Activated leukocyte cell adhesion molecule expression correlates with the WNT subgroup in medulloblastoma and is involved in regulating tumor cell proliferation and invasion.

Cluster of differentiation (CD) 166 or activated leukocyte cell adhesion molecule (ALCAM) is a transmembrane molecule known to be an intercellular adhesion factor. The expression and function of ALCAM in medulloblastoma (MB), a pediatric brain tumor with highly advanced molecular genetics, remains unclear. Therefore, this study aimed to clarify the significance and functional role of ALCAM expression in MB. ALCAM expression in 45 patients with MB was evaluated by immunohistochemical analysis of formalin-fixed paraffin-embedded clinical specimens and the relationship between ALCAM expression and pathological type/molecular subgroup, such as WNT, SHH, Group 3, and Group 4, was examined. Eight ALCAM positive (18%), seven partially positive (16%), and 30 negative (67%) cases were detected. All seven cases of the WNT molecular subgroup were ALCAM positive and ALCAM expression strongly correlated with this subgroup (P < 0.0001). In addition, functional studies using MB cell lines revealed ALCAM expression affected proliferation and migration as a positive regulator in vitro. However, ALCAM silencing did not affect survival or the formation of leptomeningeal dissemination in an orthotopic mouse model, but did induce a malignant phenotype with increased tumor cell invasion at the dissemination sites (P = 0.0029). In conclusion, our results revealed that ALCAM exhibited highly specific expression in the WNT subgroup of MB. Furthermore, we demonstrated that the cell kinetics of MB cell lines can be altered by the expression of ALCAM.

Introduction

Medulloblastoma (MB) is the most common pediatric malignant brain tumor of the cerebellum. Histologically, MB is an embryonal tumor that may differentiate into cells of neural lineages and is classified as a grade IV tumor by the World Health Organization (WHO). There are several MB histological subtypes, including classic, desmoplastic/nodular, MB with extensive nodularity (MBEN), and large cell/anaplastic. Patients with MB are clinically stratified into average or high-risk groups according to their age, metastatic status, and the presence of residual tumor following resection [1]. However, these classifications do not account for MB heterogeneity. Recently, integrated genomic analysis of MB showed that MB consists of at least four distinct molecular subgroups, WNT, SHH, Group 3, and Group 4 [2-4]. This molecular classification of MB reflects distinct demographics and clinical features, including prognosis, transcriptomes, and genetics [2-5], and have been incorporated into the WHO classification of tumors of the central nervous system as revised in 2016 [6]. This new classification also provides for additional clinical risk stratification [5, 7]. However, a further understanding of disease based on the molecular subgroups and the subsequent development of treatment strategies is necessary.

Activated leukocyte cell adhesion molecule (ALCAM) is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily. ALCAM has been identified in a wide variety of tissues and cells, such as selected epithelia, lymphoid and myeloid cells, fibroblasts, neurons, and hepatocytes and is also referred to as CD166, CD6 ligand, MEMD, SB10 antigen, and HCA [8]. ALCAM is involved in cell-cell adhesion, either by homophilic (ALCAM-ALCAM) or heterophilic (ALCAM-CD6) interaction, and is also involved in organ development, neurogenesis, hematopoiesis, and immune responses [8]. In cancer, ALCAM expression is a prognostic marker for various tumor types [9]. For example, membranous ALCAM expression and ALCAM overexpression are independent markers of poor prognosis in colorectal carcinoma and pancreatic cancer, respectively [10, 11]. Contrarily, decreased ALCAM expression in breast cancer and loss of ALCAM membrane expression in ovarian cancer have been correlated with poor prognosis [12, 13]. Therefore, the impact of ALCAM expression on prognosis seems to depend on the cancer type, and in some types of cancer, membranous versus cytoplasmic ALCAM expression should also be considered [9]. In vitro and in vivo studies have demonstrated that ALCAM is involved in migration, invasion, and stemness in several cancers [14-19]. However, the functional significance of ALCAM in cancer is not consistent, with the differences depending on both the cancer type and tumor microenvironment.

The expression of ALCAM in MB has been previously reported, but its relevance to the molecular subgroups or histological classification has not been examined [20]. Furthermore, no study has analyzed the functional role of ALCAM in MB. In the current study, we retrospectively evaluated ALCAM expression in samples from patients with MB and examined the correlation between ALCAM expression and the MB molecular subgroups and histological subtypes. In addition, we investigated the functional role of ALCAM in MB using in vitro assays and an in vivo orthotopic mouse model.

Materials and methods

Clinical samples and patient characteristics

We retrospectively recruited sample cases that a) had available specimens surgically removed between 1996 and 2020 and b) were diagnosed with MB at the original treating institute. All cases were then centrally reviewed by a senior board-certified neuropathologist (Y.K.) for inclusion in the study. Forty-five case specimens of MB and their clinicopathologic information were obtained from 11 collaborating institutions (Osaka University Graduate School of Medicine, Osaka National Hospital, Kansai Medical University, Wakayama Medical University School of Medicine, Ehime University School of Medicine, Kitano Hospital, Takatsuki General Hospital, Kansai Rosai Hospital, Osaka City University Graduate School of Medicine, Osaka City General Hospital, and Hyogo College of Medicine) in the Kansai Molecular Diagnosis Network for CNS Tumors [21]. Approval of the study was obtained from the Institutional Review Boards (IRBs) of Osaka University Graduate School of Medicine (approval number: 13244), Osaka National Hospital (approval number: 713), and all the collaborative institutes. For all cases, either written informed consent was obtained or its requirement was waived by the IRB with a public announcement on the institution website. Immunohistochemistry and data analysis were performed at Osaka University Graduate School of Medicine and genetic analysis was performed at Osaka National Hospital and The Hospital for Sick Children. MB was histologically classified based on hematoxylin-eosin (HE) and reticulin silver staining as classic, desmoplastic/nodular, MBEN, or large cell/anaplastic subtype according to the 2016 WHO classification.

Immunohistochemistry

Six-micrometer sections of formalin-fixed paraffin-embedded (FFPE) tissues were used for immunohistochemistry. Heat-induced antigen retrieval was performed using a pressure cooker in 0.01 M citrate buffer (pH 6.0) for 10 min. Sections were incubated with a primary antibody against CD166/ALCAM [EPR2759(2); Abcam, Cambridge, MA, USA; 1:100 dilution] and β-catenin (BD Bioscience, San Jose, CA, USA; 1:100 dilution) at 4 °C overnight. Histofine Simple Stain MAX-PO (Nichirei, Tokyo, Japan) was used as a secondary antibody. The antibody complexes were visualized using the Dako Liquid DAB + Substrate Chromogen System (Dako, Carpinteria, CA, USA) and the sections were then counterstained with hematoxylin.

To identify tumor cells in the orthotopic mouse model, a primary antibody to human STEM121 (Takara Bio Inc., Shiga, Japan; 1:1,000 dilution) was used with POD Conjugate Set Anti Mouse, For Mouse Tissue reagent (Takara Bio Inc.).

When we analyzed ALCAM expression, we also evaluated whether the membrane or cytoplasm of the tumor cells were stained. The immunostaining of ALCAM was evaluated as the proportion of ALCAM-positive tumor cells in a representative area of tumor in the section. The cutoff values for the subdivision of the ALCAM staining was set at < 1% for negative staining, 1–25% for partially positive staining, and > 25% for positive staining. β-catenin immunostaining of tumor cells was considered positive only in cases of nuclear staining.

For ALCAM immunohistochemical staining, the identification of optimal cutoff points for the proportion of ALCAM-positive tumor cells in the WNT molecular subgroup was evaluated using receiver operating characteristic (ROC) curves and assessment of the area under the ROC curve (AUC).

Molecular subgrouping and genetic analysis

All samples were analyzed with molecular diagnostic techniques using the nanoString nCounter system (NanoString Technologies Inc., Seattle, WA, USA) [3, 4, 22] and by Sanger sequencing. The CTNNB1 mutation hotspot region in exon 3 was amplified and sequenced using forward primer 5’-TGGAACCAGACAGAAAAGCG-3’ and reverse primer 5’-ACAGGACTTGGGAGGTATCC-3’. Cases classified by the nanoString nCounter analysis or that presented with the CTNNB1 mutation and nuclear staining of β-catenin were defined as WNT subtype.

Quantitative PCR (qPCR)

Total RNA was extracted from cultured cells and clinical samples using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) or QIAzol Lysis Reagent (Qiagen) and reverse transcribed using PrimeScript RT Master Mix (Takara Bio Inc.) following the manufacturers’ protocols. qPCR was performed using TB Green Premix Ex Taq II (Takara Bio Inc.) and the Applied Biosystems ViiA7 Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA, USA). To measure ALCAM expression, the following primer pair was used: 5’-TCCTGCCGTCTGCTCTTCT-3’ (forward) and 5’-TTCTGAGGTACGTCAAGTCGG-3’ (reverse) [19]. As an internal reference for normalization, ACTB expression was measured using the following primer pair: 5’-CACCAACTGGGACGACAT-3’ (forward) and 5’-ACAGCCTGGATAGCAACG-3’ (reverse). Expression was measured relative to Human Brain, Cerebellum Total RNA (Takara Bio) and relative quantification analyses were performed using the ΔΔCT method [23].

R2: Genomics analysis and visualization platform dataset analysis

To validate ALCAM expression in MB, the Cavalli-763 MB dataset [24] from the R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl) was used. ALCAM gene expression, patient age, histological variant, and molecular subgroup data were extracted from the dataset.

Cell lines

Four established human MB cell lines were used in this study, Daoy, D341, ONS-76, and D283. Daoy, D341, and D283 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). ONS-76 was purchased from the Japanese Collection of Research Bioresources Cell Bank. Daoy and ONS-76 were cultured as adherent cells and D341 and D283 were cultured as cell suspensions. Daoy cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS); D341 cells were cultured in Minimum Essential Medium (MEM) supplemented with 20% FBS and 1% Non-Essential Amino Acids Solution (Gibco, Grand Island, NY, USA); ONS-76 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, and D283 cells were cultured in MEM supplemented with 10% FBS. Cultured cells were maintained at 37°C in a 5% CO2 atmosphere.

Flow cytometric analysis

Cells from subconfluent monolayer cultures were suspended in phosphate-buffered saline (PBS) and incubated with phycoerythrin (PE)-conjugated mouse anti-human CD166/ALCAM antibody (3A6; BD Bioscience). The labeled cells were then analyzed on a FACS Aria III flow cytometer (BD Bioscience) according to the manufacturer’s instructions. Analysis of the flow cytometry data was performed using FlowJo, version 10 software (BD Bioscience). ALCAM fluorescence intensity is reported as the intensity ratio (IR) obtained as follows: IR = ALCAM mean fluorescence intensity / negative control mean fluorescence intensity.

Knockdown of ALCAM expression

To generate cell lines in which ALCAM expression had been stably knocked down, we used the MISSION® RNAi system and lentiviral pLKO-puro vector (Sigma Aldrich, St Louis, MO, USA). The sequences of the shRNAs were as follows: shALCAM1, 5’-CCGGCAGCCATGATAATAGGTCATACTCGAGTATGACCTATTATCATGGCTGTTTTTG-3’ and shALCAM2, 5’-CCGGCTTCGATCTAGCCCGTCATTTCTCGAGAAATGACGGGCTAGATCGAAGTTTTTG-3’. Non-target shRNA Control (Sigma Aldrich) was used as negative control shRNA. Lentivirus was produced by transfection of the lentiviral vector and the Lentiviral Packaging Mix (Sigma Aldrich) in 293T cells. Daoy and ONS-76 cells were then infected with the lentivirus expressing the shRNAs. Knockdown of ALCAM was confirmed by flow cytometry and qPCR after puromycin selection. Before performing the in vitro and in vivo assays, the 10–15% cells with lower ALCAM expression were sorted from the heterogeneous Daoy cell population in which ALCAM had been knocked down (expressing different levels of the protein) by FACS using PE-conjugated mouse anti-human ALCAM antibody.

ALCAM overexpression

The expression vector ALCAM cDNA ORF clone pCMV3-C-GFPSpark (Sino Biological, Beijing, China) was used for ALCAM overexpression. D341 cells were transfected with the ALCAM cDNA plasmid using a lipofection method and Sinofection (Sino Biological). pCMV3-C-GFPSpark Control Vector (Sino Biological) was used as negative control. Overexpression of ALCAM was confirmed by qPCR after hygromycin B selection.

Cell proliferation assays

For cell proliferation assays, Daoy and ONS-76 cells were seeded into 6-well plates (2.0 × 104 cells per well). After incubation for 48 h and 96 h, live cells were counted using a OneCell Counter (BMS, Tokyo, Japan). For D341 cell proliferation assays, cells were seeded into 12-well plates (2.0 x 105 cells in 2 ml of medium per well) and live cells counted after incubation for 96 h and 192 h.

Wound healing assays

For wound healing assays, 1.2 × 105 Daoy cells or 1.6 × 105 ONS-76 cells were seeded into 12-well plates and incubated until confluency (24 h). A wound was made in the cell monolayer by scraping with a CELL Scratcher (AGC Techno Glass Co., Shizuoka, Japan). The wound gap width was measured at 0, 24, and 48 h for the Daoy cells and 0, 12, and 24 h for the ONS-76 cells using a Nikon TMS inverted microscope (Nikon, Tokyo, Japan) and WRAYCAM camera (WRAYMER INC., Osaka, Japan). Images were analyzed using Photoshop CC (Adobe Systems Co., San Jose, CA, USA).

Orthotopic mouse model

Male NOD/Shi-scid, IL-2RγKO Jic mice (7–8 weeks; In-Vivo Science Inc., Tokyo, Japan) were used for xenograft implantation. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee at the Osaka University Medical School (approval number: 29–041). All procedures involving animals were performed according to the animal use guidelines of the Animal Experiment Committee of Osaka University. The mice were anesthetized via intraperitoneal injection with three mixed sedatives (midazolam, butorphanol tartrate, and buprenorphine) and 2 × 105 stably transduced Daoy shControl or Daoy shALCAM1 cells in 2 μL of PBS were injected (n = 11 per group) through a burr hole into the right cerebellar hemisphere using a stereotactic injector (Stoelting, Wood Dale, IL). The mice were monitored every 1–2 days and immediately euthanized by overdose-anesthesia when severe neurological symptoms such as weight loss, loss of mobility, and severe paralysis were observed. The brains and spinal cords of the mice were subsequently dissected and formalin-fixed for histopathological analysis. For each mouse, the brain was sliced using a Mouse Brain Slicer (Muromati Kikai Co., Tokyo, Japan) to obtain three cerebral and three cerebellar coronal sections and the spinal cord was sliced to obtain five coronal sections. All dissected tissues were stained with HE and immunostained for CD166/ALCAM. STEM121 was used as a cytoplasm marker. Leptomeningeal dissemination and invasion at dissemination sites to the cerebrum, brain stem, and spinal cord of each section were evaluated histologically. Invasion was defined as a lesion that was continuous from the disseminated lesion and infiltrated into the subpial parenchyma. The histological evaluation of each section was performed blindly by three independent observers and the scores obtained were averaged.

Transwell assay

The invasion ability of ALCAM-depleted Daoy cells was assessed using a BioCoat Matrigel Invasion Chamber (BD Bioscience) according to the manufacturer’s instructions. Briefly, 1.0 × 105 Daoy cells in serum-free DMEM were seeded onto the Matrigel insert membrane and control insert membrane. DMEM containing 10% fetal bovine serum was added to the lower chamber as a chemoattractant and the chambers were incubated for 22 h. Afterward, the cells on the lower surface of the membrane were stained with Diff-Quik (Sysmex Co., Hyogo, Japan) and counted. The invasion percentage was calculated as follows:

Statistical analysis

All statistical analyses were performed using JMP, version 13 software (SAS Institute, Cary, NC, USA). Analysis of the relationship between ALCAM expression and the clinicopathological and molecular genetic parameters was tested using univariate analysis with the Fisher’s exact test. Comparison of ALCAM expression between groups in the R2: Genomics Analysis and Visualization Platform was performed using Student’s t-test and Tukey-Kramer test. For in vitro and in vivo studies, the distribution of data was verified with the Shapiro–Wilk test and parametric (One-way ANOVA and Dunnet’s post hoc test) and non-parametric (Wilcoxon rank sum test) methods were used for statistical analysis of the data. Survival data from the mouse model is presented in Kaplan-Meier plots and was analyzed with the log-rank test. Data are expressed as means ± SE. For all analyses, a P value < 0.05 was considered statistically significant.

Results

ALCAM expression correlated with the WNT molecular subgroup of MB

We investigated ALCAM expression in 45 human FFPE MB specimens using immunohistochemistry. We detected eight cases positive for ALCAM staining (18%), seven partially positive cases (16%), and 30 negative cases (67%). Interestingly, ALCAM expression showed a strong significant correlation to MB molecular subgroups (P < 0.0001; Table 1). All seven WNT cases and one SHH case, which histologically belongs to the MBEN subgroup, were ALCAM positive (Table 2). There was no clear correlation between ALCAM expression and the histologic variant of MB (P = 0.32; Table 1). Factors related to the WNT subgroup, such as nuclear β-catenin expression and CTNNB1 mutation status, correlated with ALCAM expression (P < 0.0001; Table 1).

Table 1

Correlation between clinicopathological/molecular data and ALCAM expression.

	ALCAM positive (n = 8)	ALCAM negative (n = 30) & partially positive (n = 7)	P value
Age group			0.022
Infant (< 4 years)	1	7
Child (4–16 years)	3	27
Adult (> 16 yeas)	4	3
Gender			0.19
Male	4	29
Female	4	8
Molecular subgroup			< 0.0001
WNT	7	0
SHH	1	7
Group 3	0	7
Group 4	0	21
N/A	0	2
Histological variant			0.32
classic	7	29
desmoplastic/nodular	0	5
MBEN	1	0
LCA	0	1
CTNNB1 status			< 0.0001
mutation	7	1a
wild type	1	36
Nuclear β-catenin expression			< 0.0001
positive	6	0
negative	2	37

ALCAM, activated leukocyte cell adhesion molecule; N/A, data not available; MBEN, medulloblastoma with extensive nodularity; LCA, large cell/anaplastic.

aThis case had a CTNNB1 silent mutation. Statistically significant findings are in bold.

Table 2

Overview of clinicopathological/molecular characteristics and ALCAM expression in the MB cases examined.

Case No.	Age group	molecular subgroup	Histological type	CTNNB1 mutation status	Nuclear β-catenin expression	ALCAM expression (positive cell proportion)	ALCAM staininglocalization
MB22	adulta	WNT	classic	c.94G>A, p.D32N	Positive	Positive (> 75%)	Cytoplasm + partially membrane
MB29	adulta	WNT	classic	c.98C>T, p.S33F	Positive	Positive (> 50%)	Cytoplasm
MB32	adulta	WNT	classic	c.134C>T, p.S45F	Positive	Positive (> 75%)	Cytoplasm
MB34	child	WNT	classic	c.101G>A, p.G34E	Negative	Positive (> 75%)	Cytoplasm
MB43	adult	WNT	classic	c.98C>G, p.S33C	Positive	Positive (>75%)	Cytoplasm
MB9	child	WNTb	classic	c.95A>G, p.D32G	Positive	Positive (> 75%)	Cytoplasm + partially membrane
MB30	child	WNTb	classic	c.98C>T, p.S33F	Positive	Positive (> 75%)	Cytoplasm + partially membrane
MB12	child	SHH	classic	NM	Negative	Negative	N/A
MB40	adult	SHH	classic	NM	Negative	Negative	N/A
MB45	child	SHH	classic	NM	Negative	Negative	N/A
MB7	adult	SHH	desmoplastic/nodular	NM	Negative	Negative	N/A
MB25	infant	SHH	desmoplastic/nodular	NM	Negative	Negative	N/A
MB36	child	SHH	desmoplastic/nodular	NM	Negative	Negative	N/A
MB37	infant	SHH	desmoplastic/nodular	NM	Negative	Negative	N/A
MB16	infant	SHH	MBEN	NM	Negative	Positive (> 75%)	Membrane + partially cytoplasm
MB5	child	Group 3	classic	NM	Negative	Partially positive	Cytoplasm
MB13	infant	Group 3	classic	NM	Negative	Partially positive	Cytoplasm
MB11	child	Group 3	classic	NM	Negative	Negative	N/A
MB20	infant	Group 3	classic	NM	Negative	Negative	N/A
MB21	child	Group 3	classic	NM	Negative	Negative	N/A
MB35	adult	Group 3	classic	c.91C>T, p.L31Lc	Negative	Negative	N/A
MB39	child	Group 3	classic	NM	Negative	Negative	N/A
MB3	child	Group 4	classic	NM	Negative	Partially positive	Cytoplasm
MB17	child	Group 4	classic	NM	Negative	Partially positive	Cytoplasm
MB1	child	Group 4	classic	NM	Negative	Negative	N/A
MB2	child	Group 4	classic	NM	Negative	Negative	N/A
MB6	child	Group 4	classic	NM	Negative	Negative	N/A
MB8	child	Group 4	classic	NM	Negative	Negative	N/A
MB15	child	Group 4	classic	NM	Negative	Negative	N/A
MB23	child	Group 4	classic	NM	Negative	Negative	N/A
MB24	child	Group 4	classic	NM	Negative	Negative	N/A
MB26	child	Group 4	classic	NM	Negative	Negative	N/A
MB27	child	Group 4	classic	NM	Negative	Negative	N/A
MB28	child	Group 4	classic	NM	Negative	Negative	N/A
MB31	infant	Group 4	classic	NM	Negative	Negative	N/A
MB33	child	Group 4	classic	NM	Negative	Negative	N/A
MB14	infant	Group 4	classic	NM	Negative	Negative	N/A
MB19	child	Group 4	classic	NM	Negative	Negative	N/A
MB38	child	Group 4	classic	NM	Negative	Negative	N/A
MB41	child	Group 4	classic	NM	Negative	Negative	N/A
MB42	child	Group 4	classic	NM	Negative	Negative	N/A
MB44	child	Group 4	classic	NM	Negative	Negative	N/A
MB18	child	Group 4	LCA	NM	Negative	Partially positive	Cytoplasm
MB10	child	N/A	classic	NM	Negative	Partially positive	Cytoplasm
MB4	infant	N/A	desmoplastic/nodular	NM	Negative	Partially positive	Cytoplasm

Adult: > 16 years, child: 4–16 years, infant: < 4 years.

ALCAM, activated leukocyte cell adhesion molecule; IHC, immunohistochemistry; M, male; F, female; N/A, data not available; MBEN, medulloblastoma with extensive nodularity; LCA, large cell/anaplastic; NM, no mutation.

a17–21 years.

bThese cases were defined as WNT according to the mutation of CTNNB1 and nuclear staining of β-catenin.

cSilent mutation.

To validate the correlation between the immunohistochemical staining data and ALCAM expression, we performed qPCR analysis of four ALCAM-positive and 13 ALCAM-negative cases. The mean relative expression level in the ALCAM-positive cases was 10 times higher than that in the ALCAM-negative cases (P = 0.0017; Fig 1A). These findings showed that ALCAM expression was higher in the WNT subgroup of MB than in the non-WNT subgroup. Furthermore, the correlation between ALCAM protein level determined by immunohistochemical staining and ALCAM mRNA expression was confirmed.

Fig 1

(A) Correlation between ALCAM immunohistochemical staining and ALCAM mRNA expression. ALCAM-positive medulloblastoma (MB) cases showed increased levels of ALCAM compared with the ALCAM-negative cases (P = 0.0017). The relative expression in human brain (cerebellum) was arbitrarily set at 1.0. (B) Receiver operating characteristic (ROC) curve for positive immunohistochemical expression of ALCAM in WNT subgroup. Area under the curve (AUC), 0.984. (C–E) ALCAM Expression in the Cavalli-763 MB cohort from the R2 Genomics Platform. (C) The WNT subgroup of MB strongly expressed ALCAM as compared with the other subgroups. (D) There was no clear correlation between ALCAM expression levels and histologic MB subtypes. (E) In the WNT subgroup of MB cases, ALCAM expression differed between the ages ≤ 20 y and ≥ 21 y. The box-and-whisker plots show the medians (thick horizontal lines) and interquartile ranges (IQRs; boundaries of the box) and ranges. *: P < 0.0001; N.S.: not significant.

To evaluate the reliability of the WNT subgroup of MB using ALCAM immunohistochemical staining, ROC curves were prepared using the proportion of ALCAM-positive tumor cells (0, 1, 25, 50, 75, 100%), from representative tumor areas, as the independent variable (Fig 1B). Analyzing the seven cases in WNT subgroup and the 36 cases in the non-WNT subgroup, the AUC was 0.984, indicating a high accuracy, and the optimal cutoff was > 25% or > 50%.

Similar results were observed in the large cohorts of the Cavalli-763 MB dataset [24] from the R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl). Specifically, ALCAM expression was significantly higher in the WNT subgroup of MB compared to that in the SHH, Group 3, and Group 4 subgroups (P < 0.0001; Fig 1C). On the other hand, no correlation was found between ALCAM expression and histologic subtypes of MB (Fig 1D). ALCAM expression was generally higher in the WNT subgroup, but this feature was not observed in cases of patients older than 21 y (mean, 33.2; range, 23–56 y; P < 0.0001; Fig 1E).

In the seven WNT molecular subgroup cases, the tumor cells of the whole section were diffusely stained for ALCAM (Fig 2A and 2B). Most of the ALCAM-positive tumor cells stained predominantly in the cytoplasm, but a few of them showed some staining in the cell membrane (Fig 2C). In the majority of the SHH, Group 3, and Group 4 subgroup cases, no expression of ALCAM was observed in the tumor cells (Fig 2D). We also observed seven partially positive cases in which ALCAM-positive cells with cytoplasmic staining were observed in some areas of the tissue section, two Group 3 cases, three Group 4 cases, and two cases in the N/A group (Fig 2E). The MBEN histological subtype, observed in only one case, showed a characteristic staining pattern of neuropil-like tissue and differentiated neurocytic cells in the expanded lobular architecture stained positive for ALCAM (mainly in the membrane), while the small round neurocytic cells in the internodular areas were negative (Fig 2F). In contrast, in five desmoplastic/nodular cases, the tumor cells showing signs of variable neurocytic maturation in pale islands failed to show ALCAM staining, similar to the internodular areas (Fig 2G). However, in one desmoplastic/nodular case, weak ALCAM staining was observed in the tumor cells of a few pale islands. In the normal cerebellum, ALCAM staining was absent in the molecular layer, while the granular layer and white matter stained weakly (Fig 2H).

Fig 2

Immunohistochemical staining of ALCAM in medulloblastoma (MB) specimens.

(A, B) ALCAM-positive cases: the WNT subgroup showed diffuse positive staining of ALCAM. (C) Most of the ALCAM-positive cells stained predominantly in the cytoplasm. (D) An ALCAM-negative case. (E) A partially positive case of ALCAM staining: few scattered ALCAM-positive cells can be observed. (F) The MBEN histological variant demonstrated a different ALCAM expression profile: positive staining in the lobular architectural region and negative in the internodular region. (G) The desmoplastic/nodular variant showed no ALCAM expression in most of the pale nodular areas and internodular areas. (H) In normal cerebellar tissue, the expression of ALCAM is not observed in the molecular layer, but is weakly detected in the granular layer and white matter. Original magnification, ×200 (A, B, D–G), ×1,000 (C) and ×100 (H).

Expression of ALCAM and its effect on cell proliferation and migration in MB cell lines

To evaluate the expression levels of ALCAM, we performed flow cytometry analysis of four human MB cell lines. ALCAM expression differed in each of the cell lines (Fig 3A) with the lowest levels being found in D341 cells (IR = 1.1) and the highest levels in Daoy and ONS-76 cells (IR = 73.3 and 109.2, respectively). Based on these results, we selected Daoy cells as a representative cell line with high ALCAM expression to investigate the functional role of ALCAM in MB. Two ALCAM-depleted Daoy cell lines were generated by RNA interference and the reduction of ALCAM expression was confirmed by flow cytometry analysis and qPCR (Fig 3B and 3C, respectively). The comparison between ALCAM-depleted and control Daoy cells showed that ALCAM depletion decreased cell proliferation and migration (Fig 3D and 3E, respectively). Specifically, there was no difference in cell proliferation at 48 h, while a difference was observed at 96 h, with the mean numbers (± SE) of the shControl, shALCAM1, and shALCAM2 Daoy cells being 28.3 ± 0.879, 22.9 ± 0.970, and 23.1 ± 0.798 × 104 cells, respectively (Fig 3D). Additionally, in wound healing assays, shALCAM1 and shALCAM2 Daoy cells showed 32% and 26% reduced migration at 24 h, respectively, and 24% and 11% at 48 h, respectively, compared to that of the shControl Daoy cells (Fig 3E). Similar results were observed with the ALCAM-depleted ONS-76 cell lines (Fig 3F–3I). These results demonstrated that ALCAM acted as a positive regulator of proliferation and migration in Daoy and ONS-76 cells in vitro.

Fig 3

Expression of ALCAM in medulloblastoma (MB) cell lines and in vitro study of its function in Daoy and ONS-76 cells.

(A) Four MB cell lines exhibited different ALCAM expression as measured by flow cytometry. (B, C, F, G) Knockdown of ALCAM in Daoy and ONS-76 cells was verified using flow cytometry (B, F) and qPCR (C, G). (D, E, H, I) ALCAM silencing inhibits the proliferation (D, H) and migration (E, I) of Daoy and ONS-76 cells. (J) Overexpression of ALCAM in D341 cells was confirmed by qPCR. (K) ALCAM overexpression promotes the proliferation of D341 cells. Data are reported as the mean ± SE. *: P < 0.05; **: P < 0.01; ***: P < 0.0001.

Furthermore, D341 cells, which had the lowest levels of ALCAM expression, were used to establish an ALCAM-overexpressing D341 cell line using cDNA transduction. Increased ALCAM expression was confirmed by qPCR analysis (Fig 3J). Comparison of ALCAM-overexpressing D341 cells and control D341 cells revealed that enhanced ALCAM expression resulted in increased cell proliferation (Fig 3K).

Depletion of ALCAM affects invasion at dissemination sites in an orthotopic mouse model of MB

Next, we assessed the effects of ALCAM on tumor progression and dissemination in vivo. Stably transfected ALCAM-depleted or control Daoy cells were inoculated into the right cerebellar hemisphere of mice. Tumors developed in both groups of mice. During the experiment, 9 mice in the shControl were euthanized and two were found dead while 10 mice in the shALCAM group were euthanized and one mouse was found dead. Although there was no significant difference in the survival rates between the shControl and shALCAM groups (P = 0.129, log-rank test), the survival of mice injected with ALCAM-depleted cells appeared to be shorter than that of the control mice (Fig 4A). Upon pathological evaluation of the brains and spinal cords of the mice, the primary tumors were found in the cerebellum and leptomeningeal disseminated lesions were found on the surface of the cerebrum, brain stem, and spinal cord. Immunostaining of ALCAM confirmed that depletion of ALCAM was maintained in the shALCAM group (Fig 4B). In the disseminated lesions, several foci of invasive tumor cells were found into the subpial parenchyma (Fig 4C). The frequency of sections with dissemination lesions in the two groups was similar (P = 0.812; Fig 4D). However, invasion at the dissemination sites was observed more frequently in the shALCAM group compared to that in the shControl group (P = 0.0029; Fig 4D). These results indicated that ALCAM depletion may be associated with a more invasive tumor-cell phenotype in vivo. On the other hand, there was no significant difference in the invasiveness of shControl and shALCAM1 Daoy cells as per the results of the in vitro Transwell assay. The invasion percentage (± SE) of the shControl and shALCAM1 Daoy cells was 176 ± 18.5 and 206 ± 29.8, respectively (P = 0.581; Fig 4E).

Fig 4

Functional analysis of ALCAM in an orthotopic mouse model and in vitro transwell assay in Daoy cells.

(A) Kaplan–Meier survival curves of mice injected with ALCAM-silenced and control Daoy cells showed no significant difference between the two groups (P = 0.129, log-rank test). (B) Representative immunohistochemical staining of ALCAM in primary tumors in the cerebellum. (C) Representative STEM121 staining showing invasive and noninvasive tumor cells in disseminated lesions. (D) The frequency of dissemination in the ALCAM-silenced and control group was similar (P = 0.812). The frequency of invasion observed at the dissemination sites was significantly higher in the ALCAM-silenced group than that in the control group (P = 0.0029). (E) The invasion percentage of ALCAM-silenced and control Daoy cells was not significantly different, as per the results of the transwell assay (P = 0.581). Original magnification, ×100 (B, C).

Discussion

In the current study, we first showed that ALCAM expression was strongly correlated to the WNT molecular subgroup of MB. Few studies have investigated the expression of ALCAM in MB; among them, one study assessed ALCAM levels in various primary central nervous system tumors, including MB [20]. In that study, Allmendinger et al [20] showed that the positive rate of ALCAM in MB is 31%, similar to that obtained in our study. However, the authors did not examine the relation between ALCAM expression and the molecular subgroup or histological subtypes [20].

Recent studies have focused on the molecular classification of MB and the expression of several genes related to molecular subgroups has been determined in MB. However, there is no report investigating the relationship between ALCAM and the WNT subgroup. Immunohistochemically, the nuclear staining of β-catenin resulting from a CTNNB1 mutation is associated with the WNT subgroup and is a good diagnostic tool for clinical applications [25]. However, immunohistological diagnosis of the WNT subgroup of MB using β-catenin nuclear staining alone is considered insufficient [26]. ALCAM has the potential of being a new WNT-related biomarker for improving the reliability of a WNT subgroup diagnosis of MB.

Interestingly, it has been recently reported that there is a strong correlation between nuclear staining of β-catenin and ALCAM levels in adamantinomatous craniopharyngioma [27], which is closely related to the Wnt signal pathway [28]. This report and our current results suggest a relationship between the Wnt signal pathway and ALCAM expression. It is known that embryonic expression of ALCAM occurs through non-canonical Wnt/JNK signaling [29, 30]. However, it is unclear whether the activation of the Wnt pathway caused by the CTNNB1 mutation is associated with ALCAM expression.

Despite the fact that ALCAM was originally identified as a membrane protein, the localization of ALCAM staining in MB was mainly cytoplasmic and only partially observed in the membrane. This is consistent with a previous report by Allmendinger et al [20]. In fact, ALCAM is known to be expressed in the cytoplasm of many carcinomas and the pattern of expression seems to vary depending on the particular carcinoma [11, 13, 19, 20]. There is also a report that cell function is altered by the translocation of ALCAM from the membrane to the cytoplasm [13].

Regarding the immunohistochemical evaluation of ALCAM, it is necessary to understand ALCAM expression in the normal brain. As we have shown, ALCAM was slightly expressed in the granular layer of the cerebellum. Furthermore, Allmendinger et al [20] have reported that ALCAM is expressed in the normal cerebral tissue, such as hippocampus and basal ganglia, and also in reactive glial cells. In the current study, we performed Immunohistochemical evaluation of ALCAM in representative tumor areas.

In our functional study using MB cell lines, we demonstrated that in vitro silencing of ALCAM in Daoy and ONS-76 cells inhibited cell proliferation and migration. Meanwhile, the overexpression of ALCAM in D341 cells promoted cell proliferation, although cell migration could not be evaluated due to the fact that the cells grow in suspension and are not adherent cells. Our findings indicate that ALCAM may act as a positive regulator of cell proliferation in MB. However, we obtained unexpected results in the orthotopic mouse model, where the survival of mice injected with ALCAM-silenced Daoy cells appeared to be shorter than that of the control mice, even though the difference between the two groups was not statistically significant. We also found that ALCAM depletion in Daoy cells increased invasion at disseminated sites in the orthotopic mouse model. On the other hand, the increased invasiveness of ALCAM-depleted Daoy cells was not observed in the context of an in vitro transwell assay. These results seem contradictory, but the difference between ALCAM function in vitro and in vivo indicates that ALCAM may be affected by the surrounding microenvironment. Notably, our results are consistent with those from previous reports regarding other carcinomas in which depletion of ALCAM suppresses cell proliferation and migration in vitro, but the cells demonstrate invasiveness in vivo [14, 16, 19, 31]. For example, in cell lines of malignant mesothelioma and endometrioid endometrial cancer, proliferation and migration are inhibited in vitro by ALCAM silencing [16, 19]. Nevertheless, ALCAM-negativity at the invasive front of the tumor has been reported as a marker of myometrial invasion in tissue of endometrioid endometrial cancer [31]. Moreover, in a metastatic melanoma cell line, interfering with endogenous ALCAM through the expression of an amino-terminally truncated ALCAM, which disrupts ALCAM–ALCAM interactions, increases cell invasive growth in skin reconstructions [14]. The attenuation of ALCAM enhances the invasive abilities of the tumor, which is arguably caused by the reduction of ALCAM-mediated adhesion, resulting in less adhesive and more mobile tumor cells [14, 31]. Therefore, the functional role of ALCAM might vary depending on the surrounding microenvironment.

ALCAM has been shown to be a prognostic marker in several types of cancers; however, it is both a marker of good and poor prognosis depending on the cancer type [9-13]. In our study, the expression of ALCAM was observed particularly concentrated in the WNT subgroup of MB, which typically shows a good long-term prognosis in comparison to the other subgroups. Therefore, ALCAM may be regarded as a good prognostic marker in MB. Conversely, a lack of ALCAM may contribute to poor prognosis. Differences in ALCAM expression levels might be related to a factor regulating the cell kinetics between WNT MB and non-WNT MB. Accordingly, we showed that ALCAM silencing enhanced the invasiveness of disseminated lesions in the orthotopic mouse model. The invasion ability at disseminated lesions is an important negative prognostic factor of MB as most MB recurrence is caused by leptomeningeal dissemination.

A limitation of our current study was the relatively small sample size of our cohort compared to that of other large cohorts. The small sample size used for ALCAM immunohistochemical staining may have resulted in inevitable statistical bias. However, there was no significant difference in our molecular subgrouping results compared to that using another cohort [4]. It is noteworthy that our study lacked a validation study of the ALCAM immunohistochemical staining, and thus our immunohistochemical findings may be insufficient to assert ALCAM as a biomarker for the WNT subgroup of MB. However, we did perform a comparative analysis using a large cohort (Cavalli-763 MB dataset) [24] of the R2: Genomics Analysis and Visualization Platform. This comparative analysis was based on gene expression and showed a strong correlation between ALCAM expression and the WNT subgroup of MB. Consistent with this, our study showed a correlation between ALCAM protein levels based on immunohistochemical staining and ALCAM mRNA gene expression levels analyzed by RT-qPCR. As evaluation of patient age and pathological type was insufficient in our cohort, we examined this using expression data from a large cohort of the R2 database. Analysis of the R2 database cases revealed ALCAM expression was low in patients 21 y and older, even in the WNT subgroup with high ALCAM expression. Therefore, we believe that limiting the evaluation of cases to patients 20 years or younger improves the strength of the correlation between ALCAM and the WNT subgroup.

According to the results from the analysis of clinical sample in which ALCAM was strongly expressed in the WNT subgroup, it may be more appropriate to use a WNT subgroup cell line. However, cell lines belonging to the WNT subgroup are not readily available [32]. Daoy and ONS-76 cells used in our study strongly express ALCAM and have been considered to be of the SHH subgroup, while D341 and D231 cells weakly express ALCAM and are considered to be of the Group 3 and Group 3/4 subgroups, respectively [32]. In our current study, positive ALCAM immunostaining was observed in only one of the eight cases of the SHH subgroup and ALCAM expression was low. Further studies are required to determine whether ALCAM is strongly expressed in the SHH subgroup cell line, despite low expression of ALCAM in the SHH subgroup clinical sample.

Conclusions

Clinical sample analysis performed in our current study suggested that ALCAM expression in MB was strongly correlated to the molecular subgroup. Specifically, ALCAM was highly expressed in the WNT subgroup of MB compared to that in non-WNT subgroups of MB. Furthermore, the cell kinetics of MB cell lines were altered by ALCAM expression. ALCAM appeared to be involved as a positive regulator of proliferation and migration of MB tumor cells in vitro. However, in vivo studies using an orthotopic mouse model demonstrated that the attenuation of ALCAM enhanced cell invasiveness in disseminated lesions.

1 Aug 2019

PONE-D-19-17600

The expression of activated leukocyte cell adhesion molecule correlates with the WNT subgroup in medulloblastoma and is involved in the regulation of tumor cell invasion

PLOS ONE

Dear Dr Kijima,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

(1) ALCAM overexpression experiments

(2) Presentation of ROC curve and description of cut-off value for ALCAM immunohistochemistry

(3) Discussion related to ALCAM immunohistochemistry, such as (i) ALCAM immunostaining pattern, (ii) ALCAM-positive and nuclear ß-catenin-negative cases, (iii) ALCAM mRNA expression levels in medulloblastoma subgroups

(4) Discussion of weak points in this report, such as (i) small sample size and (ii) lack of validation study.  In addition, the authors need to discuss statistical power of a clinical study based on 36 cases of medulloblastoma patients.

(5) Language edition

(6) Other issues pointed out by Reviewers

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Reviewer #1: The Authors analyzed deeply the relationship between ALCAM expression and both cell proliferation and migration. There are few criticisms: they didn’t evaluated the effect of an overexpression induced in cell lines (mainly in the cell lines exhibiting a low expression level of ALCAM, D341, and D283) which should show an increasing rate of cell proliferation and migration (D341, and D28 proliferation and migration rate in normal conditions are not described). Moreover, it is well known that Daoy cell line exhibits mutated p53 which is tightly related to beta catenin pathway. The Authors could analyze the p53 mutation status also in Daoy induced MB (p53 wild type or not) to give a hypothesis to explain the different results among different experimental conditions (“in vitro” and ”in vivo”)

Reviewer #2: The aim of the present study was to reveal the functional role and significance of ALCAM expression in Medulloblastoma (MB). In the first part of the results, the authors carried out several functional in vitro and in vivo assays by silencing ALCAM using RNA interference in order to unveil the function of ALCAM in MB. In the second part of the study, the authors provide new information about ALCAM expression correlation with MB molecular and histological subtypes in a MB FFPE cohort conformed by 39 patients. As a conclusion, authors propose ALCAM as a novel MB WNT biomarker.

Reviewers Concerns:

- Authors should declare that the clinical stratification of MB tumors is stated at the 2016 Consensus Paper (Ramaswamy, V et al. Acta Neuropathol (2016) 131:821–831).

- The article seems to report two well-differentiated studies aimed at: i) the functional role of ALCAM in MB models and ii) the potential use of ALCAM expression as an IHC biomarker in MB, however, both studies are incomplete. Additional analyses are necessary to clarify the role of ALCAM as well as to propose ALCAM as a potential biomarker for the MB - WNT subgroup.

- Review of the Functional Analyses:

o Author should explain why MB cell lines were cultured in different medium and FBSi conditions. This could be a source of variability.

o Results reported in Fig1H show differences of proliferation that are hardly appreciable for the ONS-76 cell lines at 48h. Author do not specify the significance of the *** symbol.

o Authors should perform additional assays using different MB cell lines, in order to explore further the effect of ALCAM depletion on migration/invasion, and clarify the contradictory findings observed between in vitro and in vivo assays.

- Review of the IHC analysis:

o The contents of the ALCAM expression analysis section should be placed before the functional part, making it easier to understand the rational of the study and to follow the results.

o The MB FFPE cohort conformed by 36 cases used in the study is far too small to support the conclusions and includes both pediatric and adult MB cases. Only six of these are WNT MBs. The authors should prove their findings in a larger cohort.

o Table 1, ALCAM positive staining was observed in two cases (one SHH) with negative nuclear beta-catenin expression. This is critical for the study and questions the ALCAM staining as a reliable marker. Authors must verify the presence of CTNNB1 mutation.

o In addition, no biomarker studies are acceptable if the authors do not validate their results in an independent cohort.

o MB is rarely diagnosed in adults, whereas MB is the most common malignant pediatric brain tumor. However, four of the six MB WNT cases included in the FFPE cohort were adults, only two children. Adult MB is distinct from childhood MB with clear differences in the molecular variants and clinical evolution, and should thus be analysed separately. Authors do not address this issue. The cohort is biased and does not fully represent the MB WNT subgroup.

o Authors describe the IHC pattern of ALCAM in WNT-MB samples predominantly in the cytoplasm, but also the presence of some staining in the cell membrane. If authors propose the use of ALCAM as a MB-WNT biomarker, the IHC staining pattern must be described accurately (see Mezzanzanica, D et al. Clin Cancer Res 2008;14(6)). IHC consistent controls should be reported.

o Studies that propose possible biomarkers should include analyses such as ROC curves to demonstrate the reliability and validity of the results.

o The correlation between ALCAM IHC staining and ALCAM mRNA expression by qPCR was performed in a limited number of samples: 3 positive vs 6 negative for ALCAM IHC.

o The ALCAM mRNA validation in Cavalli microarray cohort (n=763) showed a large variability of ALCAM expression within subgroups and overlap across MB subgroups. ALCAM mRNA expression in MB subgroups has low specificity based on the presented results.

o Authors did not assess the correlation between ALCAM expression and the MB histological subtypes as the cohort does not has a fully representation of all histological MB subtypes. Authors should enlarge the cohort all MB histological subtypes in order to obtain robust conclusions.

o The IHC cut-off values for subdivision must be more restrictive in a biomarker study to be reliable.

- The results are insufficient to support the exposed conclusions.

- There are several typos and grammatical errors. For example: “Table1: Adult (≥ 16 yeas)” instead of years; The correct spelling is “partially” not “partial” in the staining pattern description; In page 5 line 88 “many names” is used instead of “alias”; “Subdivision” is used instead of “subgroups”).

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4 Feb 2020

Editor

(1) ALCAM overexpression experiments

Response: Related to #1-1, we have performed ALCAM overexpression experiments using the D341 cell line and showed that ALCAM promotes cell proliferation (Fig 3J and 3K).

(2) Presentation of ROC curve and description of cut-off value for ALCAM immunohistochemistry

Response: Related to #2-10, we have created ROC curves and evaluated the diagnostic performances of ALCAM Immunohistochemical staining and have identified optimal cut-off points for the diagnosis of the WNT molecular subgroup (Fig 1B).

(3) Discussion related to ALCAM immunohistochemistry, such as (i) ALCAM immunostaining pattern, (ii) ALCAM-positive and nuclear ß-catenin-negative cases, (iii) ALCAM mRNA expression levels in medulloblastoma subgroups

Response: Related to #2-9, #2-11, and #2-12, we described the immunostaining patterns in all ALCAM-positive staining cases in Table 2. Moreover, we examined the histologic subtype and age group of MB with respect to the expression of ALCAM (Fig 1D and 1E).

(4) Discussion of weak points in this report, such as (i) small sample size and (ii) lack of validation study. In addition, the authors need to discuss statistical power of a clinical study based on 36 cases of medulloblastoma patients.

Response: Related to #2-6, we have described these as limitations in the Discussion section.

(5) Language edition

Response: Related to #2-13, we have proofread the entire manuscript.

(6) Other issues pointed out by Reviewers

Response: We have responded to all reviewer questions as described below.

Reviewer #1

#1-1: They didn’t evaluated the effect of an overexpression induced in cell lines (mainly in the cell lines exhibiting a low expression level of ALCAM, D341, and D283) which should show an increasing rate of cell proliferation and migration (D341, and D28 proliferation and migration rate in normal conditions are not described).

Response: The ALCAM overexpression experiments using the D341 cell line showed that enhanced ALCAM expression increased cell proliferation. D341 cells migration could not be evaluated due to the feature that D341 cells grow in suspension and not as adherent monolayers. This has been noted in the revised manuscript.

#1-2: It is well known that Daoy cell line exhibits mutated p53 which is tightly related to beta catenin pathway. The Authors could analyze the p53 mutation status also in Daoy induced MB (p53 wild type or not) to give a hypothesis to explain the different results among different experimental conditions (“in vitro” and ”in vivo”)

Response: We analyzed the TP53 gene mutation status both in vitro in Daoy cell lines (shControl, shALCAM1, and shALCAM2) and in vivo in Daoy tumor tissues (shControl and shALCAM1). Sanger sequencing analysis of the Exon 2-11 region detected a common c.725G>T: p.C242F homozygous mutation in the TP53 gene of all samples. This was consistent with the known mutation reported in Daoy line (Saylors et al., 1991).

Reviewer #2

#2-1: Authors should declare that the clinical stratification of MB tumors is stated at the 2016 Consensus Paper (Ramaswamy, V et al. Acta Neuropathol (2016)

131V821–831).

Response: As suggested, we have added this to the Introduction of the revised manuscript and provided the proper in-text citation of this consensus paper.

#2-2: Author should explain why MB cell lines were cultured in different medium and FBSi conditions. This could be a source of variability.

Response: We used the media and FBS conditions recommended for each cell line. Culturing may not be successful for a particular cell line when conditions are changed.

#2-3: Results reported in Fig1H show differences of proliferation that are hardly appreciable for the ONS-76 cell lines at 48h. Author do not specify the significance of the *** symbol.

Response: We apologize for the error in defining the sign of the significance. We revised the description from "*: P <0.05; **: P <0.0001" to "*: P < 0.05; **: P < 0.01; ***: P < 0.0001" and revised the figure as well.

#2-4: Authors should perform additional assays using different MB cell lines, in order to explore further the effect of ALCAM depletion on migration/invasion, and clarify the contradictory findings observed between in vitro and in vivo assays.

Response: We performed an ALCAM overexpression experiment using the D341 cell line. Enhanced ALCAM expression increased cell proliferation. An orthotopic mouse model using ONS-76 cells was also evaluated, but meningeal dissemination was not well formed.

#2-5: The contents of the ALCAM expression analysis section should be placed before the functional part, making it easier to understand the rational of the study and to follow the results.

Response: As the reviewer suggested, we have restructured the appropriate sections in the revised manuscript.

#2-6: The MB FFPE cohort conformed by 36 cases used in the study is far too small to support the conclusions and includes both pediatric and adult MB cases. Only six of these are WNT MBs. The authors should prove their findings in a larger cohort.

&

In addition, no biomarker studies are acceptable if the authors do not validate their results in an independent cohort.

Response: Due to sample limitations, our cohort size was relatively small and our study lacked a validation component using ALCAM immunohistochemical staining. However, analysis from a large cohort (the Cavalli-763 MB dataset of the R2: Genomics Analysis and Visualization Platform) based on gene expression analysis showed a strong correlation between ALCAM expression and the WNT subgroup of MB. We have included the small sample size our cohort as a limitation of our study in the Discussion of the revised manuscript.

#2-7: Table 1, ALCAM positive staining was observed in two cases (one SHH) with negative nuclear beta-catenin expression. This is critical for the study and questions the ALCAM staining as a reliable marker. Authors must verify the presence of CTNNB1 mutation.

Response: As shown in Table 1, we have verified CTNNB1 mutations in most of the cases used in our study.

#2-8: MB is rarely diagnosed in adults, whereas MB is the most common malignant pediatric brain tumor. However, four of the six MB WNT cases included in the FFPE cohort were adults, only two children. Adult MB is distinct from childhood MB with clear differences in the molecular variants and clinical evolution, and should thus be analysed separately. Authors do not address this issue. The cohort is biased and does not fully represent the MB WNT subgroup.

Response: MB is pediatric brain tumor, but the age distribution differs for each molecular subgroup. Adults (> 16 y) are not uncommon in WNT, SHH, and Group 4 subgroups of MB. In the WNT subgroup, infant cases (< 4 y) are rare and cases of children (4-16 y) and adults (>16 years) occur at a 2:1 ratio (Kool et al., 2012 and Taylor et al., 2012). In our cohort, the cases of the WNT subgroup included two children (6 and 7 years old) and four adults (16, 17, 18, and 20 years old) according to the original age distribution definition (Adult: ≥ 16 years, child: < 16 and ≥ 3 years, and infant: < 3 years). We have changed the definition of age distribution in the revised manuscript to be consistent with other cohorts (Adult: > 16 years, child: 4-16 years, infant: < 4 years). The cases of the WNT subgroup now included three children (6, 7, and 16 years old) and three adults (17, 18, and 20 years old). There was no significant difference in the age distribution of the other cohorts and both the child and adult cases were positive for ALCAM immunohistochemical staining.

#2-9: Authors describe the IHC pattern of ALCAM in WNT-MB samples predominantly in the cytoplasm, but also the presence of some staining in the cell membrane. If authors propose the use of ALCAM as a MB-WNT biomarker, the IHC staining pattern must be described accurately (see Mezzanzanica, D et al. Clin Cancer Res 2008;14(6)). IHC consistent controls should be reported.

Response: As suggested, we have added the information regarding ALCAM IHC pattern to Table 2 in the revised manuscript.

#2-10: Studies that propose possible biomarkers should include analyses such as ROC curves to demonstrate the reliability and validity of the results.

&

The IHC cut-off values for subdivision must be more restrictive in a biomarker study to be reliable.

Response: We have included the accuracy of the WNT subgroup diagnosis by ALCAM immunohistochemical staining and the optimal cutoff value according to ROC curve analysis.

#2-11: The ALCAM mRNA validation in Cavalli microarray cohort (n=763) showed a large variability of ALCAM expression within subgroups and overlap across MB subgroups. ALCAM mRNA expression in MB subgroups has low specificity based on the presented results.

Response: As pointed out, in a large cohort used for validation (the Cavalli-763 MB dataset), ALCAM expression in some cases exhibited overlap between MB subgroups. As a result of additional analysis, it became clear that the cases where ALCAM expression was low in the WNT subgroup, the patients were more than 21 years old. We have described this in the Results of the revised manuscript and added it as a limitation of our study.

#2-12: Authors did not assess the correlation between ALCAM expression and the MB histological subtypes as the cohort does not has a fully representation of all histological MB subtypes. Authors should enlarge the cohort all MB histological subtypes in order to obtain robust conclusions.

Response: In our small cohort, the histologic subtype was biased and difficult to evaluate. However, there was no clear correlation between ALCAM and MB histologic subtypes in the large cohort included in the Cavalli-763 MB dataset.

#2-13: There are several typos and grammatical errors. For example: “Table1: Adult (≥ 16 yeas)” instead of years; The correct spelling is “partially” not “partial” in the staining pattern description; In page 5 line 88 “many names” is used instead of “alias”; “Subdivision” is used instead of “subgroups”).

Response: We have corrected the suggested points and re-proofread the entire manuscript for other corresponding changes that were needed.

Submitted filename: Response_to_Reviewers.docx

Click here for additional data file.

6 Mar 2020

PONE-D-19-17600R1

The expression of activated leukocyte cell adhesion molecule correlates with the WNT subgroup in medulloblastoma and is involved in the regulation of tumor cell invasion

PLOS ONE

Dear Dr Kijima,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Apr 20 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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Kind regards,

Javier S Castresana

Academic Editor

PLOS ONE

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1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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6. Review Comments to the Author

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Reviewer #1: The paper has been modified following the reviewer suggestions and criticisms and In the present revised form is now ready to publish

Reviewer #2: Activated leukocyte cell adhesion molecule expression correlates with the WNT subgroup in medulloblastoma and is involved in regulating tumor cell proliferation and invasion

Takamune Achiha, Noriyuki Kijima, Yoshinori Kodama, Naoki Kagawa, Manabu Kinoshita, Yasunori Fujimoto, Masahito Nonaka, Namiko Nishida, Takumi Yamanaka, Atsuko Harada, Kanji Mori, Junya Fukai, Naohiro Tsuyuguchi, Takehiro Uda, Kenichi Ishibashi, Yusuke Tomogane, Daisuke Sakamoto, Tomoko Shofuda, Ema Yoshioka, Daisuke Kanematsu, Masayuki Mano, Betty Luu, Michael D. Taylor, Yonehiro Kanemura, Haruhiko Kishima.

The aim of the present study was to reveal the functional role and significance of ALCAM expression in Medulloblastoma (MB). The authors provide new information of ALCAM protein levels analysed by IHC in a MB FFPE cohort conformed by 36 patient and performed an in silico correlation between ALCAM gene expression and the MB molecular and histological subtypes as well as patient age’s in a large MB cohort from the R2 genomics Platform (Cavalli et al. Cancer Cell 2017). In the second part of the study, the authors carried out several functional in vitro and in vivo assays by silencing ALCAM using RNA interference in order to unveil the function of ALCAM in MB. As a conclusion, authors offer ALCAM as a novel MB WNT biomarker.

Reviewer Concerns:

There has been a qualitative improvement in the manuscript compared to the previous version. The authors have restructured the article and now the results are easier to follow, however, the results are still insufficient to support the conclusions presented. The article is attractive and unveils an interesting role of ALCAM in MB-WNT subgroup, but it has to be reported as a study of the MB underlying biology, not as a biomarker study.

Major Comments:

- The MB FFPE cohort conformed by 36 cases used in the study is far too small to support the conclusions. The authors should prove their findings in a larger cohort. The strength of the preliminary data (IHC of 36 samples) is not sufficient to support the rationale of ALCAM as a WNT-related biomarker.

- Biomarker studies are not acceptable if authors do not validate their results in an independent cohort using the same technique. In this case, the independent validation the authors provide is an in silico validation (Cavalli et al. Cancer Cell 2017) at the gene expression level. Authors do not validate their results at the protein level using the IHC technique or other method to detect protein levels. The in silico analysis suggests a tendency but not a validation itself.

- The authors propose ALCAM IHC as an additional WNT-related biomarker to β-catenin to improve the reliability of diagnosis. However, ALCAM IHC is positive in six WNT-MB (n=6/6, 100% of all WNT analyzed) and presents an inconsistent positive result in one SHH (n=1/5, 20% of all SHH analyzed). This is not acceptable in a small cohort of 36 samples if the authors pretend to propose ALCAM as a biomarker.

- The IHC training cohort of 36 FFPE samples is small and not fully characterized:

a. 2 NA Molecular subgroup

b. 5 NA CCNB1 status

Minor Comments

- There is much more updated MB literature than the referenced: Northcott, P.A., Robinson, G.W., Kratz, C.P. et al. Medulloblastoma. Nat Rev Dis Primers 5, 11 (2019). https://doi.org/10.1038/s41572-019-0063-6

- Authors declare that ALCAM depletion is associated with a more invasive tumour cells phenotype in vivo. This could be confirmed by a transwell migration assay analysis.

- Figure 2H: ALCAM was “weakly” detected in normal cerebellum in granular layer and white matter by IHC. The partially IHC staining of ALCAM in normal tissues should be further explored in normal cerebellum as well as brain if authors propose ALCAM as a possible WNT-related biomarker.

- Positive ALCAM IHC staining of one of the five SHH tumor samples included in the study as well as high ALCAM mRNA levels in SHH cell lines (DAOY and ONS-76) should be declared and further investigated in order to understand the role of ALCAM in MB.

**********

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Reviewer #1: No

Reviewer #2: No

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27 Oct 2020

Reviewer #2

Major Comments:

#2-1:

The MB FFPE cohort conformed by 36 cases used in the study is far too small to support the conclusions. The authors should prove their findings in a larger cohort. The strength of the preliminary data (IHC of 36 samples) is not sufficient to support the rationale of ALCAM as a WNT-related biomarker.

Response:

As pointed out, a validation cohort is essential for the biomarker study as our sample size was too small. We were only able to demonstrate an association between a strong expression of ALCAM and the WNT-subgroup in MB and not prove it as a biomarker. Accordingly, we revised our manuscript and ensured we did not define ALCAM as a WNT-related biomarker. Additionally, we included the aforementioned point as a limitation of our study, in the Discussion section of the revised manuscript. In addition, to expand our cohort as much as possible, we analyzed nine additional MB cases after the extension of the case enrollment period and the addition of another collaborating institution. Among the additional MB cases, the association between the WNT subgroup and the expression of ALCAM was similar to that in the original cohort in our study. One case was a WNT subgroup case and ALCAM positive, while eight cases were non-WNT and were ALCAM negative. These new cases and their details were included in the revised manuscript; Table 1, and Table 2 were updated accordingly. The ROC curve was also revised accordingly (Fig 1B).

#2-2:

Biomarker studies are not acceptable if authors do not validate their results in an independent cohort using the same technique. In this case, the independent validation the authors provide is an in silico validation (Cavalli et al. Cancer Cell 2017) at the gene expression level. Authors do not validate their results at the protein level using the IHC technique or other method to detect protein levels. The in silico analysis suggests a tendency but not a validation itself.

Response:

As mentioned above, in this study were only able to demonstrate an association between a strong expression of ALCAM and the WNT-subgroup in MB and not prove it as a biomarker. We used IHC to evaluate the expression of ALCAM in our cohort; the large cohorts of the Cavalli-763 MB dataset we used for verification referred to mRNA gene expression. To further strengthen the correlation between ALCAM protein levels showed by IHC and ALCAM mRNA gene expression levels, we examined the eight additional MB cases included in the revised manuscript (one ALCAM-positive and seven ALCAM-negative cases) using RT-qPCR. The correlation between ALCAM protein levels based on IHC and ALCAM mRNA expression revealed by RT-qPCR was statistically significant (P = 0.0237 to P = 0.0017; Fig 1A). The manuscript was revised accordingly.

#2-3:

The authors propose ALCAM IHC as an additional WNT-related biomarker to β-catenin to improve the reliability of diagnosis. However, ALCAM IHC is positive in six WNT-MB (n=6/6, 100% of all WNT analyzed) and presents an inconsistent positive result in one SHH (n=1/5, 20% of all SHH analyzed). This is not acceptable in a small cohort of 36 samples if the authors pretend to propose ALCAM as a biomarker.

Response:

As noted above, we have revised the Discussion section and did not define the expression of ALCAM as a potential biomarker. With respect to the cohort size, again we have included as many additional cases as possible. IHC staining of ALCAM was positive in all seven of the WNT-MB cases (n = 7/7, 100%) and one of the SHH-MB cases analyzed (n = 1/8, 12.5%). The expanded results were added to the revised manuscript.

#2-4:

The IHC training cohort of 36 FFPE samples is small and not fully characterized:

a. 2 NA Molecular subgroup

b. 5 NA CCNB1 status

Response:

a. 2 NA Molecular subgroup

Due to problems with some of the samples, Nanostring analysis could not be performed and the molecular subgroup was not determined in 2 cases (MB4, MB10). However, these cases definitely belonged to non-WNT subgroups: the CTNNB1 mutation was not confirmed and β-catenin nuclear staining was negative. Furthermore, the pathological type of case MB4 was desmoplastic/nodular, suggesting that it was of the SHH subgroup. This information is provided in Table 2 of the revised manuscript.

b. 5 NA CCNB1 status

We have performed additional analysis regarding the CTNNB1 status for all cases.

Minor Comments:

#2-5:

There is much more updated MB literature than the referenced: Northcott, P.A., Robinson, G.W., Kratz, C.P. et al. Medulloblastoma. Nat Rev Dis Primers 5, 11 (2019). https://doi.org/10.1038/s41572-019-0063-6

Response:

As suggested, we have added this citation to the Introduction section of the revised manuscript.

#2-6:

Authors declare that ALCAM depletion is associated with a more invasive tumour cells phenotype in vivo. This could be confirmed by a transwell migration assay analysis.

Response:

As per the reviewer’s suggestion, we performed transwell assays using Daoy cells. There was no remarkable difference in the invasion ability of ALCAM-depleted Daoy cells in this in vitro assay compared with that of Daoy control cells (P = 0.581; Fig 4E). We considered that the increased invasiveness exhibited by ALCAM-depleted Daoy cells in the context of meningeal dissemination in vivo may be related to other factors that are not implicated in invasion in the context of in vitro transwell assays. The results of the transwell assay were added to the revised manuscript.

#2-7:

Figure 2H: ALCAM was “weakly” detected in normal cerebellum in granular layer and white matter by IHC. The partially IHC staining of ALCAM in normal tissues should be further explored in normal cerebellum as well as brain if authors propose ALCAM as a possible WNT-related biomarker.

Response:

ALCAM was slightly expressed in the granular layer of the cerebellum, as shown in the manuscript. Of note, Allmendinger et al. (2012) have previously reported that ALCAM is expressed in normal cerebral tissue, e.g. in the hippocampus and basal ganglia, as well as in reactive glial cells. We have used immunohistochemistry to evaluate the expression of ALCAM in representative tumor areas in the current study. We have revised the Discussion section of the manuscript to include a description of the expression of ALCAM in normal brain tissues.

#2-8:

Positive ALCAM IHC staining of one of the five SHH tumor samples included in the study as well as high ALCAM mRNA levels in SHH cell lines (DAOY and ONS-76) should be declared and further investigated in order to understand the role of ALCAM in MB.

Response:

As suggested, we have included this point in the Discussion section of the revised manuscript.

Submitted filename: Response to Reviewers.docx

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12 Nov 2020

PONE-D-19-17600R2

Activated leukocyte cell adhesion molecule expression correlates with the WNT subgroup in medulloblastoma and is involved in regulating tumor cell proliferation and invasion

PLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #2: All comments have been addressed

**********

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Reviewer #2: Yes

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Reviewer #2: Yes

**********

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Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The aim of the present study was to reveal the functional role and significance of ALCAM expression in Medulloblastoma (MB). The authors provide new information of ALCAM protein levels analysed by IHC in a MB FFPE cohort conformed by 45 patient and performed an in silico correlation between ALCAM gene expression and the MB molecular and histological subtypes as well as patient age’s in a large MB cohort from the R2 genomics Platform (Cavalli et al. Cancer Cell 2017). In the second part of the study, the authors carried out several functional in vitro and in vivo assays by silencing ALCAM using RNA interference in order to unveil the function of ALCAM in MB.

Reviewer Concerns:

There has been an improvement in the manuscript compared to the previous version. The authors have ensured that they did not define ALCAM as a WNT-related biomarker and the Results are now presented as an interesting correlation between ALCAM expression and the MB-WNT subgroup. The article is not as a biomarker study but an attractive exploration of the MB underlying biology and unveils an interesting role of ALCAM in MB-WNT subgroup. However, some points must be clarified.

Minor Comments:

- Authors need to clarify the finality and the description of the ROC curve analysis. It is not clear if the ROC curve analysis is meant to select ALCAM IHC positivity cut-point neither how many MB samples are used or their subgroup. Authors must remove the word “diagnosis” from the manuscript in line 325 “To evaluate the reliability of the WNT subgroup diagnosis of MB using ALCAM” as it is misleading.

**********

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Reviewer #2: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: R2_D-19-17600_20201106.pdf

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18 Nov 2020

Thank you for your valuable comment. As pointed out, we have removed the word “diagnosis” from the sentence to avoid confusion. As suggested, we have modified the description of the ROC curve analysis and added information on the number of MB cases and the subgroup. In the Methods section of the manuscript, the description of the ROC curve analysis was modified and moved from the sub-section “statistical analysis” to “Immunohistochemistry”.

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19 Nov 2020

Activated leukocyte cell adhesion molecule expression correlates with the WNT subgroup in medulloblastoma and is involved in regulating tumor cell proliferation and invasion

PONE-D-19-17600R3

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23 Nov 2020

PONE-D-19-17600R3

Activated leukocyte cell adhesion molecule expression correlates with the WNT subgroup in medulloblastoma and is involved in regulating tumor cell proliferation and invasion

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