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Ethylnitrosourea-induced gliomas: a song in the attic?
* Corresponding author: Yuji Ikeno, M.D., Ph.D.
Mailing address: Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, 4939 Charles Katz Drive, San Antonio, TX 78229, USA.
Email: ikeno@uthscsa.edu
Received: 20 June 2023 / Accepted: 21 June 2023 / Published: 28 June 2023
DOI: 10.31491/APT.2023.06.111
Abstract
It is essential to seek the underlying molecular mechanisms of glioma development, and critical to discover interventions that reduce the incidence and attenuate the growth of gliomas using a well-established in vivo experimental model because glioma is clinically one of the most difficult malignant tumors to treat. Ethylnitrosourea (ENU)-induced glioma in the rat has been extensively utilized as an experimental brain tumor model since the mid-1960s, however, the scientific value of ENU-induced glioma has been underappreciated mainly due to the recent development of transgenic mouse glioma models. Because of the pathophysiological characteristics, which are similar to the high grade human malignant gliomas, ENU-induced glioma is an excellent in vivo model to: a) examine the cell origin, development, and pathophysiology of gliomas; b) investigate anti-tumor effects of calorie restriction (CR) and its underlying mechanisms; and c) discover new preventive and/or therapeutic interventions of glioma. Further exploration of genetic changes during initiation, malignant transformation of glial cells, and progression of glioma as well as CR’s anti-tumor effects on cellular processes using cutting edge technology, e.g., spatial transcriptomics, could provide more insight and a deeper understanding of the pathophysiology of gliomas.
Keywords
Ethylnitrosourea, glioma, gliogenesis, calorie restriction, spatial transcriptomics
Ethylnitrosourea (ENU)-induced glioma
Glioma is not a common malignant tumor compared to
other types of cancers. However, it is clinically one of the
most difficult malignant tumors to treat because of its extensive infiltrative nature to surrounding central nervous
system (CNS) parenchyma, which makes complete surgical resection extremely challenging, and their resistance
to chemotherapy and other treatments. If it is not treated,
the survival for glioblastoma (most malignant glioma)
patients after diagnosis is approximately 6-9 months, and
even with treatment, glioma is one of the malignant tumors that has a poor prognosis. Therefore, it is essential
to seek the underlying molecular mechanisms of glioma
development, and it is critical to also discover interventions that reduce the incidence and attenuate the growth
of gliomas using a well-established in vivo experimental
model.
N-ethyl-N-nitrosourea (ENU)-induced glioma in the rat
has been extensively utilized as an experimental brain
tumor model since the mid-1960s [1-5]. ENU is an alkylating agent and a highly potent mutagen. When a single
dose of ENU (50 mg/kg body weight) is intravenously
injected to pregnant rats on day 15 of gestation, all offspring from ENU injected pregnant rats develop glioma.
The continuous profile of tumor development of ENUinduced glioma has been well characterized which makes
the ENU-induced glioma an excellent in vivo model to
seek the mechanisms of glioma development and examine
the effects of interventions such as calorie restriction (CR).
ENU-induced glioma starts as hyperplasia, characterized
by a few or several abnormal cells forming a cluster with
no destructive nature. At 10-12 weeks of age, a cluster of
abnormal cells shows further growth (larger than that for
hyperplasia, but less than 500 μm in diameter) with higher
cell density and a mild destructive nature to surrounding CNS parenchyma, and progress as a microtumor (< 1
mm), medium sized (1-2 mm), and gross tumor (2 mm <)
with age. Histologically, a gross tumor is classified into
two types: a) oligodendroglioma, which shows isomorphic proliferation of small round cells similar to a human oligodendroglioma; and b) anaplastic glioma showing
cellular atypism and pleomorphism, and structural change
such as necrosis, hemorrhage, and endothelial proliferation. Specific cell type and origin of glioma induced by
ENU is still controversial because of the existence of glial
fibrillary acidic protein (GFAP) positive cells within the
tumor, which has a cellular morphology that resembles an
oligodendrocyte. However, most investigators agree that
major neoplastic cells are immature glial cells committed
to an oligodendrocyte lineage [2-5] and maintain GFAP
expression potential [2, 3, 6].
Effects of CR on ENU-induced glioma development
Since the original discovery by McCay and colleagues,
CR has become well known for anti-aging effects [7-
9]. Anti-aging actions of CR is correlated to a reduction
in tumor incidence and growth. CR’s anti-tumor effects
have been further demonstrated using several experimental model systems, including spontaneous lymphomas in
p53-deficient mice [10], breast cancer in DBA mice [11],
spontaneous tumors in Fischer 344 (F344) rats [12], transplantation of cultured cells or tumors [13], and induced
carcinogenesis [14-19]. However, previous studies have
been unable to fully uncover the exact underlying mechanisms of the anti-tumor effects of CR. In addition, some
of the experimental models previously used have several
weaknesses as follows: a) as the incidence of each spontaneous tumor is not high (15-20%), a large number of
experimental animals are required to examine the particular tumor or organ; b) the time course of tumor onset and
growth during the lifespan of the animal is not established
in spontaneous tumors; c) the interactions among mutation, oncogenes and transformation is not established in all
of the spontaneous and induced tumors; and d) carcinogen
challenge or tumor cell transplantation after CR induction
could be due to the difference in sensitivity against the
carcinogen and transplanted tumor cells, and normal cell
proliferation activities between CR and ad libitum (AL)
animals.
To further test the effects of CR on tumor development
and seek the possible underlying mechanisms of antitumor actions of CR, our laboratory has utilized the ENUinduced glioma in rats [20]. The development of tumors,
especially chemically induced tumors, is considered a
multistage process that can be divided into three distinct
stages, i.e., initiation, promotion, and progression [21].
In the ENU-induced glioma, initiation of tumors is controlled by the injection of ENU at day 15 of gestation,
but monitoring the incidence and growth of tumors over
time can provide insight into the effects of CR on promotion and progression. The advantages of this model are its
extremely high rate (100%) of tumor induction and the
certainty of the occurrence of multiple tumors per brain.
Additionally, the continuous profile and time course of
tumor progression in this experimental model have been
well documented as described above. Thus, this model is
an ideal in vivo model to critically evaluate whether CR
attenuates tumor incidence and/or growth as well as to
seek underlying mechanisms to attenuate glioma development in vivo.
Our results showed that the number of gliomas did not
change with age in the AL groups; however, the average size of the gliomas was significantly larger in the old
group compared to that of the younger groups. Immunohistochemical analysis showed increased accumulation of
lipid peroxidation products, oxidized protein, glycated end
products, heme oxygenase-1 (HO-1), and thioredoxin 1
(Trx1) with the growth of gliomas. The CR group reduced
both number and size of gliomas, and tumors exhibited
less accumulation of oxidative damage, decreased formation of glycated end products, and a decreased presence
of HO-1 and Trx1 compared to the AL group. Gliomas of
the CR group also showed less proliferating cell nuclear
antigen (PCNA) positive and more single-stranded DNA
(ssDNA) positive cells which are correlated to the suppressed tumor growth. Furthermore, the anti-tumor effects
of CR were associated with decreased hypoxia inducible
factor-1 alpha (HIF-1α) levels in normal brain tissue. Our
results demonstrated the anti-tumor effects of CR in gliomas, which were accompanied by reduced accumulation
of oxidative damage, decreased formation of glycated end
products, decreased presence of HO-1 and Trx1, reduced
cell proliferation, increased apoptosis, and decreased levels of HIF-1α [20].
ENU-induced glioma meets modern technology: in search of mechanisms
In spite of much endeavor, the exact mechanism of malignant transformation and multi-step carcinogenesis process
of ENU-induced glioma has not been fully uncovered.
This is most likely due to technical limitations to follow
the target cells of ENU during the carcinogenesis and accompanied molecular/biochemical changes.
Recent advancement of transcriptome experiments provides researchers with a very powerful tool to explore
molecular signatures that play important roles in tumor
development/progression. In particular, spatial transcriptome analysis provides extremely important information
for cancer research because this technique can map the
analytes, e.g., RNA data from their spatial localization on
tissue sections, which allows us to further analyze the molecular changes associated with malignant transformation
and cancer progression [22].
As described above, development of ENU-induced glioma
started as hyperplasia followed by sequential progress of
early neoplastic proliferation (ENP), microtumor, medium
sized and gross tumor (oligodendroglioma and anaplastic
glioma). Using this cutting edge technology, i.e., spatial
transcriptomics, we will be able to obtain the molecular
signatures that allow us to trace the transformed glial
cells and discover the gene expression changes that play
important roles in each step of ENU-induced glioma development (from malignant transformation of glial cells to glioma progression) and also determine the responsible
molecular changes by CR’s anti-tumor actions.
Conclusion
The scientific value of ENU-induced glioma has been underappreciated and underutilized mainly due to the recent development of transgenic mouse glioma models and the clinical importance of glioblastoma, which is the most common malignant glioma in humans. Although the histopathological features of ENU-induced glioma are different from the human malignant glioma, i.e., the glioblastoma, some of the pathophysiological characteristics, e.g., proliferative activity, invasiveness, vascularization, and blood-brain barrier disturbances, are similar to the high grade human malignant gliomas. Therefore, ENU-induced glioma is an excellent in vivo model to: a) examine the cell origin, development, and pathophysiology of gliomas; b) investigate anti-tumor effects of CR and its underlying mechanisms; and c) discover the new preventive and/or therapeutic interventions of glioma. Further exploration of genetic changes during initiation, malignant transformation of glia, and progression of glioma as well as CR’s anti-tumor effects on cellular processes using cutting edge technology, e.g., spatial transcriptomics, could provide more insight and a deeper understanding of the pathophysiology of gliomas. More importantly, results from the further study of ENU-induced glioma could be clinically significant for developing a new intervention to attenuate the occurrence and growth of gliomas in humans.
Declarations
Acknowledgements and financial support
I would like to express my sincerest gratitude to Dr. Takayoshi Ikeda for his invaluable guidance and support throughout the research work on ENU-induced glioma and pathology training when I was in the First Department of Pathology, Nagasaki University School of Medicine. This research was supported by the San Antonio Nathan Shock Center Pathology Core (NIH Grant AG13319).
Conflicts of interest
Yuji Ikeno is a member of the Editorial Board of Aging Pathobiology and Therapeutics. All authors declare no conflicts of interest and were not involved in the journal’s review or decision of this manuscript.
Ethical approval and informed consent statement
Not applicable.
Availability of data and materials
Not applicable.
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