio

95% Confidence
interval

p value

1.221–3.222

0.006

1.964

1.207–3.198

0.007

0.413–1.083

0.102

Hazard ratio

95% Confidence
intervalAge (< 70 vs. ≥ 70 years)

1.983

Sex (male vs. female)

0.669

Tumor location (colon vs. rectum)

0.716

0.415–1.238

0.232

Tumor size (< 4.0 vs. ≥ 4.0 cm)

1.285

0.789–2.092

0.314

Histological grade (differentiated vs. undifferentiated)

1.303

0.524–3.237

0.569

Depth of invasion (T1/2 vs. T3/4)

2.768

0.678–11.299

0.156

Lymph node metastasis (absent vs. present)

1.163

0.725–1.864

0.532

Lymphatic invasion (ly0/1 vs. ly2/3)

1.559

0.626–3.879

0.340

Vascular invasion (v0/1 vs. v2/3)

1.755

1.096–2.810

0.019

1.698

1.055–2.734

0.029

AMIGO2 expression (low vs. high)

2.016

1.237–3.284

0.005

1.781

1.087–2.918

0.022

a

Histological grade, depth of invasion, lymphatic invasion and vascular invasion were determined by the same grade as described in Table 1

Goto et al. Diagnostic Pathology(2022) 17:16

Page 9 of 12

Table 3 Univariate and multivariate analysis of factors affecting liver metastases in CRC patients (Logistic regression model)
Variablesa

Univariate analysis

Multivariate analysis

Odds ratio

95% Confidence
interval

p valueAge (< 70 vs. ≥ 70 years)

0.764

0.393–1.484

0.427

Sex (male vs. female)

0.485

0.242–0.970

0.041

Tumor location (colon vs. rectum)

0.734

0.345–1.561

0.422

Tumor size (< 4.0 vs. ≥ 4.0 cm)

1.490

0.743–2.986

0.261

Histological grade (differentiated vs. undifferentiated)

1.205

0.369–3.936

0.757

Depth of invasion (T1/2 vs. T3/4)

1.839

0.383–8.834

0.447

Lymph node metastasis (absent vs. present)

2.115

1.076–4.158

0.030

Lymphatic invasion (ly0/1 vs. ly2/3)

1.532

0.756–3.101

0.236

Vascular invasion (v0/1 vs. v2/3)

3.422

1.710–6.879

0.001

AMIGO2 expression (low vs. high)11.8485.433–25.837

0.000b

Odds ratio

95% Confidence
interval

p value

0.443

0.197–0.998

0.049

12.2545.511–27.247

0.000caHistological grade, depth of invasion, lymphatic invasion and vascular invasion were determined by the same grade as described in Table 1
p = 5.146E-10cp = 7.930E-10b

CRC than in those with AMIGO2 low-expressing tumors (p = 7.930E-10, Table 3).
A Kaplan-Meier survival analysis was performed tocompare which of the two anti-AMIGO2 monoclonal
antibodies could more accurately determine the clinically worse outcome of CRC patients based on AMIGO2
expression levels. Overall survival tended to have a poor
prognosis in patients with high AMIGO2 levels (Fig. 5 A).
Most notably, in an analysis, rTNK mAb (p = 0.004), but
not sc mAb (p = 0.107), predicted worse overall survival
in patients with high AMIGO2 expression than in thosewith low AMIGO2 expression (Fig. 5 A). Moreover, ahigh expression of AMIGO2 resulted in short diseasespecific survival, which is common to both antibodies,but rTNK mAb (p = 0.000044) was detected at a significantly higher level compared to sc mAb (p = 0.001;
Fig. 5B).
The above findings showed that immunohistochemical
detection of high AMIGO2 expression in CRC patientswith rTNK mAb serves as a superior diagnostic biomarker and allows for prediction of poor prognoses bydetecting liver metastasis compared to commerciallyavailable antibodies.

Discussion
In this study, we produced a rat rTNK mAb specific forhuman AMIGO2, and confirmed that it detects
AMIGO2 but does not cross-react with other AMIGO
family molecules; that is, AMIGO1 and AMIGO3. Bycomparing the results previously reported for commercially available sc mAb with rTNK mAb using the same
CRC tissues, the following five new facts and the usefulness of rTNK mAb were clarified: (i) the detection rateof liver metastases in CRC patients with high AMIGO2
expression in primary tumors improved from 47.5% for
sc mAb [11] to 65.3% for rTNK mAb (Fig. 4 A); (ii) the
association between AMIGO2 expression and liver metastasis by multivariate analysis resulted in predictability
with sc mAb (p = 1.707E-5) [11]; but it was significantly
predictable with rTNK mAb (p = 7.930E-10, Table 3);(iii) AMIGO2 expression and overall survival were notstatistically significant when detected with sc mAb (p =0.107), but was significantly correlated with worse prognosis when detected with rTNK mAb (p = 0.004,
Fig. 5 A); (iv) in disease-specific survival, high AMIGO2
expression resulted in a poor prognosis, which can also
be detected with sc mAb (p = 0.001), whereas it had amuch higher correlation with rTNK mAb (p = 0.000044,
Fig. 5B); and (v) factors affecting liver metastasis in
multivariate logistic regression analysis using sc mAb
were AMIGO2 expression (p = 1.707E-5), lymph nodemetastasis, vascular invasion, and sex [11]. In contrast,
analysis using rTNK mAb eliminated the risk of lymph
node metastasis and vascular invasion, leaving only
AMIGO2 expression (p = 7.930E-10) and sex (p =0.049). The above findings showed that by using rTNK
mAb, AMIGO2 expression serves as a superior prognostic immunohistochemical biomarkers, especially for thedetection of liver metastases and worse prognosis in
CRC patients, compared to commercially available sc
mAb.
Intriguingly, the use of rTNK mAb revealed that the
AMIGO2 protein was N-glycosylated. Since it has beenreported that the AMIGO1 protein, which is mainly
expressed in the central nervous system, is N-glycosylated [26], the AMIGO family proteins may be includedin those proteins in which half of all mammalianGoto et al. Diagnostic Pathology(2022) 17:16

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Fig. 5 Comparison of two AMIGO2 antibodies in AMIGO2 expression and survival in CRC patients. Cumulative survival rates were assessed using
the Kaplan-Meier plot method. Differences were analyzed using the log-rank test. Overall survival (A) and disease-specific survival (B) are shown

proteins are glycosylated as post-translational modifications [27]. In tumor tissues, abnormal glycosylation, that
is, alterations to glycan epitopes, such as truncated Oglycans; altered N-glycan branching; increased sialylation; and fucosylation have been observed [23, 28–30].
These specific glycosylations have been shown to accelerate malignancies in tumor cells, such as signal transduction [31], growth [32], tumor immunity [33],epithelial-mesenchymal transition [34], motility [31], andmetastasis [35], especially in CRC [36]. As a typical example of glycosylation-mediated liver metastasis in CRC
patients, primary tumors expressing sialyl LewisX (sLeX)
specifically bind or adhere to activated hepatic vascular
E-selectin [31, 37–39]. We believed that CRC cells expressing AMIGO2 selectively form liver metastases byspecifically binding to hepatic endothelial cells expressing AMIGO family molecules in a homophilic/heterophilic manner. To comprehensively understand themechanism of liver metastasis due to AMIGO2 expression, it is necessary to examine how N-glycosylation of
the AMIGO2 protein is associated with liver metastasis.
Since there are eight putative N-glycosylation sites at 58,
104, 281, 288, 345, 373, 381, and 384 asparagine residues[22] in AMIGO2 that undergo N-glycosylation, we aregoing to investigate the degree of these N-glycosylationand liver metastatic activity.
The association between organ carcinogenesis and
AMIGO2 expression has been reported in various typesof cancer, including gastric cancer [13, 14], pancreaticcancer [20], colorectal cancer [11, 19], breast cancer[12], ovarian cancer [17], endometrial cancer [21],melanoma [15, 16, 40], and pituitary neuroendocrine tumors [18]. Since AMIGO2 expression is involved not
only in tumor development but also in tumor progression including acquisition of metastasis, AMIGO2 may
be a new molecule that controls cancer stem cell-likefunction. Elucidating the relationship between AMIGO2
expression and organ carcinogenesis and its malignant
tumor progression not only improves the prognosis ofcancer patients, but also determines target molecules fortreatment and prevention. To proceed with these studies, it is conceivable to make great use of the specificantibody produced in this study.
In conclusion, we succeeded in establishing a monoclonal
antibody that specifically recognizes human AMIGO2 anddemonstrated its clinical applicability by immunohistochemistry. Pathological diagnosis by AMIGO2 expression using
the rTNK mAb for surgically resected or biopsied primary
CRC may be a promising new evaluation tool for predicting
liver metastasis and poor prognosis in CRC patients.
Abbreviations
AMIGO: amphoterin-induced gene and open reading frame; CRC: colorectal
cancer; mAb: monoclonal antibody; sc mAb: anti-AMIGO2 mousemonoclonal antibody clone sc-373699; rTNK mAb: anti-AMIGO2 rat
monoclonal antibody clone rTNK1A0012

Supplementary information
The online version contains supplementary material available at https://doi.
org/10.1186/s13000-021-01176-2.
Additional file 1. Flowchart of the experimental method forestablishing a monoclonal antibody specific for human AMIGO2.

Goto et al. Diagnostic Pathology(2022) 17:16

Additional file 2. The monoclonal antibodies with highimmunoglobulin titers were selected by enzyme-linked immunosorbent
assay (ELISA). From December 25, 2017 to January 22, 2018.
Acknowledgements
We would like to thank Yukako Sumida, Eri Kaneda, and Genki Hichiwa at
Tottori University, and Toko Kurosaki, Michika Fukino, Maya Hiratsuka, Masami
Morimura, Maki Takami, Kazuhisa Honma, Haruka Takayama and Shoko
Takehara at Trans Chromosomics Inc. for their technical support. This
research was partly performed at the Tottori Bio Frontier managed by Tottori
prefecture and Tottori Pharmaceutical Innovation Center managed by
Chromosome Engineering Research Center, Tottori University.
Authors’ contributions
KG, MOsa, RI, HI performed immunohistochemistry and quantification; RI, RS
performed in vitro experiments and biochemical analysis; KG wrote originaldraft; HT, HS, YK, HK, MOsh designed & arranged generation of monoclonal
antibodies; KG, MOsa, MY, YF, FO contributed to formulation of theexperimental design; KG, AT, MY YF performed statistical analysis; KG, MOsa, RI,
AT, HS, YK, MY, HK, HI, MOsh, YF, FO contributed to interpretation anddiscussion of the results; FO designed & arranged the whole experiments, andwrote the manuscript. The authors read and approved the final manuscript.

Page 11 of 12

2.

3.
4.

5.

6.

7.

8.
Funding
This work was supported in part by a Grant-in-Aid for Scientific Research C
to FO (20K07447), a Grant-in-Aid for Early-Career Scientists to AT (20K16303)
from the Japanese Ministry of Education, Culture, Sports, Science and Technology, CREST from Japan Science and Technology Agency to YK(JPMJCR18S4), and Basis for Supporting Innovative Drug Discovery and Life
Science Research of Japan Agency for Medical Research and Development
to YK (JP21am0101124).

10.

Availability of data and materials
All data generated or analyzed during this study are included in thispublished article.

11.

9.

Declarations
12.
Ethics approval and consent to participate
The experimental protocol was conducted according to the guidelines of
the Declaration of Helsinki, approved by the Tottori University Hospital
Institutional Review Board (ID: 17A142 on June 26, 2018), and approved by
the Committee of the Institute for Animal Experimentation of Tottori
University (ID: 17-Y-28 on September 11, 2017).

13.

14.
Consent for publication
Not applicable.
Competing interests
MOsh is a CEO, employee and shareholder of Trans Chromosomics, Inc. HT is
employees of Trans Chromosomics, Inc. The other authors declare no
financial or other interests regarding this manuscript that might beconstrued as a conflict of interest
Author details
1
Division of Experimental Pathology, Tottori University Faculty of Medicine,
86 Nishicho, 683-8503 Yonago, Japan. 2Division of Gastrointestinal andPediatric Surgery, Department of Surgery, School of Medicine, Tottori
University Faculty of Medicine, 683-8503 Yonago, Japan. 3Chromosome
Engineering Research Center (CERC), Tottori University, 683-8503 Yonago,
Japan. 4Trans Chromosomics, Inc., 683-8503 Yonago, Tottori, Japan. 5Divisionof Genome and Cellular Functions, Tottori University Faculty of Medicine,683-8503 Yonago, Japan.

15.

16.

17.

18.

19.

Received: 24 September 2021 Accepted: 26 November 2021
20.
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