Background
Medulloblastoma (MB) is the most common pediatric brain malignancy that frequently arises below 10 years of age [
1,
2]. Approximately 20–30% of patients remain incurable, and high dose radiation and chemotherapy frequently lead to significant long-term sequelae [
3]. Progress in molecular diagnostics has revealed that MB is classified into 4 subgroups: WNT, SHH, Group 3 (G3) and Group 4 (G4) [
1,
2,
4]. The prognosis of each subgroup ranges from being excellent in WNT MB to intermediate in SHH and G4, to poor in G3 MB [
1,
4]. As subgroup-specific prognostication and personalized medicine are in demand, clinically applicable subgrouping has become essential [
3,
5‐
7]. Practical molecular subgrouping has been developed by multiple researchers via screening subgroup-specific signature genes using various tools, such as nanoString nCounter [
3,
4,
6].
The significance of lymphocytes and tumor-associated macrophages (TAMs) in the tumor microenvironment has been perpetually examined for more than a decade; however, their comprehensive role is rather elusive [
8‐
12]. TAMs release growth factors, cytokines, and inflammatory mediators into the environment and are classified according to their functional phenotype [
13‐
16]. The current paradigm of macrophage polarization is undergoing reassessment. It has been commonly accepted that classically activated M1 macrophages suppress tumor growth and progression by production of reactive oxygen species (e.g., nitric oxide), whereas alternatively activated M2 macrophages promote tumor growth and progression by releasing growth factors (e.g., epidermal growth factor, fibroblast growth factor 1, vascular endothelial growth factor A) [
9,
13‐
16]. The literature has often described conflicting roles of TAMs in various cancers due to the complexity of the tumor microenvironment and diverse contributing factors, such as immune responses, tumor stages, and types of tumors [
11,
13,
17‐
20].
Despite the molecular insights provided by MB subgroups, relatively little is known about the role of tumor microenvironment with respect to MB and its subgroups [
8]. A previous report on the characterization of immunophenotype in pediatric brain tumors suggests that MB is less infiltrated with T lymphocytes and displays an immunosuppressive M2 phenotype compared to other pediatric brain tumors [
8]. A recent study demonstrated that TAM recruitment is subgroup-specific in MB, suggesting that the expression of TAM-associated genes was significantly higher in the SHH subgroup [
3]. This finding indicates that SHH MB has a distinct tumor microenvironment, which may have important pathophysiological and therapeutic implications. However, the roles of TAMs and their activation phenotypes are inconclusive because the previous study did not present the prognostic connotations of TAMs in SHH MB [
3].
In the present study, we investigate the correlation between TAM recruitment in SHH MB with prognosis. We identified that M1 macrophage recruitment rather than total TAM recruitment correlates more strongly with a reduced overall survival outcome within the SHH subgroup. Considering the commonly accepted role of macrophage polarization in various human cancers (M1 tumor-suppressing and M2 tumor-promoting roles), the negative prognostic implication of M1 macrophages in SHH MB is intriguing and requires further investigation.
Methods
Patients and samples
The Institutional Review Board (IRB) of the Seoul National University Hospital (SNUH) approved the study protocol (IRB approval No. 1610–027-797). To identify SHH MB, 48 snap-frozen MB tissues were retrieved from the Brain Bank of the Department of Neurosurgery, Seoul National University Hospital. Tissue samples were collected from 141 MB patients who underwent surgery at Seoul National University Children’s Hospital (SNUCH) from 1999 to 2015. The molecular subgroups of the samples were partially verified via immunohistochemistry (IHC) using representative markers [
4]. To solidify the molecular subgroup, a nanoString-based RNA assay was performed on these samples. Previously, we provided MB tissues to Dr. M. Taylor from the Hospital for Sick Children (Toronto, Canada) for analysis, and the molecular subgroups were provided for these cases through nanoString [
10].
We collected 32 SHH MBs from two sources: cases newly tested for subgrouping (n = 16) and cases with subgroup information from Toronto (n = 16). Among the 32 known SHH MB patients, 25 patients had available formalin-fixed paraffin-embedded (FFPE) tissues. Two FFPE tissue samples were removed from selection due to small tissue size or the inability to undergo a complete experiment; 23 SHH MB samples were finally recruited from our institution. An additional 7 SHH MB FFPE tissue samples were received from Yonsei University. In total, 30 SHH MB were analyzed in the present study. Subgroups other than SHH were randomly selected with respect to FFPE tissue availability as control groups to validate the correlation between TAM infiltration and the prognosis of the subgroups (WNT = 3, Group 3 = 2, Group 4 = 17).
Subgrouping
Molecular subgroups were identified through gene profiling using nanoString nCounter [
6]. Total RNA was extracted from snap-frozen patient tissue samples (
n = 48) using the miRNeasy kit according to the manufacturer’s protocol (Life Technologies, Carlsbad, CA, USA). Procedures related to hybridization, detection and scanning were performed as recommended by nanoString Technologies (Seattle, WA, USA). The collected data were normalized in
R, and an algorithm for class prediction analysis was provided by Dr. M. Taylor (Toronto, Canada) [
6]. The subgroup of additionally received FFPE tissue samples from Yonsei University, which were identified via immunohistochemistry (IHC), was provided by Dr. SH Kim (Seoul, Korea). For the SHH subgroup, IHC generally yields stable and concordant results with nanoString.
Immunohistochemistry
Macrophage recruitment was investigated using immunohistochemistry (IHC) on FFPE tissue samples (
n = 45). Human tonsil tissue was used as a positive control (Additional file
1: Figure S1).The recruitment of activated macrophages was identified using the following antibodies: CD68 for total macrophages, CD86 for M1-activation, and CD163 for M2-activation (Additional file
2: Table S1). Five hot spots were randomly selected in each paraffin section, and positive cells among the 300 counterstained cells were counted using the ImageJ Cell Counter plugin [
21]. The mean value of the five hot-spots count was used in the following statistical analyses. Researchers engaged in the present experiment were blinded from all clinical data, including subgroup, through data collection.
Immunofluorescence
To confirm the independent localization of M1 and M2 macrophages, an immunofluorescence (IF) assay was performed on FFPE tissue samples. The retrieved blocks were sectioned at 4 μm using a microtome and transferred to silane-coated slides by the SNUH pathology lab. The slides were deparaffinized in xylene and rehydrated through a graded ethanol series. To retrieve antigen, the slides were microwaved in 10 mM sodium citrate buffer (pH 6.0) for 3 min, with a 15 s cooling interval after 2 min. The slides were washed three times in phosphate-buffered saline (PBS) with 0.1% bovine serum albumin (BSA) for 5 min each and then permeabilized (1× PBS/ Timerasol: 95 mg/L, saponin: 0.6 g/L, normal goat serum: 1%) for 15 min. The slides were subsequently blocked in blocking solution (1 × PBS/ Timerasol: 95 mg/L, saponin: 0.35 g/L, normal goat serum: 3.5%) for 30 min at room temperature [
22]. The primary antibody was prepared in a modified blocking solution (1 × PBS/ Timerasol: 95 mg/L, saponin: 0.1 g/L, normal goat serum: 1%), with adequate dilution and incubated overnight at 4 °C. The secondary antibody was similarly diluted accordingly and applied for 1 h at room temperature.
Clinical data
Clinical data, including sex, age at diagnosis, pathology, degree of surgical resection, presence of leptomeningeal seeding at presentation, applied treatment modalities, progression, and survival, were collected independently of the researchers conducting the experiments. Progression-free survival (PFS) refers to the time interval from the day of initial surgery to the date when tumor progression was radiologically identified or the date of the last follow-up [
10]. Overall survival (OS) refers to the time interval from the day of initial surgery to the date of patient death or the date of the last follow-up [
10].
All 32 patients with SHH MB received chemotherapy. The chemotherapy regimens changed from 1999 to 2015. Prior to 2006, the Children’s Cancer Group (CCG) 9921 regimen (3 patients) or the 8 in 1 (6 patients) regimen were applied, and from 2006, the KSPNO (Korean Society for Pediatric Neuro-Oncology) protocols for infant or child MB were applied (14 patients). Eleven patients were aged < 3 yrs. at diagnosis, and radiation therapy (RT) was delayed for these patients. Overall, 20 patients received RT. The RT doses were adapted to the risk status of each patient: the standard risk group: craniospinal axis 19.8–23.4 Gy, tumor bed boost up to 54 Gy; the high risk group: craniospinal axis 28.8–36 Gy, tumor bed boost up to 54 Gy. The three patients for whom RT was delayed did not receive RT. One patient was lost to follow-up prior to initiating RT, while another patient died at 11 months with rapid disease progression, and another patient was cured with chemotherapy alone.
Statistical analysis
Subgroup prediction analysis was conducted in
R. IBM SPSS Statistics version 23 was used to perform common statistical analyses, including χ
2, bivariate Pearson’s correlation, Cox regression analysis, survival analysis, and the log-rank test as previously described [
10]. Appropriate indications are provided in the text and supplementary data.
Discussion
We demonstrate an unconventional correlation between subgroup-specific recruitment of TAM in SHH MB and prognosis. We confirmed subgroup-specific augmentation of M1 and M2 macrophages in SHH MB and compared this result with relevant prognostic factors. Survival analyses and Cox-regression analysis showed that M1 rather than M2 infiltration correlates better with worse OS and PFS in SHH MB, with relative risk values of 11.918 and 6.022, respectively.
The SHH MB subgroup, as suggested by its name, is thought to be driven by alterations in the Sonic-hedgehog signaling pathway [
4]. The SHH pathway plays a crucial role in cerebellar development, inducing the proliferation of neuronal precursors [
1,
4]. Individuals with germline or somatic mutations in the SHH pathway, such as
PTCH,
SMO,
SUFU,
GLI1, and
GLI2, are predisposed to MB [
1,
4]. Moreover, SHH MB has an intermediate prognosis among the 4 subgroups but, interestingly, is saturated with the highest number of TAMs, as demonstrated in the present and the previous one [
1,
3]. A dichotomous age distribution (< 4 years and > 16 years) is another hallmark of SHH subgroup; the present study showed that the age distribution within SHH MB did not significantly correlate with activated macrophage recruitment [
1].
The recognition of microenvironment in tumor biology has escalated over the past few decades, and this emphasis has led researchers to characterize contributing factors, including immunophenotypes, in various cancers [
8]. However, these studies are often limited to phenotypic characterization and lacked prognostic connotation. A previous study investigated TAM recruitment in MB and proposed subgroup-specific recruitment in SHH MB [
3]. We sought to verify this phenomenal recruitment in MB by a different method. Indeed, we found corroborating results showing augmented TAM recruitment in SHH MB and confirmed its unique microenvironment. Aside from M2 macrophages, we further characterized M1 macrophages in SHH MB and investigated the prognostic connotation of their recruitment.
In the present study, high M1 macrophages correlated with poor prognosis in SHH MB patients. This result apparently contradicts the common view of tumoricidal M1 macrophages. In many cancer types, M1 macrophage infiltration is associated with better prognosis [
23‐
25]. However, recent studies suggest that the dichotomous M1/M2 classification is oversimplified, and the role of TAM in tumors is still controversial [
14,
26]. We cannot provide a conclusive role for M1 macrophages in SHH MB because the causality of the worse prognosis associated with M1 macrophages has not been investigated. However, few plausible hypotheses can be made from the present results: 1) high M1 macrophage recruitment assists growth and progression of SHH MB contrary to its role in other cancers, 2) M1 macrophages are highly recruited to enhance the tumoricidal effect in aggressive group of SHH MB, but this mechanism alone was insufficient to fight the particular malignancy, or 3) high M1 recruitment is an epiphenomenon, and these cells are simply recruited by other SHH MB initiators and do not directly affect prognosis. Interestingly, the literature suggests multiple perspectives. The loss of nitric oxide synthase2 (NOS2) in the Ptch1
+/-SHH MB mouse model was reported to promote development of medulloblastoma [
27]. NOS2 is a key enzyme that produces nitric oxide in M1 macrophages in response to pathogens [
26]. This suggests good prognostic role of M1 macrophages, which supports the second hypothesis. However, direct production of interferon-γ, a known stimulatory cytokine of M1 macrophages, in the developing brain was reported to activate the SHH pathway and cerebellar dysplasia. [
28]. This activation may suggest that M1 macrophages are coincidentally recruited in response to the abnormal source of IFN-γ in the developing brain, not in recognition of MB or to destroy it. Such conflicting perspectives may also suggest a context-dependent role for TAM.
The small number of patients is a major limitation of the present study. The heterogeneity of the treatment administered to the patients may also confound the results, although all patients followed modernized treatment protocols in terms of risk stratification, chemotherapy regimen, and RT doses. Further validation in a comparable MB cohort is required to consolidate the role of TAM in SHH MB.
Acknowledgments
The authors would like to thank the reviewers and editors for detailed analysis of the present manuscript and constructive comments and suggestions.