Tumor microenvironment (Part I): Tissue integrity in a rat model of peripheral neural cancer

Abstract

ICAM-1 (intercellular adhesion molecule 1) and MPZ (myelin protein zero) are thought to be a factor in the integrity of nerve tissues. In this report, we attempted to trace the expression of ICAM-1, responsible for cell-to-cell adhesion, and of MPZ, the main constituent of myelin sheath, in malignant tissues of the sciatic nerve (SN) in inbred male Copenhagen rats. AT-1 Cells (anaplastic tumor 1) were injected in the perineurial sheath, and tissues of the SNs were collected after 7, 14 and 21 days and compared to a sham-operated group of rats (n = 6 each). Tissues were sectioned and histologically examined, under light microscope, and stained for measuring the immunoreactivity of ICAM-1 and MPZ under laser scanning microscope. The cancer model was established, and the tumor growth was confirmed. ICAM-1 showed severe decreases, proportional to the growing anaplastic cells, as compared to the sham group. MPZ revealed, however, a distinct defensive pattern before substantially decreasing in a comparison with sham. These results support the notion that malignancies damage peripheral nerves and cause severe axonal injury and loss of neuronal integrity, and clearly define the role of ICAM-1 and MPZ in safeguarding the nerve tissues.

Full text

Heliyon 10 (2024) e33932
Available online 1 July 2024
2405-8440/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
Research article
Tumor microenvironment (Part I): Tissue integrity in a rat model
of peripheral neural cancer
Ahmad Maqboul
a
,
b
,
*
, Bakheet Elsadek
b
a
Department of Anesthesiology and Operative Intensive Care Medicine, Charit´
e–School of Medicine Berlin, Corporate Member of Freie Universit¨
at
Berlin and Humboldt Universit¨
at zu Berlin, Berlin, Germany
b
Department of Biochemistry and Molecular Biology, College of Pharmacy, Al-Azhar University, Asyût, Egypt
ARTICLE INFO
Keywords:
Anaplastic tumor 1
AT-1
Cancer cells
CD54
Copenhagen rat
ICAM-1
Intercellular adhesion molecule 1
MPZ
Myelin protein zero
Neural cancer
P0
Protein 0
Tissue integrity
TME
Tumor microenvironment
ABSTRACT
ICAM–1 (intercellular adhesion molecule 1) and MPZ (myelin protein zero) are thought to be a
factor in the integrity of nerve tissues. In this report, we attempted to trace the expression of
ICAM–1, responsible for cell-to-cell adhesion, and of MPZ, the main constituent of myelin sheath,
in malignant tissues of the sciatic nerve (SN) in inbred male Copenhagen rats. AT–1 Cells
(anaplastic tumor 1) were injected in the perineurial sheath, and tissues of the SNs were collected
after 7, 14 and 21 days and compared to a sham-operated group of rats (n =6 each). Tissues were
sectioned and histologically examined, under light microscope, and stained for measuring the
immunoreactivity of ICAM–1 and MPZ under laser scanning microscope. The cancer model was
established, and the tumor growth was conrmed. ICAM–1 showed severe decreases, proportional
to the growing anaplastic cells, as compared to the sham group. MPZ revealed, however, a distinct
defensive pattern before substantially decreasing in a comparison with sham. These results sup-
port the notion that malignancies damage peripheral nerves and cause severe axonal injury and
loss of neuronal integrity, and clearly dene the role of ICAM–1 and MPZ in safeguarding the
nerve tissues.
1. Introduction
The neuron doctrine has established that nerve cells construct the nervous system individually forming syncytial networks. Each
neuron is designed with bundles of axons, and each axon is surrounded by myelin sheaths, with its distinctive Schwann cells [1]. In our
project, we are trying to explore the patterns of expressing different classes of proteins participating in neuronal invasion, nerve
damage, and regeneration and immune response within and distant from the complex tumor microenvironment (TME). These classes
include neurotrophins, cytokines, cytochromes and epoxyeicosanoids, apoptotic, oncogenic and transcription factors, glia and
astrocyte markers, and ion channels. Therefore, we rst transplanted AT–1 (anaplastic tumor 1) cells in Copenhagen rats, which
enabled us to establish the model of induced peripheral neural cancer, and to generate tissue samples constantly and reproducibly from
the malignant lumps. These tissues were used to deeply explore the TME and to investigate the regulation of the underlying proteins
responsible for cell adhesion, myelination, and neural growth and survival. Here, in the context of tissue integrity, we have examined
ICAM–1 (intercellular adhesion molecule 1; CD “cluster of differentiation” 54) and MPZ or P0 (myelin protein zero). ICAM–1, on one
* Corresponding author. Department of Anesthesiology and Operative Intensive Care Medicine, Charit´
e – School of Medicine Berlin, Charit´
eplatz
1, 10117, Berlin, Germany.
E-mail address: ahmad@maqboul.com (A. Maqboul).
Contents lists available at ScienceDirect
Heliyon
journal homepage: www.cell.com/heliyon
https://doi.org/10.1016/j.heliyon.2024.e33932
Received 19 April 2024; Received in revised form 27 June 2024; Accepted 1 July 2024

Heliyon 10 (2024) e33932
2
hand, endorses, as its name indicates, adhesion of the cells to each other and to the extracellular matrix, organizes intracellular re-
sponses, and recruits activated leukocytes in cases of inammation [2]. MPZ, on the other hand, is the main constituent and the most
abundant protein of the myelin sheaths. Its functions include myelination, insulation and protection of the axons, and propagation of
the nerve impulses [3]. In earlier studies on animal peripheral nerves, malignant cells were inoculated in the locality of the sciatic
nerve (SN) [4,5]. Here we bypassed the effect of the surrounding tissues by directly injecting the anaplastic cells inside the envelope
surrounding the SN. This permitted a more objectivity in investigating different neurotrophic factors and receptors arising from the
continuously growing tumor.
2. Materials and methods
Copenhagen rats (COP/CrCrl): Copenhagen rats have been enrolled due to their MHC (major histocompatibility complex) of the
RT1
av1
haplotype which permitted malignant cells with a hundred-percent-growing rate [6]. To exclude any possible effects of sex
hormones, and although the AT-1 cells are estrogen- and androgen-receptors negative, we preferred to recruit only inbred male rats [7,
8]. Including female rats, however, might have had no increased variability according to a recent report [9]. The animals have been
purchased from Charles River Laboratories International, Inc., Germany, through the Research Institutes for Experimental Medicine
(FEM), Charit´
e-School of Medicine Berlin. They have been sent (n =6 for each group) approximately weighing 250 g and have been
bred with numbers from 2 to 4 rats in each cage. Nutritional and water supply have been continually provided with a light source of
two light and dark cycles a day. The experiments were performed in a random manner, and the allocation codes have been kept out of
the examiner’s hands throughout the experiments’ times. The animals were to be euthanized upon the detection of any indicators of
discomfort or distress. For this goal, an isolated space, with a transparent compartment supplied with 1.8–5.3 L/min CO
2
(around
twenty percent ow rate), was set up [10]. However, none of the animals, after the optimization of the model, were excluded from the
experiments.
Cancer cell line and antibodies: AT-1 Cells, originating from Dunning R3327, are described by being non-metastasizing with an
incredibly fast progression in the absence of the androgens hormones [8]. Its main source, i.e. Dunning R3327, was rst detected as a
naturally occurring tumor in an old male of Copenhagen rat. It was seen at a specimen from the fty fourth generation of its previous
culture 2331 [6,11,12]. AT–1 Cell line was supplied from ECACC, UK (Catalogue number: 94101449). The anti–ICAM–1 antibody
(mouse; Catalogue number: ab2213) was purchased from Abcam plc, Cambridge, UK and the anti–MPZ (chicken; Catalogue number:
AB9352) from Millipore Corporation, MA, USA. Alexa Fluor® 594 (H +L) donkey anti-mouse IgG (Catalogue number: A-21203), and
Alexa Fluor® 488 goat anti-chicken IgY (Catalogue number: A-11039) were bought from Thermo Fisher Scientic™ GmbH, Germany.
Fig. 1. Schematic representation of the experimental design. AT-1 Cells were injected within the SNs’ perineurial sheath of Copenhagen rats.
Different groups were recruited for each timepoint. A sham-operated group was also recruited, in all steps, for comparison purposes. Three paths
were taken to perform these experiments. The morphological changes of the extracted SNs were photographed (1). Tissues of the SNs were sliced,
stained by H&E, and visualized under light microscope (2). For the third path, SN-tissues have been taken away using a different protocol, incubated
with the primary and then the uorochrome-tagged-secondary antibody. Fluorescence captured by confocal laser microscope has been further
processed, and the measured intensities were statistically analyzed (3).
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Heliyon 10 (2024) e33932
3
Cancer cells inoculation: AT–1 Cells were ourished in RPMI 1640, with the addition of 2 mM L-glutamine, 250 nM dexamethasone
and 10% foetal bovine serum, kept at ve percent CO
2
and thirty seven Celsius degrees, and counted with a Hemacytometer slide
(Bright-Line™) by means of a simple microscope. In order to perform the surgery, the animals were hypnotized by an O
2
inhalation
with isourane. The SN was uncovered by making a small opening between the gluteal muscle and the biceps after sterilization of the
right back limb using iodine and alcohol. Cell culture was prepared in PBS (phosphate buffered saline) with a pH adjusted to 7.4 (1.0 ×
10
6
cells in 10
μ
L PBS) and gradually injected beneath the nerve covering by the aid of a Hamilton® syringe (GASTIGHT® 1702LT
Series, 25
μ
L; Sigma-Aldrich Chemie GmbH, Germany). Throughout the initial seven days, animals showed an unprompted ache with a
changing grade of inammatory reactions in the place of surgery, which is attributed, upon euthanasia, to a very apparent tumor
inltration outside the nerve and in compact adherence to the internal tissues. Therefore, we reduced the amount to 0.5×10
6
cells per
10
μ
L PBS. The progression of the tumor was declined with neither an incursion to the adjacent tissues nor exaggerated adverse events.
This number of cells was used for all cancer groups (cf. pancreatic cells in a concentration of 1.0×10
5
per microliter buffer [13]). To
exclude the effects that may arise from the surgical procedure we injected a group of animals with the vehicle, i.e. PBS, and used it as a
control. The post-surgical analgesic used, metamizole sodium, was injected intraperitoneal, and also inserted in bottles of water for the
following 72 h. Repetitive inspections were conducted every 24 h to conrm regular movements and decient signs of discomfort, e.g.
bending or twisting, lacked consumption, and neglected cleaning actions (Fig. 1).
Tissue collection: Tissues were collected from sham-operated and cancer-inoculated animals, after 7, 14, and 21 days, in two
separate procedures. The rst for the investigation of morphology and light microscopy and the second for the study of immunou-
orescence (Fig. 1).
For morphology and light microscopy: Rats were anaesthetized with isourane, and the SNs, around 2.5 cm long, were extracted into
Eppendorf® Tubes (Eppendorf AG, Germany), instantly submerged within liquid nitrogen, placed in a −80 ◦C refrigerator, and further
checked for the development of neoplasms and the continuity of swelling. One to one-and-half centimeters of tumorous tissue were cut
from the SNs. Nerve tissues were sliced into longitudinal sections. The prepared slides were histologically visualized by means of
hematoxylin and eosin (H&E) and an inverted light microscope (Axiovert 25, Carl Zeiss, Germany) assembled with a cooled charge-
coupled device (CCD) camera (AxioCam HRc).
For immunouorescence staining: Rats were anaesthetized with isourane and a 100 mL PBS solution, with an adjusted pH of 7.4, was
diffused via the heart throughout the circulation, followed by 300 mL of a four percent-paraformaldehyde solution in 0.1 M buffer (w/
v). The SNs, around 2.5 cm long, were collected in Eppendorf® Tubes, also in paraformaldehyde, and left at room temperature for 90
min. The nerve tissues were rinsed with the buffer and kept in a ten percent sucrose solution in buffer at a 4 ◦C refrigerator to the
following morning. Tissues were, then, immersed within a “optimal-cutting-temperature” material and retained at −20 ◦C.
Fig. 2. A Copenhagen rat (COP/CrCrl) and morphological changes in the extracted SNs immediately captured after surgery. (A) A Copenhagen rat
with its characteristic white color and brown hood. (B) Photos of exposed SNs, around 2.5 cm long, from both ipsilateral (upper photo) and
contralateral (lower photo) sides of the same animal. (C–E) Photos of exposed SNs from sham-operated (upper) and cancer injected (lower) animals.
The continuous proliferation of the malignant cells within the SNs in the cancer-inoculated rats after seven (C), fourteen (D), and twenty one (E)
days are observed. Scale bars =1 cm. (For interpretation of the references to color in this gure legend, the reader is referred to the Web version of
this article.)
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Heliyon 10 (2024) e33932
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Approximately one centimeter of tumorous tissue was cut from the SNs. Tissue cubes were sliced into sections from ve to 7
μ
m
thickness by means of a cryostat (CryoStar™ NX70, Thermo Fisher Scientic™ GmbH, Germany). Tissue slides were incubated for 1 h
with a PBS solution containing Triton X-100 (0.3 percent), BSA (bovine serum albumin, 1 percent), horse serum (5 percent) and donkey
serum (5 percent). Slices were attached to slides covered with gelatin and kept at 25 ◦C with the intended antibodies for the next
morning. Tissue slides were rinsed and retained with the secondary antibodies labeled with Alexa Fluor® 488 or Alexa Fluor® 594.
Tissues were visualized by means of a confocal microscope (Zeiss® LSM 510, Germany). To maximize the signal and reduce back-
ground and noise, we used photo-stable uorophores of high-quantum yield, a CCD camera, clean and close coverslips, and a mini-
mally uorescent mounting medium.
Software-aided image analysis: Fiji (ImageJ) is the program used in this study because of its capabilities of automated unbiased
image analysis (ImageJ User Guide 1.46r) [14,15]. PIVs (pixel intensity values) in the ROIs (regions of interest) were estimated by
transforming the captured uorescent micrographs from a LSM (laser scanning microscope)- to a TIF (tagged image le)-type, pro-
grammed scaling the eight-bit photos, producing binaries, and subtracting the noises surrounding the ROIs. PIVs were generated and
Fig. 3. Histopathological examination of longitudinal sections of SN stained by hematoxylin and eosin. (A1 and A2) A light micrograph from sham-
operated animal tissues (left-hand column, ×20) and its magnied insert (right-hand column, ×40) reveal smooth bundles and a curvy shape of
bers which portray regular SNs. (B1–D1 and B2–D2) SN Tissues from cancer-injected animals, on days seven, fourteen and twenty one, respec-
tively, display an inltration of malignant cells with a gradual enlargement of Schwann cells nuclei and a degradation of nerve bers. Scale bars =
100
μ
m.
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Heliyon 10 (2024) e33932
5
accumulated in a separate le of the CSV (comma-separated values) type for statistical analysis (see Supplementary File).
Statistical data analysis: GraphPad Prism® (GraphPad Software, Inc., USA) was implemented in statistical assays. Data were
presented as [means (standard errors)] and decimals were rounded to two signicant digits [16]. To compare between the studied
groups, PIVs were analyzed by one-way ANOVA (analysis of variances) and the Dunnett’s post hoc test. If PIVs were not normally
distributed, the variations of the mean ranks were assayed by the Kruskal-Wallis and the Dunn’s tests. Statistical results of P <0.05
were concluded as considerable.
3. Results
SNs’ Morphology: We evaluated the macroscopical and microscopical changes, as a result of AT-1 cells injection, in the SNs.
Tumors proliferated in the ipsilateral and not in the contralateral SNs with observed gradual thickening and weight gain. Tumor
invasion also was not encountered in the sham-operated group (Fig. 2 (A – E)).
Fig. 4. Immunouorescence images of ICAM–1 in the neurons [on the left side] and the corresponding regions of interests identied by ImageJ
software [on the right side] in sham (A) and cancer-induced animals after 7 (B), 14 (C) and 21 (D) days of AT–1 cells inoculation in SN of
Copenhagen rats. The images show a gradual loss of immunoreactivity, which is represented by the staining of the whole nerve bers, from one (B)
to two (C) and then three (D) weeks of cancer invasion as compared to sham (A). Scale bars =50
μ
m.
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Heliyon 10 (2024) e33932
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Histology of tumor tissues: SNs have been checked for signs of cancer development, tissue disintegration, and inltration of im-
mune cells. Micrographs of the SNs in sham-injected group showed ordinary fascicles with a distinctive wavy shape and bar-like bers.
However, the brocytes and the Schwann cells’ nuclei revealed no obvious distinction. In cancer-inoculated rats, SNs were inltrated
by mononuclear cells. In addition, the gradual growth of the tumor from day 7 to day 21 was indicated by loss of integrity (Fig. 3 (A –
D)).
Decreased expression of ICAM–1: The visualization of the uorescent micrographs, on the left-hand column, shows a decline of
ICAM–1 positive immunoreactive (+IR) nerve bers after 7 days of cancer cells inoculation, followed by a dramatic decrease to the
halfway on day 14, which tries to regenerate on day 21, as compared to sham (Fig. 4 (A–D)).
The corresponding photos, on the right-hand column, clearly represent the difference in uorescence, i.e. the numbers and areas
identied as ROIs, between the sham-operated group in a comparison to the one-week group of tumor cells’ injection, and further to
the two- and three-weeks groups (Fig. 4 (A–D)).
These observations were conrmed by measuring the PIVs of ICAM–1 +IR nerve bers showing a decrease on the seventh day
[95.4 % (1.1)], severely going to a point of an equidistance after 14 [66.8 % (0.79)] and 21 [79.2 % (2.8)] days as compared to sham
[100 % (1.6)]. The difference between the values after 14 [66.8 % (0.79)] and 21 [79.2 % (2.8)] days are also signicant (FIG. 5).
Modied expression of MPZ: The immunouorescent photos of MPZ +IR bers behaved, however, in a different way. They
remarkably showed a greater amount of MPZ +IR bers after one week, which became substantially fewer after the second and third
weeks. The results displayed a substantial rise in the animal group on the 21-days in a comparison with that 14-days one. The cor-
responding photos, on the right-hand column, show the increased regulation in uorescence in the one-week group of tumor cells’
injection as compared to the sham group, followed by severe decreases in the two- and three-weeks groups. These visual identications
were in an agreement with the assessed PIVs as exhibited from the corresponding ROIs in the photos on the right-hand column (Fig. 6
(A–D)).
The uorescence of the MPZ +IR bers in the one-week group of tumor growth has been very intensely increased on day 7 [124 %
(1.9)] then decreased for the most on days 14 [72.8 % (0.7)] and 21 [72.2 % (1.6)] as compared to sham [100 % (1.9)]. However, there
was no change in the intensity values between the groups of the second- and third week of cancer injection. (Fig. 7).
4. Discussion
The TME in neural cancer: Models of perineural cancer invasion were previously induced through the injection of invading
malignant cells in the surroundings of that peripheral nerve [4,5]. Limitations, nevertheless, resulting from existent immune cells may
arise. Therefore, and in order to avoid this obstacle, we have designed the study in a way that cancer cells were injected in situ giving an
intimate contact to nerve bers.
Disintegration and demyelination of the nerve bers, damage of the axons, and occupation by the invading anaplastic cells. In
the current study, photos represent an evidence for the continuous development of malignant lumps in the SN (Fig. 2). Light mi-
croscope photos revealed a penetration of mononuclear cells within the nerve bundles, and clearly identied internal neural structures
and veried the degradation of nerve tissues characterized by gradual disintegration of the structural arrangement of the fascicles,
thinning of the bers, and damage and persistent dystrophy of the axons. The current study conrmed the occurrence of lesions in the
myelin sheaths exposing the axons leading to their damage and the subsequent replacement of the nerve tissue with a growing lump of
tumor cells.
Automated determination of ROIs and measurement of pixel intensity values: The novelty in the current report lie in the
application of an automated method for measurement of pixel intensity values, for regions of interest (ROIs) in immunouorescent
micrographs, reecting the expression of the studied proteins, ICAM-1 and MPZ, in the TME. The automation and the precision in
Fig. 5. Quantifying PIVs of ICAM–1 +IR bers in SNs of vehicle- and cancer-injected groups (on days 7, 14 and 21). ICAM–1 positive immuno-
reactive nerve bers decreased meaningfully on day 7, then a severely on days 14 and 21. (****) P <0.0001.
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Heliyon 10 (2024) e33932
7
determining the ROIs, which is traceable from the micrographs and their corresponding output photos, have given substantially ac-
curate and time-saving measurements when compared to other traditional methods (Figs. 4 and 6). The lack of bias and vagaries is,
above all, of a greater importance.
Loss of ICAM−1’s adhesive function in rat’s model of peripheral neural cancer. The continual decrease in ICAM−1’s production,
a protein distinguished by Rothlein et al., in 1986 [17], as compared to sham, conrms its main function in adhesion, and its failure to
maintain this role once its stores were depleted and the cell machinery was disturbed. The inoculation of tumorous cells, in our in vivo
cancer model, causes the recruitment of tissue-resident macrophages and other circulating inammatory mediators to the invasion
site, nally leading to a decrease in ICAM−1’s production (cf. different responses in mammalian cell lines) [18–20]. This may give our
model an advantage over other studies on cell lines, as responses to different stimuli, in different cell types or tissues and even in
different reactive regions within the same tissue, cannot be identical. ICAM-1’s increased expression on day 21, as indicated by higher
PIVs of +IR nerve bers, which is signicant from day 14, may, however, be understood as a trial from these tissues to regenerate and
to resist the loss of adhesiveness caused by the invading malignant cells. This rise is still a decline when compared to the higher levels of
Fig. 6. Immunouorescence micrographs of MPZ in the neurons [on the left side] and the corresponding regions of interests identied by ImageJ
software [on the right side] in sham (A) and cancer-induced animals after 7 (B), 14 (C) and 21 (D) days of anaplastic tumor cells inoculation in SNs of
Copenhagen rats. The images show an increase of immunoreactivity, which is represented by the more bright staining of the whole nerve bers after
one week (B), followed by severe decreases after two (C) and three (D) weeks of cancer invasion as compared to sham (A). Scale bars =50
μ
m.
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Heliyon 10 (2024) e33932
8
the sham and, even, of the 7 days’ group.
Similar expression proles of ICAM-1 in other tissues and cells. Our experimental model on ICAM-1 in peripheral neural cancer is
consistent with studies, on other types of tissues and cells, showing its declined expression. In breast cancer’s tissues and cells, ICAM-1
exhibited declined levels as compared to normal breast epithelia or benign breast cells. ICAM-1’s production have been stimulated by
tumor necrosis factor alpha (TNF
α
). This was not the case with colony stimulating factor (CSF), interleukins 2 and 6 (IL-2 and IL-6), and
interferons alpha and gamma (IFN
α
and IFNγ) [21]. It was also reported that ICAM-1’s production have been decreased in stomach
cancer which is correlated to lymphatic metastasis [22]. The decreased frequency of ICAM-1-expressing malignant cells was correlated
to their metastasizing capabilities [23]. Another study has shown that ICAM-1 is exclusively expressed in aggressive tumor cells linked
to the development of tertiary lymphoid structures (TLS) such as triple negative breast cancer (TNBC) and HER2 (human epidermal
growth factor receptor 2), and that an encouraged ICAM-1’s expression, in a number of cell lines, was observed by the
pro-inammatory cytokines TNF
α
, IL-1β (interleukin 1β) and IFNγ [24].
Different expression proles of ICAM-1 in other tissues and cells. In contrast to our results, ICAM-1 was up-regulated in the following
models. ICAM-1’s production was enhanced in glioblastoma through the incubation of TAMs (tumor associated macrophages) in a
hypoxic conditions with the addition of a HIF (hypoxia-inducible factor)-stabilizing drug. This increased expression has been
concluded to promote tumorigenesis and glioblastoma aggressiveness. In context of this conclusion, and as a therapeutic approach,
tumor cells were intracranially injected into ICAM-1 decient mice leading to a longer survival with a lower overall tumor volume
comparet to the wild type [25]. ICAM-1 was also overexpressed in bevacizumab-resistant glioma stem cells in hypoxic circumstances.
The hypoxia-induced overexpression of ICAM-1 was linked to the up-regulation of p-STAT3 (phosphorylated signal transducer and
activator of transcription). Overwhelming ICAM-1 restrained cancer invasiveness in different models [26]. ICAM-1 was also overex-
pressed in TNBC. Guo and colleagues have designed a model of a MRI (magnetic resonance imaging)-probe depending on ICAM-1
antibody-coupled particles leading to an improved ICAM-1’s production [27].
MPZ’s defensive role in regenerating the myelin sheaths and protecting the neural axons. The myelinating protein showed an
early protective response trying to insulate the nerve bers and minimize axonal exposure. This trial delayed the damage for one week,
which didn’t succeed after two weeks, with the continuous growth of the aggressive malignant cells, to protect the axons. This
conclusion is in line with studies referring to MPZ as the most abundant protein in myelin guaranteeing tissue integrity [28]. The
immunostaining of MPZ reveals the characteristic shape of the nerve bers which changes in agreement with its increased then
decreased expression with the development of malignant tumors.
MPZ’s defferential expression patterns. It is well established that mature Schwann cells express the neuronal markers, MPZ, and
others, insulating and protecting large nerve axons with myelin sheaths [29]. Transgenic mice expressing active Fyn (a tyrosine kinase)
displayed an elevated MPZ’s production as compared to control [30]. Whole genome microarray analyses for samples of urinary
bladder cancer and controls identied MPZ, with other 16, as common differentially expressed genes [31]. The demyelination of
RSC96 (rat Schwann cells) characterized by a decreased expression of MPZ was also caused by the platinum-based chemotherapeutics
cisplatin and carboplatin but not oxaliplatin [32]. A CGH (comparative genomic hybridization) analysis for a 6-member family from
three sequential generations, diagnosed with demyelination and neural inammation, revealed MPZ’s enhanced dosage. Therefore,
tissue integrity of myelin was correlated to a signicant MPZ’s dosage function and an elevated manufacturing of MPZ mRNA [33].
Loss of tissue integrity and failure of neural regeneration. Our report conrms that nerve tissues have been degenerated, and
axons severely damaged, as a result of the progressive tumor lesions. These lesions are irreversible. It was noted, on one side, that the
expression of ICAM−1 was down-regulated without presenting any other role in opposing the growth of the AT−1 cells. It was
observed, on the other side, that the expression of MPZ has been increased as a trial to rebuild the myelin sheath to protect the axon and
allow nerves to regenerate. As the trial did not succeed, the increased expression of MPZ showed a severe decay. These all indicate a
Fig. 7. Quantifying PIVs of MPZ +IR bers in control and cancer-injected rats’ SNs (on days seven, fourteen, and twenty one). MPZ positive
immunoreactive nerve bers showed a substantial rise after one week, and then severe declines at the end of the second and third weeks. There was
non signicant (ns) change between groups of the 14 and the 21 days. (****) P <0.0001.
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Heliyon 10 (2024) e33932
9
permanent proliferation and an eternal replication of cancer cells, and a failed neural regeneration.
Prospective studies. The late rise of the expression level of ICAM-1 requires more examination as a target protein with potential
anti-tumorigenesis and anti-proliferative therapeutic magnitudes. An investigation of the molecular mechanisms underlying the link
between the declined expressions of ICAM-1 and MPZ from one side, and the regulations of related cytokines, and apoptotic and
oncogenic factors, needs also to be conducted within and distant from this TME.
Ethics statement
The project was approved by the State Ofce for Health and Social Affairs (LAGeSo, Berlin, Germany) according to the guidelines of
the Charit´
e - School of Medicine Berlin (Project code: G 0314/13) and in agreement with ARRIVE 2.0 guidelines (https://
arriveguidelines.org/).
Funding
This work was funded by the Cultural Affairs and Missions Sector, Ministry of Higher Education of Egypt, for two years; and the
Foundation of Prof. K.H. Ren´
e Koczorek of Germany, grant No.: IA89838780, for one year. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Data availability
The raw data are submitted as a Supplementary File. Further enquiries can be directed to the corresponding author.
CRediT authorship contribution statement
Ahmad Maqboul: Writing – original draft, Visualization, Validation, Software, Methodology, Investigation, Funding acquisition,
Formal analysis, Data curation, Conceptualization. Bakheet Elsadek: Writing – review & editing, Supervision, Project administration,
Funding acquisition, Conceptualization. The research question of this study is a part of the PhD proposal registered by A. M. at the
Charit´
e - School of Medicine, Berlin, Germany.
Declaration of competing interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to
inuence the work reported in this paper.
Acknowledgement
Time had taken a highly expensive toll in health, in trust, in humanity, and in money. The rst author acknowledges the great
support of his family members during this time. The authors thank their colleagues, from Charit´
e - School of Medicine, Berlin, Ger-
many, and Al-Azhar University, Asyût, Egypt, who contributed to this work.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e33932.
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