5.2

7.4

ArticleA Novel High-Throughput
Screening Method for a
Human Multicentric
Osteosarcoma-Specific
Antibody and Biomarker Usinga Phage Display-Derived
Monoclonal Antibody
Takuma Hayashi, Naoki Yamamoto, Gene Kurosawa, Kaori Tajima, Mariko Kondo,
Noriko Hiramatsu, Yu Kato, Miho Tanaka, Hisateru Yamaguchi, Yoshikazu Kurosawa etal.

https://doi.org/10.3390/cancers14235829

cancers
ArticleA Novel High-Throughput Screening Method for a Human
Multicentric Osteosarcoma-Specific Antibody and Biomarker
Using a Phage Display-Derived Monoclonal Antibody
Takuma Hayashi 1 , Naoki Yamamoto 2,3, * , Gene Kurosawa 3 , Kaori Tajima 1 , Mariko Kondo 4 ,Noriko Hiramatsu 2 , Yu Kato 2 , Miho Tanaka 5 , Hisateru Yamaguchi 6 , Yoshikazu Kurosawa 7 ,Harumoto Yamada 1 and Nobuyuki Fujita 1
1
2

3

4
5

6
7

*

Citation: Hayashi, T.; Yamamoto, N.;
Kurosawa, G.; Tajima, K.; Kondo, M.;
Hiramatsu, N.; Kato, Y.; Tanaka, M.;
Yamaguchi, H.; Kurosawa, Y.; et al. A
Novel High-Throughput Screening
Method for a Human Multicentric
Osteosarcoma-Specific Antibody andBiomarker Using a Phage
Display-Derived Monoclonal
Antibody. Cancers 2022, 14, 5829.

Department of Orthopedic Surgery, Fujita Health University, Toyoake 470-1192, Japan
Support Office for Bioresource Research, Research Promotion Headquarters, Fujita Health University,
Toyoake 470-1192, Japan
International Center for Cell and Gene Therapy, Research Promotion and Support Headquarters,
Fujita Health University, Toyoake 470-1192, Japan
Perseus Proteomics Inc., Tokyo 153-0041, Japan
Center for Joint Research Facilities Support, Research Promotion and Support Headquarters,
Fujita Health University, Toyoake 470-1192, Japan
Yokkaichi Nursing and Medical Care University, Yokkaichi 512-8045, Japan
Institute for Comprehensive Medical Science, Fujita Health University, Toyoake 470-1192, Japan
Correspondence: naokiy@fujita-hu.ac.jp; Tel.: +81-562-93-2317

Simple Summary: Human multicentric osteosarcoma (HMOS) is a variant of human osteosarcoma(HOS) in which multiple bone lesions occur simultaneously or asynchronously before lung metastasis.
HOS is associated with a poor prognosis. Using the human antibody phage library, we obtained
95 types of antibody clones against HMOS cell lines and developed a fluorescence probe-based
enzyme-linked immunosorbent assay (ELISA) that enables high-throughput screening of the reactivityof these antibodies. The lysate of the HMOS cell line was incubated with the selected antibody, andthe antigen–antibody complex was recovered using magnetic beads. Liquid chromatography-mass
spectrometry (LC/MS) analysis of the sample bands extracted from gel electrophoresis detected
CAVIN1/PTRF specifically in HMOS cells. This novel high-throughput screening assay using
antibody–drug conjugate targeting for these specific proteins is promising for clinical applications.

https://doi.org/10.3390/
cancers14235829
Academic Editor: Frank Traub
Received: 4 October 2022
Accepted: 23 November 2022
Published: 26 November 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affiliations.

Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons

Abstract: Osteosarcoma is a malignant tumor that produces neoplastic bone or osteoid osteoma.
In human multicentric osteosarcoma (HMOS), a unique variant of human osteosarcoma (HOS),
multiple bone lesions occur simultaneously or asynchronously before lung metastasis. HMOS isassociated with an extremely poor prognosis, and effective treatment options are lacking. Using
the proteins in our previously generated HMOS cell lines as antigens, we generated antibodies
using a human antibody phage library. We obtained antibody clones recognizing 95 independent
antigens and developed a fluorescence probe-based enzyme-linked immunosorbent assay (ELISA)
technique capable of evaluating the reactivity of these antibodies by fluorescence intensity, allowing
simple, rapid, and high-throughput selection of antibody clones. These results were highly correlatedwith those using flow cytometry. Subsequently, the HMOS cell lysate was incubated with theantibody, the antigen–antibody complex was recovered with magnetic beads, and the protein bands
from electrophoresis were analyzed using liquid chromatography-mass spectrometry (LC/MS).
CAVIN1/polymerase I transcript release factor was specifically detected in the HMOS cells. In
conclusion, we found via a novel high-throughput screening method that CAVIN1/PTRF is an
HMOS-specific cell membrane biomarker and an antigen capable of producing human antibodies.
In the future, antibody–drug conjugate targeting of these specific proteins may be promising forclinical applications.

Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Cancers 2022, 14, 5829. https://doi.org/10.3390/cancers14235829

https://www.mdpi.com/journal/cancers

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Keywords: phage library; multicentric osteosarcoma; cell surface antigen; CAVIN1/Polymerase I
and transcript release factor (CAVIN1/PTRF)

1. Introduction
Human osteosarcoma (HOS) is a malignant tumor that produces neoplastic boneor osteoid, with a high prevalence among teenagers. It is considered the most common
malignant primary bone tumor [1]. There has been an increase in the number of patientswith cancer as the population ages. Mortality directly associated with bone metastasis ofa malignant tumor is rare; however, bone metastasis severely compromises the patient’s
quality of life through pain, hypercalcemia, and limited mobility [2]. The prognosis remains
poor in cases with ineffective chemotherapy, and lung metastasis is the primary cause of
mortality. Lung metastasis has been observed in approximately 25% of HOS [1]. The fiveyear survival rate for HOS has reached 60–70% owing to recent advances in chemotherapy,
diagnostic imaging, and surgical therapy [3,4]. However, from a clinical perspective,
prevention of bone metastasis was associated with further improvements in survival andquality of life.
Metastasis of malignant tumors is known to have tissue directivity. Breast, prostate,
and lung cancers are examples of tumors that readily metastasize. The “seed and soil”
hypothesis has been put forward, which states that organ-selective metastasis of a tumor
requires a mutual relationship between the biological environment of the tumor cells andthe target organ [5]. Metastasis of a malignant tumor undergoes several steps: proliferationof tumor cells in the primary lesion, vascular infiltration, dissociation from the primarysite, binding to the target organ, and proliferation at the new site [6]. Many researchers
have investigated the factors that govern the affinity of tumors to bone. They reported the
involvement of various factors in bone metastasis, including bone morphogenetic protein,interleukin, and cadherin [2,7–11].
Two types of HOS metastasis have been reported: HOS without bone metastasis
and osteosarcoma with bone metastasis. HOS with bone metastasis showed increased
expression of CD31, which is involved in migration between endothelial cells, and bonemorphogenetic protein (BMP), which induces and activates various mesenchymal cells that
affect bone formation. On the other hand, the expression of the Met/hepatocyte growth
factor (Met/HGF) receptor, which promotes HOS differentiation, was increased in HOS
without bone metastasis [12]. HOS cells have been reported to express αvβ3 integrin
as a ligand for CD31 [13,14]. The homophilic interaction between CD31–CD31 and theheterophilic interaction between CD31–αvβ3 integrin suggested that CD31 expression may
promote HOS cell adhesion to and migration between endothelial cells [12]. BMPs belong
to the transforming growth factor-β (TGF-β) superfamily of proteins. BMPs, named after
their ability to induce ectopic bone formation, have been implicated in BMP signaling in
the development and progression of musculoskeletal carcinoma [15]. BMP ligands andreceptors are expressed in most HOS cell lines, but BMP signaling activity is not decreasedin HOS compared to benign bone-forming tumors, suggesting that BMP signaling activity
is not a major factor in the pathogenesis of HOS. BMP signaling in HOS may be associatedwith a worse prognosis. At the cellular level, BMP signaling appears to mediate migratory
effects in HOS and chondrosarcoma cell types, possibly via integrin β1 interaction andactivation [16]. Vascular endothelial growth factor (VEGF) expression and high vascular
distribution within HOS may correlate with a poor prognosis, suggesting a higher rate ofcancer recurrence and a higher frequency of distant metastases in high VEGF groups [17].
In a particular variant of HOS, multiple bone lesions occur simultaneously or asynchronously before lung metastasis. This variant is referred to as human multicentricosteosarcoma (HMOS), which has been reported to have an incidence of 1.5–3.6% among
all HOS cases [18–21]. While the pathological characteristics of HMOS remain mostly
unclear, the common notion is that it is a specific form of multiple bone metastasis fromCancers 2022, 14, 5829

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tumor cells with a strong affinity to the bone. We previously reported a case of HMOS
in which we harvested tissues from a lesion site on the left humerus, assumed to be theprimary lesion, and a lesion on the sternum that had occurred later during chemotherapy.
We established HMOS cell lines from the two lesions [22]. We then compared the two celllines and observed a higher expression of the chemokine CXCR4, which is believed to be
involved in HOS metastasis, in the secondary (HMOS-P) as compared to the primary lesion
cell line (HMOS-A). This suggests that the secondary lesion likely metastasized from theprimary lesion [22].
The human antibody library termed “Antibody Intended for Multiple Screening”(AIMS) and the related methodology suite have been well established by the Division of
Antibody Project of the Fujita Health University, and have been used in several studies
to develop cancer therapeutic antibodies [23–27]. The AIMS human antibody librarycomprises approximately 1 × 1011 clone types obtained from human B-lymphocytes using
the phage display method. With this library, an antibody against a specific target antigen or
an unknown antigen may be isolated. The antibodies are fully human and are expected tohave clinical applications as anti-tumor drugs [24]. However, when obtaining an antibody
using the phage display system, there may be several hundreds of antibody types for one
type of cell. A lot of time and effort is required to investigate each antibody individually.
In this study, we performed a comprehensive antibody screening of HMOS-P surface
antigens using a phage display method. We established an antibody selection method
using a fluorescence probe-based enzyme-linked immunosorbent assay (ELISA), which
has a higher throughput than the conventional analytical method of flow cytometry (FCM).
We further aimed to identify, isolate, and characterize those antibodies strongly expressedwith bone affinity in HMOS cell lines but weakly expressed in HOS cell lines. We thus
attempted to identify potential novel biomarkers for HMOS diagnosis.
2. Materials and Methods
2.1. Cell Lines
We established two new HMOS cell lines (Supplementary Figure S1), one of which
was derived from the anterior (i.e., primary) lesion after an incisional biopsy of the left
humerus (designated as HMOS-A), and the other was derived from one of the posterior(i.e., secondary) lesions of the sternum (designated as HMOS-P) [22]. To compare the celllines, we used the following HOS cell lines: MNNG-HOS (EC87070201; European Collectionof Authenticated Cell Cultures: ECACC, Salisbury, UK), MG63 (EC86051601; ECACC),
and Saos2 (EC89050205; ECACC). MNNG-HOS, MG63, and Saos2 cells were cultured inaccordance with the culture conditions of ECACC. HOS is defined as malignant tumors thatform osteoids. Per this definition, NOS-1 (RCB1032) and NOS-2 (RCB1033) HOS cell linesthat form osteoids in vitro or in xenograft models were used to validate HMOS-specificproteins. NOS-1 and NOS-2 cells were provided by the Riken BioResource Research Center(RIKEN BRC) through the National BioResource Project of the Ministry of Education,
Culture, Sports, Science and Technology (MEXT), Japan. Mycoplasma contamination in thecell lines was inspected using a mycoplasma detection kit (EZ-PCRTM Mycoplasma Test Kit,
Biological Industries, Cromwell, CT, USA), according to the manufacturer’s instructions.
This study was approved by the Ethics Committee of Fujita Health University (approval
number: 15-181).
2.2. Antibody Library and Screening
The human antibody library (AIMS7 phage library) and the screening method of
the Division of Antibody Project of Fujita Health University Institute for Comprehensive
Medical Science were used [23,27]. Briefly, cultured HMOS-P cells were washed andincubated with 1 × 1013 colony-forming units (CFU) of AIMS human antibody library
phage at 4 ◦ C for 4 h with constant rotation. The final concentration of the reaction liquid
was then divided into two parts, each of which was layered onto 0.6-mL organic solvent

Cancers 2022, 14, 5829

4 of 15(dibutyl phthalate:cyclohexane = 9:1) and centrifuged. The precipitated cells and phagecomplex were snap-frozen in liquid nitrogen and thawed at 37 ◦ C to yield the phage.
The obtained phage was added to E. coli DH12 cultured in yeast extract-tryptone
culture medium at 37 ◦ C for 1 h. The phage-infected E. coli was cultured for 12–15 h
at 30 ◦ C. A 5-mL aliquot of the culture solution was mixed with KO7 helper phage andcultured. This culture solution was then centrifuged at 8000 rpm (11,800× g) for 10 min,the supernatant was collected, mixed with polyethylene glycol solution (20% polyethylene
glycol 6000, 2.5 M NaCl), and centrifuged to precipitate the phage. The phage was thensuspended in phosphate-buffered saline (PBS) to provide the first screening phage. Overall,four screening phages were collected, and the set of antibodies obtained from the fourthscreening was used for further experiments (Supplementary Table S1).
Ninety-five clones were selected for the fourth HMOS-P screening. All isolated clones
encoding antibodies were sequenced and classified according to the data summarized in
IgBLAST [28]. According to the classification of respective VH and VL nucleotide sequences,candidate monoclonal antibodies were selected for analysis.
2.3. Analysis of Fluorescence Probe-Based ELISAHMOS-P cells (4 × 104 ) were seeded onto a 96-well assay plate (Corning Inc., Corning,
NY, USA) and cultured for 24 h. The primary antibody, i.e., the phage solution obtained
from the antibody screening (antibody solution), was incubated with the cells for 1 h at
37 ◦ C. The cells were washed with PBS and incubated with the secondary antibody, antimouse cp3 polyclonal antibody (1:370 dilution), for 30 min at 37 ◦ C. The cells were thenwashed with PBS and incubated with the third antibody, Alexa 488 anti-mouse IgG (Molecular probes; 1:1000 dilution), for 30 min at 37 ◦ C. The cells were washed twice with PBS,
and the fluorescence intensity was measured using ARVO X (PerkinElmer Inc., Waltham,
MA, USA). Fluorescence intensity was measured at 25 sites per well, and values ≥ 10,000
arbitrary units (AU) were considered positive. Using the “positive” antibodies, ELISA
analyses were repeated in a similar fashion using HMOS-A, MNNG-HOS, MG63, andSaos2 cell lines. The analyses were performed five times in each case, and the fluorescenceintensity was compared using the median value from the five replicates.
2.4. Flow Cytometry Analysis
The antibodies judged to be positive by ELISA were used as the primary antibodies.
Cultured HMOS-A, HMOS-P, MNNG-HOS, MG63, and Saos2 cells were dissociated with
TrypLETM Select (Thermo Fisher Scientific K.K., Waltham, MA, USA) and washed with PBS.
The primary antibodies selected based on the ELISA screens (10, 12, 17, 19, 43, and 77) werediluted at 1:10 and incubated with the cells for 20 min at 4 ◦ C. The cells were then washedwith PBS. The cells were then incubated with the secondary anti-rabbit cp3 polyclonal
antibody (1:370 dilution) and the third Alexa 488 anti-rabbit IgG (1:1000 dilution) for 20 min
each at 4 ◦ C. The cells were washed with PBS in the dark and analyzed using FACScanTM(Becton Dickinson and Company, Franklin Lakes, NJ, USA). Each measurement of theindividual primary antibodies was repeated thrice, and the positive rate with respect to thenegative control and the GeoMean value of fluorescence intensity were calculated using
CellQuestTM Pro (Version 5.2.1, Becton Dickinson and Company, Waltham, MA, USA).
2.5. Immunohistochemistry
Paraffin sections of the HMOS posterior lesion site and HOS, from five patients, were
immunohistochemically stained using hematoxylin and eosin (HE) and primary antibodies
10, 12, 17, 19, 43, and 77. Before staining, the antigen was retrieved by immersing theparaffin sections in a citric acid buffer with pH 6.0 and heated twice at 90 ◦ C for 20 min in a
microwave. The sections were incubated with a 1:10 dilution of each of the six “positive”
primary antibodies for 1 h, washed with PBS, and incubated with the secondary antimouse cp3 polyclonal antibody (1: 370 dilution) at 37 ◦ C for 30 min. The sections were
washed with PBS and incubated with Histofine® Simple StainTM MAX-PO MULTI (Nichirei

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Corporation, Tokyo, Japan) at 37 ◦ C for 30 min. The sections were then stained with liquid
3,3′ -diaminobenzidine tetrahydrochloride (DAB) + Substrate Chromogen System (Dako),and cell nuclei were stained with hematoxylin. Finally, the sections were observed undera BX51 upright fluorescence microscope fitted with a DP71 digital camera (Olympus
Corporation, Tokyo, Japan).
2.6. Immunoprecipitation with Magnetic Beads and Electrophoresis
The HMOS-P cell line was lysed using the ULTRARIPA® Kit (BioDynamics Laboratory
Inc., Tokyo, Japan) to prepare cytoplasmic and cell membrane lysates. The lysate was individually incubated with antibody number 12, and an anti-rabbit cp3 polyclonal antibody
was added as the secondary antibody. The antigen–antibody conjugates were adsorbed byincubating with µMACS™ Protein A/G MicroBeads (Miltenyi Biotec BV & Co. KG, Gladbach, Germany). This reaction solution was then incubated with a MultiMACS™ Protein
A/G kit (Miltenyi Biotec BV & Co. KG), and the antigen–antibody complex was washedand concentrated by immunoprecipitation using magnetic beads. The antigen–antibody
complex was then cleaved by enzymes, released from the magnetic beads, and eluted fromthe column.
The cell lysate, the negative control (with only the antibody), and the antigen–antibody
complex incubated with the cell lysate were subjected to sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE). The gels were stained with SYPRO® Ruby Protein Gel
Stain (Thermo Fisher Scientific K.K.), and bands were detected using FLA3000G (FUJIFILM
Healthcare Laboratory Co., Ltd., Tokyo, Japan). Finally, the Coomassie-stained gels wereused to visualize the regions where the antigen–antibody complex was detected.
2.7. Protein Digestion and LC/MS Analysis
The gel was cut into smaller bands that reacted differently than the bands detectedby electrophoresis of the negative control, i.e., with antibody only. After washing the
gel fragments, they were de-stained with 50 mM ammonium bicarbonate (NH4 HCO3 )containing 50% acetonitrile (MeCN). The gel fragments were dehydrated with acetonitrile and dried in a vacuum evaporator. Cysteine residues were reduced using 10 mM
DTT/50 mM NH4 HCO3 , followed by alkylation with 8 µL of 500 mM iodoacetamide (IAA)
in 50 mM NH4 HCO3 . The gel fragments were washed and dehydrated again, and dried gel
fragments were digested with 200 ng of trypsin (Promega Corporation, Madison, WI, USA)in 50 mM NH4 HCO3 at 37 ◦ C for 16 h. The trypsinized gel extract with 50% MeCN/0.1%
trifluoroacetic acid (TFA) was pooled and desalted using GL-Tip SDB (GL Sciences Inc., Tokyo,
Japan). The samples were processed using a solution eluted with 80% MeCN/0.1% TFA.
The samples were analyzed by liquid chromatography (LC, EASY-n LC 1000, Thermo
Fisher Scientific) coupled to a mass spectrometer (MS, Orbitrap Fusion™ Tribrid™ Mass
Spectrometer, Thermo Fisher Scientific). The AcclamPepMap100 trapping column (#164946,
Thermo Fisher Scientific) and EASY-Spray LC columns (#ES800A, Thermo Fisher Scientific)
were used. The 60-min gradient program was performed with water containing 0.1%
formic acid (FA) and MeCN with 0.1% FA at a flow rate of 300 nL/min. Data analysis was
performed using Proteome Discoverer™ version 2.2 (Thermo Fisher Scientific) with MASCOT (Matrix Science, Boston, MA, USA) as the search engine and SwissProt HomoSapience(v2017-10-25) as the database. The Human Protein Atlas (www.proteinatlas.org (accessedon 1 November 2022)) was a freely available interactive resource, and was used as areference for the survey of candidate proteins resulting from the analysis [29].
2.8. Confirmation with Purified Protein and Commercial Antibody
Purified commercially available purified CAVIN1/PTRF (LifeSpan Biosciences, Inc.,
Seattle, WA, USA) was immobilized on a microplate and incubated with antibody number 12. After washing with PBS, an anti-mouse cp3 polyclonal antibody was added. Afterwashing with PBS, an anti-mouse goat IgG-HRP (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA, USA) HRP-labeled secondary antibody was added. After washing with PBS,

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the absorbance at 450 nm was measured with a microplate reader (Benchmark, Bio-Rad
Laboratories, Inc., Hercules, CA, USA) using a tetramethylbenzidine staining kit (Nacalai
Tesque, Inc., Kyoto, Japan). Instead of the primary antibody, the reaction with PBS was
measured as a blank.
As a confirmatory experiment, immunostaining of HMOS and HOS tissues (tissues
from three patients) was performed using the commercial antibody against CAVIN1/PTRF(Proteintech Group, Inc., Rosemont, IL, USA), which was diluted at 1:100 and incubatedat 37 ◦ C for 30 min. The sections were washed with PBS and incubated with Histofine®
Simple StainTM MAX-PO MULTI at 37 ◦ C for 30 min. The sections were stained with aliquid DAB+ Substrate Chromogen System, and cell nuclei were stained with hematoxylin.
In addition, osteoid-producing HOS cells (NOS-1, NOS-2) and HMOS-P cells were analyzed
in triplicate by FCM using CytoFLEXTM (Beckman Coulter, Inc., Brea, CA, USA). The cells
were incubated with 1:50 dilution of antibody against CAVIN1/PTRF for 20 min at 4 ◦ C.
The second antibody, Alexa 488 anti-rabbit IgG (1:1000 dilution), was incubated for 20 min
at 4 ◦ C.
Western blot (WB) analysis was performed as follows. Total proteins of HMOS-P and
NOS-1 were extracted using a RIPA Lysis Buffer System (Santa Cruz Biotechnology). The
proteins were mixed with loading SDS sample buffer (Thermo Fisher Scientific) containing 5% 2-mercaptoethanol, electrophoresed through 12.5% e-PAGEL (ATTO Corporation,
Tokyo, Japan), and transferred onto Immobilon-P Transfer PVDF Membranes (Merck KGaA,
Darmstadt, Germany). The membranes were blocked with 0.3% non-fat dry milk/TBS0.1% Tween20 for 30 min and probed overnight with target antibodies. The following
antibodies were used: anti-CAVIN1/PTRF and β-actin (1:1000, Proteintech). The membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5000,
Proteintech), and the protein bands were visualized using Clarity Western ECL Substrate(BIO-RAD). The Amersham™ ImageQuant™ 800 system (Cytiva, Tokyo, Japan) was used
for chemiluminescence detection and gel imaging.
2.9. Statistical Analysis
Each experiment was performed in triplicate. Data are presented as the mean ± standard
deviation (SD) and were analyzed using Welch’s t-test and Spearman’s rank correlation
coefficient. Statistical Package for Social Science (SPSS) Statistics 24 (IBM, New York, NY,
USA) was used to perform the statistical analyses. A p-value < 0.05 was considered toindicate statistical significance.
3. Results
3.1. Antibody Selection at Fluorescence Probe-Based ELISA
Among the generated antibody clones recognizing 95 independent antigens, antibody
numbers 10, 12, 17, 19, 43, and 77 exhibited a fluorescence intensity ≥ 10,000 AU with
HMOS-P and were therefore judged as positive (Figure 1). Antibody reactions using
the ELISA method were performed on all cell types using the six antibodies (Figure 2).
All antibodies exhibited stronger fluorescence intensity, indicating greater reactivity with
HMOS-A than with HMOS-P. Antibody number 10 was positive for all cells except for
Saos2 cells. Antibody numbers 12, 17, 19, and 77 were positive only for HMOS-A and
HMOS-P, and the fluorescence intensity was less than 10,000 AU in the other HOS cell lines.
Antibody number 43 was positive for HMOS-A, HMOS-P, and Saos2. The cell lines tested
negative for mycoplasma infection.

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Figure 1. Measurement of fluorescence intensity of the 95 clones prepared by the fourth screening of the secondary human multicentric osteosarcoma (HMOS-P) using fluorescence probe-based
enzyme-linked immunosorbent assay (ELISA). Among these, six clones showed a fluorescence≥intensity≥ 10,000 arbitrary units (AU).
≥Figure 2. Comparison of antibody reactivities toward various cell lines. The six clones were also
incubated with HMOS-P, the primary human multicentric osteosarcoma (HMOS-A), MNNG-HOS,
MG-63, and Saos2, and the fluorescence intensity was measured using ELISA. All antibodies showedstronger fluorescence intensity, indicating greater reactivity with HMOS-A than with HMOS-P.
Antibody numbers 10 and 43 were positive for MNNG-HOS. In MG63 cells, only antigen number 10
was positive, whereas only antigen number 43 was positive in Saos2 cells.

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3.2. Flow Cytometry
The positivity rate of FCM for HMOS-A, HMOS-P, MNNG-HOS, MG63, and Saos2
cells relative to their respective negative controls are listed in Table 1. The reactivity of eachantibody with HMOS-P is depicted in FCM histograms (Figure 3a–f), and the GeoMean
values for the fluorescence intensity of each cell type in FCM were compiled (Figure 3g).
Table 1. Positivity rate of flow cytometry (FCM) for HMOS-A, HMOS-P, MNNG-HOS, MG63, andSaos2 cells.
Cell LineNo. 10No. 12

No. 17

No. 19No. 43

No. 77

HMOS-A
HMOS-P
MNNG-HOS
MG63
Saos2

90.3 ± 1.1
77.5 ± 2.1
96.8 ± 0.9
76.4 ± 1.5
22.3 ± 1.9

88.9 ± 1.5
78.8 ± 2.3
3.4 ± 0.2
1.0 ± 0.1
0.6 ± 0.2

88.8 ± 1.5
77.1 ± 1.9
0.3 ± 0.1
0.2 ± 0.1
68.2 ± 3.6

76.1 ± 1.4
63.2 ± 2.2
0.2 ± 0.1
0.2 ± 0.1
0.7 ± 0.2

92.1 ± 1.0
77.8 ± 2.0
91.1 ± 1.4
41.5 ± 6.3
96.1 ± 0.7

92.9 ± 1.1
82.9 ± 1.7
45.4 ± 5.1
24.7 ± 2.1
14.4 ± 1.7

Figure 3. Correlation between ELISA-based and FCM fluorescence intensities at HMOS-P. FCM
histograms of antibody numbers 10 (a), 12 (b), 17 (c), 19 (d), 43 (e), and 77 (f) incubated with HMOS-P.
The FCM fluorescence intensity (GeoMean) for all cell lines was investigated. According to the resultsof flow cytometry of the HOS cell lines, most antibodies against antibody numbers 12, 17, 19, and 77
were negative (the values highlighted in blue were judged to be negative). In the HMOS cell line,
most cells of antibody numbers 12, 43, and 77 were positive (g). The correlation between ELISA
fluorescence intensity (median) and FCM fluorescence intensity (GeoMean) for all cell lines wasstrong. The correlation coefficient was r = 0.805 (h).

The correlation between ELISA (median) and FCM fluorescence intensities (GeoMean)
was investigated for all cell lines using Spearman’s rank correlation coefficient. We observed
a significant correlation, with a correlation coefficient of 0.805 (p < 0.05).

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3.3. Immunohistochemistry
The immunostaining results using tissue samples from the HMOS posterior lesion
site exhibited strong positive staining of the cells with antibody numbers 10, 12, 17, and
77. Antibody numbers 19 and 43 also revealed positive reactions, albeit with weakerstaining. Using HOS tissue samples, a weak reaction was observed with antibody number
10; however, the other antibodies were negative (Figure 4).

Figure 4. Immunohistochemistry of cancer tissues. Staining of similar paraffin sections of HMOS
(upper row) and HOS (lower row). The cell lines exhibited strong positive staining with antibody
numbers 10, 12, 17, and 77. The staining was also positive for antibody numbers 19 and 43 but
relatively weaker. Immunohistochemical staining shows positive cells (yellow arrowheads). These
results were comparable to those of the ELISA fluorescence intensity. Scale bar, 50 µm.

3.4. Identification of Antigen Reacting with Antibody
Based on the results of ELISA fluorescence intensity (median), FCM fluorescenceintensity (GeoMean), and immunohistochemistry, we selected antibody number 12, which
was highly reactive in HMOS and weakly reactive in all HOS. Subsequently, antibody
number 12 was added to the cell lysates. In antibody numbers 12, a band of immune
complexes, in which an antigen and a secondary antibody were bound to the antibody, wasCancers 2022, 14, 5829

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detected (Figure 5a,b). LC/MS analysis of this band revealed that antibody 12 corresponded
to CAVIN1/PTRF, respectively. Bands detected in the IP that were different or stronger
than those detected in the negative control (with only the antibody) were also analyzed,
but no specific proteins were detected in the MASCOT data analysis.

Figure 5. SDS-PAGE of the antigen–antibody complex and confirmation analysis with purified
protein. Using immunoprecipitation through magnetic beads, the band incubated separately withthe antibody was detected. Number 12 (a); M, marker; L, cell lysate solution; N, negative; IP,
positive samples recovered by immunoprecipitation. Figure 5a is inverted in black and white andshows an enlarged view of more specific bands. This band (yellow arrowhead) was analyzed by
Liquid chromatography-mass spectrometry (LC/MS) (b). The reactivity of commercially available
CAVIN1/PTRF with antibody number 12 was confirmed. The protein had a stronger reactivity withthe purified protein compared to that with the negative control, suggesting the occurrence of anantigen–antibody reaction (* p < 0.001) (c).

Therefore, to confirm the reactivity of antibody number 12, its absorbance was compared against commercially available purified protein using ELISA. The absorbance ofantibody number 12 against the CAVIN1/PTRF antigen was 1.860 ± 0.121, and the antigen
was detected. The non-solidified negative was 0.943 ± 0.098, which was determined to besignificantly different by Welch’s t-test (p < 0.001; Figure 5c).
Immunostaining with CAVIN1/PTRF antibody revealed positive cells in HMOS tissues but no positive cells in HOS tissue. FCM using CAVIN1/PTRF antibody revealed
all-positive HMOS-P cells, slightly positive NOS-1 cells (6.8%), and all-negative NOS-2
cells. The WB analysis revealed a specific and strong band of CAVIN1/PTRF at 50 kDa in
HMOS-P. On the other hand, a very weak band was observed in HOS at 50 kDa. The WB
results were very similar to the FCM results (Figure 6).

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Figure 6. Immunostaining, FCM, and Western blot (WB) as a validation experiment for
CAVIN1/PTRF. Tissue immunostaining of HMOS and HOS (upper row, red/yellow arrowheads
indicate positive cells) and FCM of HMOS-P and NOS-1 cells (lower row) using the commercial antibody for CAVIN1/PTRF. The WB analysis result showed a specific and strong band (red arrowhead)of CAVIN1/PTRF. A very weak band at approximately 43 kDa, as calculated molecular weight, wasalso detected (yellow arrowhead). M, marker; 1, cell lysate of HMOS-P solution; 2, cell lysate of HOS
solution. Scale bar = 50 µm.

4. Discussion
In this study, we comprehensively analyzed HMOS-P surface antigens using a phagedisplay method and established a high-throughput antibody selection method using fluorescent ELISA, which is simpler than conventional FCM-based methods. We identified one
protein (CAVIN1/PTRF) strongly expressed in the HMOS cell lines. In validation analysis
using a specific commercial antibody that reacted with the identified protein, the antibody
reacted specifically with HMOS tissue and not with the new HOS cell line. The cellular
protein CAVIN1/PTRF, which was identified in this study, has not been described in HMOS
or HOS. This protein is anticipated to develop into a novel molecular target marker for
HMOS, for which there is neither a test nor an efficient treatment.
Human antibodies are expected to have clinical applications as anti-tumor drugs [24].
HMOS-A showed a higher positivity rate than HMOS-P cells; therefore, these cells wereused to generate the antibody. This suggests that the antigens (proteins) recognized by thesix antibodies were more strongly expressed in HMOS-A than in HMOS-P. This differencein the expression levels might be attributed to chemotherapy; notably, HMOS-A cells wereobtained prior to chemotherapy, and HMOS-P cells were obtained from metastatic lesions
that developed during chemotherapy treatment. Consequently, the expression levels in
HMOS-P may have been slightly lower due to chemotherapy. However, the result of thisselection is a shared protein across HMOS stages, as it was expressed in both HMOS-A and
HMOS-P from primary and metastatic lesions, respectively. When FCM and ELISA were
performed using the six antibody types, a strong correlation was observed between ELISA
fluorescence intensity (median) and FCM fluorescence intensity (GeoMean) in HMOS-A
and HMOS-P cells. This indicates that the ELISA-based fluorescence measurements fromthe developed method are likely valuable for screening phage antibodies. These resultspresent a high-throughput method for selecting human antibodies that react with highly
expressed proteins.
Antibody number 12 recognized CAVIN1/PTRF. CAVIN1/PTRF is a caveolar structural protein. Previously, CAVIN1 was termed Cav-P60 and reported [30,31] to formcaveolae, an Ω structure on the cell membrane, with Caveolin-1. Caveolae is a cell memΩ

Cancers 2022, 14, 5829

12 of 15brane microdomain involved in regulating intracellular signaling pathways. Caveolae
formation and maintenance were initially thought to be primarily due to the caveolin 21 kD
protein. Three homologous genes are associated with caveolae expression in mammaliancells: Cav1, Cav2, and Cav3. The resultant proteins share a common topology. Caveolin
is synthesized as a monomer and transported to the Golgi apparatus. Caveolins then
combine with lipid rafts to form oligomers. These oligomerized caveolins form caveolae.
The presence of caveolae results in localized changes in membrane morphology [32]. PTRF
is a soluble protein containing a nuclear localization signal and a PEST domain imported
into the cell nucleus by nuclear transport [33]. Moreover, PTRF (Cavin1) forms a trimer
with Cavin2 and Cavin3 in caveolae formation and interacts with other membrane-boundproteins such as EHD2 and caveolae [34]. PTRF interacts with ZNF148 [35], and ZNF148
interacts with p53 [36]. Furthermore, the role of PTRF in cellular senescence reportedly
depends on its interaction with caveolin-1, which appears to be regulated by targeting the
caveolae and phosphorylation of PTRF, a regulator of cellular senescence acting throughthe p53/p21 and caveolae pathways [37]. Caveolin-1 reportedly functions as a tumor
suppressor in sarcomas [38–40]. To the best of our knowledge, there have been no priorreports on the action of CAVIN1 in HOS.
The tissue specificity of CAVIN1/PTRF is relatively low, but it is expressed in mesenchymal cells, especially in adipose tissue, and has been reported as a prognosticallyunfavorable marker in urothelial cancer, ovarian cancer, and colorectal cancer. Since
CAVIN1/PTRF is a central component of the CAVIN complex, and CAVIN1/PTRF is essential for caveolae complex formation [41], it interacts with a variety of proteins and genes.
Caveolin-1 (Cav1) and CAVIN1/PTRF are components of caveolae, and Cav1 isoforms
alpha and beta and BMP receptors BR1a and BR2 have been reported to co-localize at thecell surface [42]. Furthermore, the localization of BMP receptors in membrane domains has
been associated with distinct endocytosis pathways that specifically affect Smad-dependentand Smad-independent signaling cascades [43].
In Ewing’s sarcoma, hypermethylation was identified in the body of the PTRF geneand in the CpG located in the S-Shore, correlating with transcriptional repression status.
When the membrane caveolae of Ewing’s sarcoma cells are restored by the reintroductionof CAVIN1/PTRF, the MDM2/p53 complex is disrupted, resulting in p53 activation andinduction of apoptosis [40].
In addition, the lysophosphatidic acid (LPA) receptor LPAR1 is highly expressed incommon osteosarcoma cells. It was found that when osteosarcoma cells come into contact
with platelets in the bloodstream, platelets are activated, and the invasive potential isenhanced via LPA produced and released from the activated platelets, resulting in lungmetastasis. This indicates that LPAR1 antagonist administration may inhibit pulmonary
metastasis of osteosarcoma [44]. CAVIN1/PTRF, which is specifically expressed in HMOS
as revealed in this study, should also be investigated with antagonists.
The phage library method allowed us to rapidly narrow down the functional molecules
in HMOS. The human antibody against CAVIN1/PTRF detected in this study may beclinically applicable as an anti-tumor drug. However, our study has some limitations. Our
method is not suitable for the analysis of exclusively expressed intracytoplasmic proteins
and we did not verify gene expression using RNA sequencing analysis. We were unable
to perform confirmation outside of our own experience with HMOS cases, as HMOS is avery rare disease, and no cell lines were established from HMOS-derived cells. Although
research on biomarkers has progressed remarkably, our method proves that it is possible
to analyze not only genes but also expressed proteins, and furthermore, that the detected
proteins can be recognized as antigens and antibodies can be produced. It has been very
time-consuming and laborious to select and produce antibodies and to identify the antigens
recognized by these antibodies. We believe that our newly developed method overcomes
these weaknesses. In addition, we will continue to explore the possibilities for clinicalapplication of antibody–drug conjugate for CAVIN1/PTRF, which were found in this study,in the treatment of HMOS or the prevention of bone metastasis. Because we investigated aCancers 2022, 14, 5829

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rare case of HMOS, the antibody needs to be validated in a number of other HMOS cases
for potential application in testing and treatment approaches.
5. Conclusions
We performed a comprehensive antibody screening of HMOS-P surface antigens
using the phage display method and developed an antibody selection technique using
fluorescence-based ELISA. The results of this ELISA technique exhibited a strong correlation
with those of the conventional FCM method, which allowed the identification of a humanantibody that reacts specifically with HMOS tissue samples. CAVIN1/PTRF was observedto specifically bind to HMOS cell lines. We believe that this protein may be used as potential
biomarker in the diagnosis or treatment of HMOS.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cancers14235829/s1, Figure S1: Two novel HMOS cell lines;
Table S1: Phage Library Screening for HMOS-P.
Author Contributions: Conceptualization, T.H., N.Y., H.Y. (Harumoto Yamada) and N.F.;
methodology, N.Y., G.K., M.K., N.H., Y.K. (Yu Kato), M.T., H.Y. (Hisateru Yamaguchi)
and Y.K. (Yoshikazu Kurosawa); software, H.Y. (Hisateru Yamaguchi); validation, N.Y. and N.F.;
formal analysis, N.Y., H.Y. (Hisateru Yamaguchi) and N.F.; investigation, T.H., N.Y., G.K., K.T.,
M.K., N.H., Y.K. (Yu Kato), Y.K. (Yoshikazu Kurosawa) and H.Y. (Harumoto Yamada); resources,
N.Y.; data curation, N.Y.; writing—original draft preparation, T.H., N.Y. and N.F.; writing—review
and editing, T.H., N.Y., G.K., K.T., M.K., N.H., Y.K. (Yu Kato), M.T., H.Y. (Hisateru Yamaguchi),
Y.K. (Yoshikazu Kurosawa), H.Y. (Harumoto Yamada) and N.F.; visualization, N.Y.; supervision, T.H.,
N.Y., Y.K. (Yoshikazu Kurosawa) and N.F.; project administration, N.Y. and N.F.; funding acquisition,
N.F. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by JSPS KAKENHI (grant number JP23592201). This research
was supported by JST START University Ecosystem Promotion Type (Supporting Creation of Startup
Ecosystem in Startup Cities), Grant Number JPMJST2183, Japan.
Institutional Review Board Statement: The study was conducted in accordance with the Declarationof Helsinki, and approved by the Institutional Review Board of Fujita Health University (protocol
code 15-181, 9 June 2017).
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We would like to thank Masato Morikawa for editing and reviewing thismanuscript for English language and technical support. We also thank Kaoru Saijo (RIKEN BRC)
who assisted with the cell subdivision.
Conflicts of Interest: The authors declare no conflict of interest.

References
1.
2.
3.

4.
5.
6.
7.

Sun, X.; Niu, G.; Yan, Y.; Yang, M.; Chen, K.; Ma, Y.; Chan, N.; Shen, B.; Chen, X. Phage display-derived peptides for osteosarcoma
imaging. Clin. Cancer Res. 2010, 16, 4268–4277. [CrossRef] [PubMed]
D’Amico, L.; Roato, I. The Impact of Immune System in Regulating Bone Metastasis Formation by Osteotropic Tumors. J. Immunol.
Res. 2015, 2015, 143526. [CrossRef] [PubMed]
Senerchia, A.A.; Macedo, C.R.; Ferman, S.; Scopinaro, M.; Cacciavillano, W.; Boldrini, E.; Lins de Moraes, V.L.; Rey, G.; de Oliveira,
C.T.; Castillo, L.; et al. Results of a randomized, prospective clinical trial evaluating metronomic chemotherapy in nonmetastatic
patients with high-grade, operable osteosarcomas of the extremities: A report from the Latin American Group of Osteosarcoma
Treatment. Cancer 2017, 123, 1003–1010. [CrossRef] [PubMed]
Guan, M.; Wang, J.; Yang, L.; Zhao, Z.; Lu, K.; Zhao, L.; Xiao, J.; Li, Z.; Shi, Z. Targeting osteosarcoma vasculature with peptide
obtained by phage display. Contemp. Oncol. 2014, 18, 165–170. [CrossRef]
Fidler, I.J.; Yano, S.; Zhang, R.D.; Fujimaki, T.; Bucana, C.D. The seed and soil hypothesis: Vascularisation and brain metastases.
Lancet Oncol. 2002, 3, 53–57. [CrossRef]
Ramaswamy, S.; Ross, K.N.; Lander, E.S.; Golub, T.R. A molecular signature of metastasis in primary solid tumors. Nat. Genet.
2003, 33, 49–54. [CrossRef]
Breuksch, I.; Weinert, M.; Brenner, W. The role of extracellular calcium in bone metastasis. J. Bone Oncol. 2016, 5, 143–145. [CrossRef]

Cancers 2022, 14, 5829

8.
9.
10.
11.
12.
13.
14.
15.
16.
17.

18.
19.
20.
21.
22.

23.

24.

25.
26.
27.

28.
29.
30.
31.
32.
33.

34.

14 of 15

Satcher, R.L.; Pan, T.; Cheng, C.J.; Lee, Y.C.; Lin, S.C.; Yu, G.; Li, X.; Hoang, A.G.; Tamboli, P.; Jonasch, E.; et al. Cadherin-11 inrenal cell carcinoma bone metastasis. PLoS ONE 2014, 9, e89880. [CrossRef]
Pulido, C.; Vendrell, I.; Ferreira, A.R.; Casimiro, S.; Mansinho, A.; Alho, I.; Costa, L. Bone metastasis risk factors in breast cancer.
Ecancermedicalscience 2017, 11, 715. [CrossRef]
Holen, I.; Lefley, D.V.; Francis, S.E.; Rennicks, S.; Bradbury, S.; Coleman, R.E.; Ottewell, P. IL-1 drives breast cancer growth andbone metastasis in vivo. Oncotarget 2016, 7, 75571–75584. [CrossRef]
Mi, F.; Gong, L. Secretion of interleukin-6 by bone marrow mesenchymal stem cells promotes metastasis in hepatocellular
carcinoma. Biosci. Rep. 2017, 37, BSR20170181. [CrossRef] [PubMed]
Arihiro, K.; Inai, K. Expression of CD31, Met/hepatocyte growth factor receptor and bone morphogenetic protein in bonemetastasis of osteosarcoma. Pathol. Int. 2001, 51, 100–106. [CrossRef] [PubMed]
Buckley, C.D.; Doyonnas, R.; Newton, J.P.; Blystone, S.D.; Brown, E.J.; Watt, S.M.; Simmons, D.L. Identification of alpha v beta 3 asa heterotypic ligand for CD31/PECAM-1. J. Cell Sci. 1996, 109 Pt 2, 437–445. [CrossRef]
Stuiver, I.; Ruggeri, Z.; Smith, J.W. Divalent cations regulate the organization of integrins alpha v beta 3 and alpha v beta 5 on thecell surface. J. Cell Physiol. 1996, 168, 521–531. [CrossRef]
Bami, M.; Mavrogenis, A.F.; Angelini, A.; Milonaki, M.; Mitsiokapa, E.; Stamoulis, D.; Soucacos, P.N. Bone morphogenetic protein
signaling in musculoskeletal cancer. J. Cancer Res. Clin. Oncol. 2016, 142, 2061–2072. [CrossRef] [PubMed]
Nguyen, A.; Scott, M.A.; Dry, S.M.; James, A.W. Roles of bone morphogenetic protein signaling in osteosarcoma. Int. Orthop.
2014, 38, 2313–2322. [CrossRef] [PubMed]
Lammli, J.; Fan, M.; Rosenthal, H.G.; Patni, M.; Rinehart, E.; Vergara, G.; Ablah, E.; Wooley, P.H.; Lucas, G.; Yang, S.Y.
Expression of Vascular Endothelial Growth Factor correlates with the advance of clinical osteosarcoma. Int. Orthop. 2012,36, 2307–2313. [CrossRef]
Parham, D.M.; Pratt, C.B.; Parvey, L.S.; Webber, B.L.; Champion, J. Childhood multifocal osteosarcoma. Clinicopathologic andradiologic correlates. Cancer 1985, 55, 2653–2658. [CrossRef]
Corradi, D.; Wenger, D.E.; Bertoni, F.; Bacchini, P.; Bosio, S.; Goldoni, M.; Unni, K.K.; Sim, F.H.; Inwards, C.Y. Multicentricosteosarcoma: Clinicopathologic and radiographic study of 56 cases. Am. J. Clin. Pathol. 2011, 136, 799–807. [CrossRef]
Bacci, G.; Fabbri, N.; Balladelli, A.; Forni, C.; Palmerini, E.; Picci, P. Treatment and prognosis for synchronous multifocal
osteosarcoma in 42 patients. J. Bone Jt. Surg. Br. 2006, 88, 1071–1075. [CrossRef]
Hameed, S.; Vijayan, S.; Naik, M.; Rao, S. Multicentric osteosarcoma. Singap. Med. J. 2012, 53, e214–e217.
Yamamoto, Y.; Yamamoto, N.; Tajima, K.; Ohno, A.; Washimi, Y.; Ishimura, D.; Washimi, O.; Yamada, H. Characterization ofhuman multicentric osteosarcoma using newly established cells derived from multicentric osteosarcoma. J. Cancer Res. Clin.
Oncol. 2011, 137, 423–433. [CrossRef] [PubMed]
Akahori, Y.; Kurosawa, G.; Sumitomo, M.; Morita, M.; Muramatsu, C.; Eguchi, K.; Tanaka, M.; Suzuki, K.; Sugiura, M.; Iba,
Y.; et al. Isolation of antigen/antibody complexes through organic solvent (ICOS) method. Biochem. Biophys. Res. Commun. 2009,
378, 832–835. [CrossRef]
Kurosawa, G.; Sumitomo, M.; Ukai, Y.; Subere, J.; Muramatsu, C.; Eguchi, K.; Tanaka-Hashiba, M.; Sugiura, M.; Ando, M.; Sato,N.; et al. Selection and analysis of anti-cancer antibodies for cancer therapy obtained from antibody phage library. Cancer Sci.
2011, 102, 175–181. [CrossRef] [PubMed]
Kurosawa, G.; Sugiura, M.; Hattori, Y.; Tsuda, H.; Kurosawa, Y. Classification of 27 Tumor-Associated Antigens by Histochemical
Analysis of 36 Freshly Resected Lung Cancer Tissues. Int. J. Mol. Sci. 2016, 17, 1862. [CrossRef] [PubMed]
Kurosawa, G.; Kondo, M.; Kurosawa, Y. A variety of human monoclonal antibodies against epidermal growth factor receptor
isolated from a phage antibody library. Biochem. Biophys. Res. Commun. 2016, 480, 94–100. [CrossRef] [PubMed]
Kurosawa, G.; Akahori, Y.; Morita, M.; Sumitomo, M.; Sato, N.; Muramatsu, C.; Eguchi, K.; Matsuda, K.; Takasaki, A.; Tanaka,M.; et al. Comprehensive screening for antigens overexpressed on carcinomas via isolation of human mAbs that may betherapeutic. Proc. Natl. Acad. Sci. USA 2008, 105, 7287–7292. [CrossRef]
Ye, J.; Ma, N.; Madden, T.L.; Ostell, J.M. IgBLAST: An immunoglobulin variable domain sequence analysis tool. Nucleic Acids Res.
2013, 41, W34–W40. [CrossRef]
Uhlen, M.; Fagerberg, L.; Hallstrom, B.M.; Lindskog, C.; Oksvold, P.; Mardinoglu, A.; Sivertsson, A.; Kampf, C.; Sjostedt, E.;
Asplund, A.; et al. Proteomics. Tissue-based map of the human proteome. Science 2015, 347, 1260419. [CrossRef]
Govender, P.; Romero, F.; Shah, D.; Paez, J.; Ding, S.Y.; Liu, L.; Gower, A.; Baez, E.; Aly, S.S.; Pilch, P.; et al. Cavin1; a regulator oflung function and macrophage phenotype. PLoS ONE 2013, 8, e62045. [CrossRef]
Tillu, V.A.; Lim, Y.W.; Kovtun, O.; Mureev, S.; Ferguson, C.; Bastiani, M.; McMahon, K.A.; Lo, H.P.; Hall, T.E.; Alexandrov, K.; et al.
A variable undecad repeat domain in cavin1 regulates caveola formation and stability. EMBO Rep. 2018, 19, e45775. [CrossRef]
Lajoie, P.; Nabi, I.R. Lipid rafts, caveolae, and their endocytosis. Int. Rev. Cell Mol. Biol. 2010, 282, 135–163. [CrossRef]
Aboulaich, N.; Vainonen, J.P.; Stralfors, P.; Vener, A.V. Vectorial proteomics reveal targeting, phosphorylation and specificfragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes. Biochem. J.
2004, 383, 237–248. [CrossRef]
Kovtun, O.; Tillu, V.A.; Ariotti, N.; Parton, R.G.; Collins, B.M. Cavin family proteins and the assembly of caveolae. J. Cell Sci. 2015,
128, 1269–1278. [CrossRef]

Cancers 2022, 14, 5829

35.

36.
37.
38.

39.

40.

41.

42.
43.
44.

15 of 15

Hasegawa, T.; Takeuchi, A.; Miyaishi, O.; Xiao, H.; Mao, J.; Isobe, K. PTRF (polymerase I and transcript-release factor) is
tissue-specific and interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor whichbinds to the two mouse type-I collagen gene promoters. Biochem. J. 2000, 347 Pt 1, 55–59. [CrossRef] [PubMed]
Bai, L.; Merchant, J.L. ZBP-89 promotes growth arrest through stabilization of p53. Mol. Cell. Biol. 2001, 21, 4670–4683. [CrossRef]
Bai, L.; Deng, X.; Li, J.; Wang, M.; Li, Q.; An, W.; Deli, A.; Cong, Y.S. Regulation of cellular senescence by the essential caveolar
component PTRF/Cavin-1. Cell Res. 2011, 21, 1088–1101. [CrossRef] [PubMed]
Wiechen, K.; Diatchenko, L.; Agoulnik, A.; Scharff, K.M.; Schober, H.; Arlt, K.; Zhumabayeva, B.; Siebert, P.D.; Dietel, M.; Schäfer,R.; et al. Caveolin-1 is down-regulated in human ovarian carcinoma and acts as a candidate tumor suppressor gene. Am. J. Pathol.
2001, 159, 1635–1643. [CrossRef] [PubMed]
Faggi, F.; Chiarelli, N.; Colombi, M.; Mitola, S.; Ronca, R.; Madaro, L.; Bouche, M.; Poliani, P.L.; Vezzoli, M.; Longhena, F.; et al.
Cavin-1 and Caveolin-1 are both required to support cell proliferation, migration and anchorage-independent cell growth inrhabdomyosarcoma. Lab. Investig. 2015, 95, 585–602. [CrossRef]
Huertas-Martinez, J.; Court, F.; Rello-Varona, S.; Herrero-Martin, D.; Almacellas-Rabaiget, O.; Sainz-Jaspeado, M.; Garcia-Monclus,
S.; Lagares-Tena, L.; Buj, R.; Hontecillas-Prieto, L.; et al. DNA methylation profiling identifies PTRF/Cavin-1 as a novel tumor
suppressor in Ewing sarcoma when co-expressed with caveolin-1. Cancer Lett. 2017, 386, 196–207. [CrossRef]
Hill, M.M.; Bastiani, M.; Luetterforst, R.; Kirkham, M.; Kirkham, A.; Nixon, S.J.; Walser, P.; Abankwa, D.; Oorschot, V.M.; Martin,S.; et al. PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function. Cell 2008, 132, 113–124.
[CrossRef] [PubMed]
Nohe, A.; Keating, E.; Underhill, T.M.; Knaus, P.; Petersen, N.O. Dynamics and interaction of caveolin-1 isoforms with BMPreceptors. J. Cell Sci. 2005, 118, 643–650. [CrossRef] [PubMed]
Hartung, A.; Bitton-Worms, K.; Rechtman, M.M.; Wenzel, V.; Boergermann, J.H.; Hassel, S.; Henis, Y.I.; Knaus, P. Different routes
of bone morphogenic protein (BMP) receptor endocytosis influence BMP signaling. Mol. Cell. Biol. 2006, 26, 7791–7805. [CrossRef]
Takagi, S.; Sasaki, Y.; Koike, S.; Takemoto, A.; Seto, Y.; Haraguchi, M.; Ukaji, T.; Kawaguchi, T.; Sugawara, M.; Saito, M.; et al.
Platelet-derived lysophosphatidic acid mediated LPAR1 activation as a therapeutic target for osteosarcoma metastasis. Oncogene
2021, 40, 5548–5558. [CrossRef] [PubMed]