Page 140 - MI-2-3

Microbes & Immunity Characterizing low-grade CNS tumors

and their prognostic and therapeutic relevance, the Formalin-fixed tissues were processed with
present study incorporated Ki-67, MMP-2, VEGFR2, embedding and sectioned at 10 µm using a microtome
CD11b, Iba1, and DNMT1 for characterization. (WESWOX, India). Sections were mounted on glass slides,
deparaffinized through alcoholic dehydration, stained
In the present work, we analyzed histopathology and
molecular hallmarks of three types of low-grade CNS with hematoxylin and eosin (Merck, India), and observed
under a bright field microscope (TS100F Eclipse, Nikon
tumors: spinal myxopapillary ependymoma, fibroblastic Corp., Japan). Images were captured using a CCD camera
meningioma, and diffuse astrocytoma. Low-grade CNS (DS-Fi2-U3, Nikon Corp., Japan).
tumors pose considerable clinical challenges due to
their unpredictable progression and the lack of reliable Tumor type and grade were determined by a
prognostic biomarkers. Present diagnostic modalities collaborating pathologist based on histological features,
often fail to predict which tumors will undergo malignant following the 2016 WHO classification of CNS tumors.
transformation, when this transition might occur, and A total of 10 low-grade tumor samples were obtained: three
how aggressively the disease may evolve. Consequently, spinal myxopapillary ependymomas, four fibroblastic
therapeutic strategies remain suboptimal, adversely meningiomas, and three diffuse astrocytomas. These
affecting patient outcomes. tumors are frequently underdiagnosed and are typically
identified only after progression to higher-grade lesions,
Moreover, the tumor microenvironment and cellular which limits the number of available samples. Additional
dynamics that drive progression from indolent to clinical data included magnetic resonance imaging (MRI)
aggressive phenotypes remain poorly characterized, scans with T1- and T2-weighted contrast-enhanced
representing a critical knowledge gap in neuro-oncological imaging and magnetic resonance spectroscopy (MRS),
research. Accordingly, this study aimed to: (i) conduct a focusing on N-acetylaspartate (NAA) peaks and choline/
comparative analysis of key molecular ontogenic profiles creatine ratios. The use of post-operative human tumor
between glial and non-glial low-grade CNS tumors to tissues adhered to ethical guidelines approved by the
reveal physiological differences; (ii) determine whether Institutional Ethical Committee, IPGME&R, Kolkata (vide
these molecular hallmarks represent viable, subtype- Memo No. Inst/IEC/553, dated January 15, 2014).
specific therapeutic targets; and (iii) explore whether these
molecular characterizations could inform the development 2.2. Silver/gold (SG) staining of paraffin-embedded
of targeted therapies with improved efficacy and reduced tissue
off-target toxicity. Silver staining and gold toning were performed on
Although limited by a small sample size, this study 10 µm paraffin-embedded tissue sections. After fixation
offers a unique contribution to understanding the and deparaffinization, sections were stained using freshly
molecular landscape of low-grade CNS tumors. It may prepared ammoniacal silver carbonate (CDH, India),
aid in diagnosis, risk stratification, and therapeutic followed by rinsing in 10% formalin. Subsequently, the
decision-making. Ultimately, this approach seeks to bridge sections were toned in gold chloride (Loba Chemie,
fundamental tumor biology with clinical application, India) and then fixed in sodium thiosulfate (MERCK,
offering potential pathways for early intervention strategies India). Microscopic analysis was carried out using the
that could prevent malignant transformation and enable same imaging system as described in Section 2.1. Images
more effective targeted therapies. were acquired using the NIS Element-BR software (Nikon
Corp., Japan) to identify electron-dense macrophages and
2. Methodology microglia.
2.1. Tumor samples, histology, and metadata 2.3. Tissue preparation for immunofluorescence
Post-operative CNS tumor tissues were collected from microscopy
the Bangur Institute of Neurosciences, Institute of Post Tissues were fixed in 4% paraformaldehyde (MERCK,
Graduate Medical Education and Research (IPGME&R), India), washed, and stored in phosphate-buffered saline
Kolkata, India. Samples were preserved in (i) 10% (PBS; CDH, India) at 4°C. Specimens were sectioned at
buffered formalin (CDH, India) for histopathological and a thickness of 10 µm, and separate immunofluorescence
immunohistochemical analysis, (ii) 4% paraformaldehyde staining protocols were applied using the following
(MERCK, India) for immunofluorescence studies, and (iii) antibodies: glial fibrillary acidic protein (GFAP)-Alexa
serum-free Dulbecco’s Modified Eagle Medium (DMEM) Fluor 488-conjugated monoclonal antibody (mAb) (Cat.
(Gibco Invitrogen, United States of America [USA]) for No. 561449, BD Pharmingen, USA), CD11b-FITC mAb
live cell isolation and flow cytometry. (Cat. No. 101205, BioLegend, USA), and Iba1 mAb

Volume 2 Issue 3 (2025) 132 doi: 10.36922/MI025190040