Doxorubicin-provoked increase of mitotic activity and concomitant drain of G0-pool in therapy-resistant BE(2)-C neuroblastoma
Doxorubicin-provoked increase of mitotic activity and concomitant drain of G0-pool in therapy-resistant BE(2)-C neuroblastoma
Isabell Hultman 0 1
Linnea Haeggblom 1
Ingvild Rognmo 0 1
Josefin Jansson Edqvist 0 1
Evelina Blomberg 0 1
Rouknuddin Ali 0 1
Lottie Phillips 0 1
Bengt Sandstedt 0 1
Per Kogner 0 1
Shahrzad Shirazi Fard 0 1
Lars AÈ hrlund-Richter 0 1
0 Department of Women's and Children's Health, Karolinska Institutet, Stockholm. Sweden, 2 Department of Oncology and Pathology, Karolinska Institutet , Stockholm. Sweden
1 Editor: Javier S. Castresana, University of Navarra , SPAIN
In this study chemotherapy response in neuroblastoma (NB) was assessed for the first time in a transplantation model comprising non-malignant human embryonic microenvironment of pluripotent stem cell teratoma (PSCT) derived from diploid bona fide hESC. Two NB cell lines with known high-risk phenotypes; the multi-resistant BE(2)-C and the drug sensitive IMR-32, were transplanted to the PSCT model and the tumour growth was exposed to single or repeated treatments with doxorubicin, and thereafter evaluated for cell death, apoptosis, and proliferation. Dose dependent cytotoxic effects were observed, this way corroborating the experimental platform for this type of analysis. Notably, analysis of doxorubicin-resilient BE(2)-C growth in the PSCT model revealed an unexpected 1,5fold increase in Ki67-index (p<0.05), indicating that non-cycling (G0) cells entered the cell cycle following the doxorubicin exposure. Support for this notion was obtained also in vitro. A pharmacologically relevant dose (1μM) resulted in a marked accumulation of Ki67 positive BE(2)-C cells (p<0.0001), as well as a >3-fold increase in active cell cycle (i.e. cells positive staining for PH3 together with incorporation of EdU) (p<0.01). Considering the clinical challenge for treating high-risk NB, the discovery of a therapy-provoked growthstimulating effect in the multi-resistant and p53-mutated BE(2)-C cell line, but not in the drug-sensitive p53wt IMR-32 cell line, warrants further studies concerning generality and clinical significance of this new observation.
Data Availability Statement: All relevant data are
within the paper.
Funding: This work was supported by the Cancer
Research Foundations of Radiumhemmet (Dnr
121262 and 141393) to LAR, The Swedish
Childhood Cancer Foundation (Dnr 2015-0168) to
LAR, Robert Lundbergs minnesstiftelse to IH, and
Stiftelsen Anna Brita och Bo Castegrens Minne to
SSF and LH. The funders had no role in study
Childhood cancers show fundamental differences to most common adult solid tumours in
their cancer-causing genetics, cell biology, and importantly also their local tissue
]. Neuroblastoma (NB) is the most common extracranial solid tumour during
infancy, half of which are clinically manifested before the age of 18 months [
evidence for early stage microscopic tumour-like NB lacking metastasis in young infants supports
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
the notion of an origin already during the prenatal phase [
]. The diagnosis comprises a
spectrum of embryonic tumours of the peripheral sympathetic nervous system and shows a high
degree of intra- and inter-tumoural heterogeneity on both genetic and phenotypic levels, as
well as unique abilities to spontaneously regress/differentiate or develop metastatic phenotypes
]. Metastasis and relapse are the main causes of death, which make it imperative to
understand metastatic dissemination and intra-tumour variations for therapy responsiveness. An
influence from the microenvironment on clonal dominance likely contributes to the disparity
between primary and metastatic tumours seen in many patients, as well as inter-tumour
heterogeneity between patients with the same tumour type [
]. Modelling of tumour micro
colonisation reflects in this context the ability of cell subpopulations to comply or adapt to new
environments (i.e. subsets of cells either present in a minority at diagnosis or develop during
therapy), a feature with great impact on metastasis and clinical prognosis .
Several in vivo NB-models are available, including among others subcutaneous or
orthotopic xenografts, as well as genetically modified models to simulate tumour induction and
growth, ideally confined to the relevant tissue environment [
]. Patient-derived xenograft
(PDX) models generated by injection of fresh human tumours to mice is widely considered a
clinically more predictive alternative compared to serially transplanted tumour lines [
Specifically for NB, orthotopic PDX has been suggested as the model of choice for studying
invasion and metastasis ([
] and ref therein).
Progress will come from deciphering the complex cross talk between the primary tumour,
its immediate microenvironment, and metastatic niches. A comprehensive program to
systematically evaluate anti-tumour agents for childhood cancers in various models for significant
clinical activity (The Pediatric Preclinical Testing Program; supported by The National Cancer
Institute) noted that the dominant difference between the gene expression of xenograft models
and their human counterparts was the signature contributed by stromal cells [
these findings it is noteworthy that a large study on colorectal cancer demonstrated that when
gene expression patterns in human tissue environment from patient material were compared
to results in a PDX model the analysis in mouse stroma showed significantly altered
predictions on clinical response to therapy [
]. Further, convincing data today link processes of
cancer progression to induction of cellular potency [
]. At the same time there is an
increasing insight regarding differences between human and mouse species for the signalling
pathways controlling the induction of cellular potency [14±16].
Developmental and species aspects are thus of importance when analysing the relevant
signalling between NB and the host. A driving momentum behind the here presented approach is
that compared to current animal xeno-models, a homologous embryonic setting may provide
a favourable micro-environmental setting for studies and preclinical evaluation of embryonic
tumours and their response to chemotherapy. Tzukerman, Skorecki and co-workers were first
to demonstrate the use of non-malignant human experimental teratoma as a more optimal
niche for intercellular interaction and a tool in cancer research investigating the stromal
response in human tumour cell growth [17±21]. The model represents increasingly chaotic
embryonic processes, comprising compartmentalised tissues or organoid-like development
including stages immediately preceding the positioning of adrenal sympatical progenitors in
embryonic mesenchyme [
]. This led us to test the PSCT milieu for in vivo support of
tumours of embryonic origin, establishing the NB-PSCT model ([
] and reviewed in
). The embryonic nature of the model makes the approach especially applicable for
socalled `embryonic childhood cancers' originating early in life.
Here we apply the human embryonic PSCT experimental platform to explore
chemotherapy-responsiveness of two well-characterised NB tumours.
2 / 16
Material and methods
This study was performed in strict accordance with permissions from the Local Ethics
Committee at Karolinska Institute (114/00) and from the regional ethics committee (Stockholm
Northern Animal Review Board; Dnr N101/13; N118/14). All surgery was performed under
approved protocols for anaesthesia and all efforts were made to minimize suffering.
BE(2)-C and IMR-32 were obtained from ATCC (Manassas, VA, USA). Cell line
authentications were performed using STR analysis.
BE(2)-C is a clonal subline of the multidrug-resistant and p53-mutated NB tumour line
SK-N-BE(2) which was derived 1972 from a metastatic site of the bone marrow from a
22-month old boy after repeated courses of chemotherapy and radiotherapy. IMR-32 was
derived from a metastatic site in abdominal mass of a 13 months old boy. Both cell lines have
been demonstrated to exhibit a poorly differentiated phenotype in vivo and genetic features
typical for high-risk NB [
]. NB-cells were cultured in Thermo Scientific HyClone RPMI
1640 medium supplemented with 10% fetal bovine serum, 1% L-glutamine (Invitrogen,
Carlsbad, CA, USA), and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA), at 37ÊC, 5%
CO2 with high humidity.
Doxorubicin (doxo) (Sandoz; Ebewe) was diluted in 1xPBS (DPBS GIBCO ™ Life technology)
to indicated doses and administered by intra peritoneal injections (PSCT model), or added to
indicated concentrations in cell culture medium (in vitro analysis). Control (mock) treatment
was performed with diluent (1xPBS). The in vitro IC50-value of doxo for IMR-32 (0.02 μM) is
400 times lower than for BE(2)-C (8.0 μM), classifying them as doxo-sensitive and
PSCT in vivo model
Pluripotent stem cell induced experimental teratoma (PSCT) was generated in NOD SCID
gamma (NSG) mice from diploid bona fide hESC as described [
23, 24, 26
]. In brief; 8±12 weeks
old NSG male mice received an injection of 105 HS181 cells (46XX) under the testicular capsule
(one side) [
]. When the growth reached a diameter over 9mm, 2x106 NB cells in logarithmic
growth were injected in a 50μl medium suspension. Animal hosts were randomised into treatment
groups (5±6 animals per group) and doxo therapy started 14 days after transplantation of NB cells.
PSCT including NB growth was harvested at indicated time points, formalin-fixed, paraffin
embedded and processed as previously described [
]. The blocks were consecutively cut
at 4μm or 10μm and stained with Hematoxylin&Eosin (HE) for histological orientation and
Positive engraftment of IMR-32 was verified by fluorescent in situ hybridization (FISH)
using probes specific for human chromosome X (spectrum orange) and Y (spectrum green)
(Vysis CEP X/Y DNA 30±16: nr 7J2050 Abbott). The presence of human Y-chromosome was
taken as evidence for tumour cells, growing in female (46XX) teratoma environment (PSCT).
The simultaneous signal for the X-chromosome was used as internal control of the assay.
Positive engraftment of BE(2)-C, lacking a stable Y-chromosome centromere, was verified by FISH
using probe for detection of amplified NMYC (spectrum orange) (Vysis LSI N-MYC so 3219;
nr 5J5001 Abbott).
3 / 16
The histological slides were scanned in a Hamamatsu 2.0 high-resolution scanner and
analysed using the NDP.view2 software. The percentage of positive cells was determined through
blind assessment by two well-trained researchers. The immunohistochemistry (IHC) results
were verified using positive control slides from tissues known to express the antigen of interest.
Negative controls included using tissues known to be negative for the marker, omitting primary
antibody and the use of isotype control antibody. A list of used antibodies is presented in Table 1.
Proliferation was assessed using IHC for the expression of Ki67 [
]. Early apoptosis was
assayed using IHC staining for cleaved caspase 3 (c-casp3). Cell death was assayed by
morphological appearance of mitotic catastrophe, i.e. multiple micronuclei and nuclear fragmentation
visualized by DAPI staining [
In vitro analysis
NB cells in logarithmic growth were suspended and dispersed into petri dishes containing
glass-coverslips (VWR #631±0149). For BE(2)-C: 2x105 cells, and for IMR-32: 1x106 cells, were
added per plate (Day 0). A minimum of three separate experiments were performed, each
experiment in duplicates.
Doxo was added to a concentration of 1μM (Day 1). For mock-treatment, the same amount
of 1xPBS was added. Forty-eight hours later (Day 3), the cells were fixed for 15 min in ice-cold
freshly prepared 4% paraformaldehyde (then washed and stored in 1xPBS at +4ÊC); or
alternatively received a second treatment (1μM doxo or 1xPBS) given 1 hour after replacement of
medium (allowing culture condition to recover before adding the chemotherapy). The cells
were then cultured for another 48 hours before fixation (Day 5). Forty-eight hours before
fixation 1μM of EdU was added to each plate.
Immunocytochemistry was performed accordingly; following fixation, cells were incubated
in TNB Buffer: 0.5g of blocking reagent (#FP1020) to 100 ml TBS buffer (Tris/NaCl pH 7.4)
for 30 min at room temperature. Cells were then incubated with the 1Âab diluted in 0.3%
TX100, 0.1% NaN3 in 1xPBS over night at +4ÊC. Following washing, cells were incubated with 2
Âab diluted in TNB buffer, for 2 h at room temperature. The cells were washed and mounted
with Prolong Gold anti-fade with DAPI to visualize nuclei. Stained cells were analysed using a
Metafer1 Slide Scanning Platform.
A list of the antibodies and kits used is presented in Table 1.
Collected data was analysed using one-way analysis of variance (Anova). Bonferroni correction
or TukeyÂs multiple comparison post-hoc test was applied to adjust for multiple testing. The
Vectastain Universal Elite
DAB-ABC, or goat anti rabbit Cy3.5 1:300
Vectastain Universal Elite ABC; or goat anti
rabbit Cy3.5 1:300
Goat anti rabbit AF488 1:1000
According to manufacturerÂs NA
According to manufacturerÂs NA
4 / 16
software GraphPad Prism 6.0 or 7.03 was used for testing normal distribution and generation
of graphs. The in vitro data was analysed in RStudio and percentage of positive cells followed
by the mean (±SEM) for each combination of labelling was calculated. All scores presented in
percentages were transformed into rationalized-arcsine-units [
], the transformation of data
makes the distribution normal, thereby reducing problems related to the use of a
A. In vivo analysis
Micro colonisation of BE(2)-C and IMR-32 in the PSCT model. Tumour growth could
be observed two weeks after transplantation of BE(2)-C or IMR-32 cells to the PSCT model, in
line with previous reports (24). Micro-colonies were initiated by migrating NB-cells showing
tropism to loose mesenchyme in immediate proximity of blood vessels, the preferred cellular
context/niche supporting micro-colonisation (Fig 1) (24).
Doxo-induced cell death of BE(2)-C and IMR-32 in the PSCT model. Next, doxo was
administered to the mouse host by single or repeated intraperitoneal injections as described
below and illustrated in Tables 2 and 3 and Fig 2. Considering the reported multi-resistance
and high doxo-IC50 value of BE(2)-C , we titrated intraperitoneal regimens with host
sub-lethal doses of doxo, aiming to attain significant levels of cytotoxicity (Table 2). A dose of
4mg/kg did not result in significant levels of cell death in BE(2)-C cells. Raising the dose to
8mg/kg resulted in a significantly higher mitotic catastrophe compared to mock-treatment.
Repeated doses of 4 + 4mg/kg given with a 48 hour interval increased the frequency of mitotic
catastrophe in BE(2)-C cells (Table 2, Fig 2). Besides mitotic catastrophe we were interested in
investigating early apoptosis. Positive staining for c-casp3 was used as an indication of early
apoptosis. The 8mg/kg dose resulted in a significantly higher value compared to
mock-treatment in BE(2)-2 cells. Similar levels of c-casp3 were observed after repeated doses (4 + 4mg/
kg) as with a single dose (8mg/kg) (Table 2). Based on these findings, the 8mg/kg and 4
+ 4mg/kg regimens were chosen for further studies.
Administration of 8mg/kg doxo resulted in 12±12% cells presenting mitotic catastrophe
and 14±9.9% cells staining positive for c-casp3 in IMR-32 cells. This was not significantly
different from mock-treatment (Table 3, Fig 2). Repeating the dose (4 + 4mg/kg doxo given with
a 48 hour interval) resulted in 28±15% presenting mitotic catastrophe (not significantly
different from mock-treatment: 4.3±0.2%) and 39±12% cells staining positive for c-casp3
(mocktreatment: 9.6±7.5%; p<0.05) (Table 3) in IMR-32 cells. Notably, a high variability in therapy
response between individual PSCTs was observed for these tumours (Fig 2), affecting the
Effects on proliferation of BE(2)-C and IMR-32 in the PSCT model. BE(2)-C tumours
in PSCT exhibited a Ki67-index of 43 ± 6.1%, similar to our earlier findings [
]. A single
administration of 8mg/kg doxo did not result in altered Ki67-index (Table 3 and Fig 2),
However, repeated treatment (4+4mg/kg) resulted in a significant increase of Ki67-index; from
43% to 64% (p<0.01): (Table 3 and Fig 3).
IMR-32 tumours in PSCT exhibited a Ki67-index of 63±8.3%, similar to previous findings
], and this proliferative index was not significantly altered by either of the administered
doses of doxo, when compared to control (Table 3, Fig 3).
Effects on the PSCT microenvironment. PSCT-tissues with high mitotic activity were
tested for doxo-induced toxicity, as an indication of toxic side effects in adjacent
non-malignant tissues. The Ki67-index in proliferative neural epithelium was not significantly affected
following treatment with 8mg/kg or 4+4mg/kg doxo (Table 4). Low levels of early apoptosis
5 / 16
Fig 1. Micro colonisation of IMR-32 tumours in the PSCT model. (A) Schematic illustration; NB cells were injected
into an arbitrary position centrally in the PSCT cellular mass, resulting in multiple micro-colonisations from migrating
NB cells. (B) A representative FFPE section of a PSCT with four IMR-32 colonies indicated (red borders). (C) IMR-32
colony surrounded by loose mesenchyme. LM = loose mesenchyme; NE = neural epithelium; C = cartilage;
M = muscle; Blue arrows = vessels. Size bars: B:5mm, C:500μm.
PLOS ONE | https://doi.org/10.1371/journal.pone.0190970
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(c-casp3) were indicated in NE following either treatment regimes (3.3±1.7% and 3.0±3.0%,
respectively; Table 4). Frequencies of mitotic catastrophe however showed no significant
differences in treated compared to mock-treated tumours (Table 4).
Embryonic cartilage and embryonic muscle are additional examples of components
exhibiting strong proliferation in PSCT [
]. Fig 4 illustrates a typical staining for c-casp3 in
embryonic cartilage and embryonic muscle, 96 hours after administration of doxo, illustrating
low or absent apoptosis.
B. In vitro analysis
Tables 5 and 6 summarise the in vitro data for BE(2)-C and IMR-32, respectively.
Doxo-induced cell death. The doxo-resistant phenotype of BE(2)-C and doxo-sensitivity
of IMR-32 was confirmed by TUNEL assay (Tables 5 and 6). Frequencies of early apoptosis
(ccasp3) were not significantly affected by single or repeated exposure to doxo in BE(2)-C cells
(Table 5). For IMR-32 cells, a single dose did not change the low frequency of c-casp3 positive
cells, but a repeated exposure yielded a significant reduction of this marker (Table 6).
Effects on Ki67-index in vitro. Exposure of BE(2)-C cells to 1μM doxo resulted 48 hours
later in a significant increase in the percentage of Ki67 positive cells (97±1.4 compared to
mock-treatment 36±5.5; p<0.0001). Similar results were obtained after repeated doses, 1+1μM
doxo, given with a 48-hour interval (p<0.0001,Table 5).
In IMR-32 cells, levels of Ki67 positive cells following 1μM doxo was not statistically
different significant compared to mock-treatment (Table 6). However a significant reduction was
observed following a repeated dosing (1+1μM doxo given with a 48-hour interval) (p<0.05,
7 / 16
Fig 2. The effects of doxo on BE(2)-C and IMR-32 tumours in the PSCT model. Fraction of cells (%; mean±SD)
expressing indicated markers following indicated treatments. mock = PBS; 4+4 = Repeated dose 4+4mg/kg doxo with
48h interval; 8 = single dose 8mg/kg doxo. Effects measured 96h after first administration of doxo. Data based on 5
PSCT per group. For statistical analysis, see text.
Induction of cell cycle arrest. To further analyse the cell cycle profiles of BE(2)-C and
IMR-32 following doxo exposure we analysed the incorporation of 5-ethynyl-2-deoxyuridine
(EdU), and/or fractions of cells staining positive for PH3 (Tables 5 and 6).
For BE(2)-C cells (Table 5); a single dose of 1μM doxo resulted in a decreased proportion of
EdU positive cells (79±6.1 compared to mock-treatment 99±1.3, p<0.0001), as well as an
increased proportion of PH3 positive cells (28±6.3% compared to mock-treatment 5.1±2.9%;
p<0.01). The fraction of double positive (PH3+/EdU+) cells increased to 19±6.3%, compared
to mock-treatment 5.1±3.0% (p<0.01). The remaining PH3 positive cells were EdU negative,
i.e. growth arrested.
Repeating the dose (ie. 1+1μM doxo) with a 48-hour interval resulted in an almost complete
ablation of EdU positive cells compared to mock-treatment (p<0.0001). The majority of PH3
positive cells were now EdU negative and only a minute fraction of PH3/EdU double positive
cells (0.4±0.4%) was found, a significantly lower percentage compared to mock-treatment (8.9
For IMR-32 cells (Table 6); a single dose of 1μM doxo resulted in decreased levels of EdU
positive cells (18±4.5% compared to mock-treatment 69±24%, p<0.0001). There was no
difference in the percentages of PH3 positive cells in mock versus treated, which were both low.
Similar to BE(2)-C cells repeated dose of 1+1μM doxo resulted in a decreased fraction of EdU
positive cells (12±3.1% compared to mock-treatment 81±18%, p<0.001) for IMR-32. There
was no difference in the percentages of PH3 positive cells in the mock-treated versus
doxotreated groups, where both values were low.
Chemotherapy (CT) is crucial for survival in high-risk NB and doxo is one of the most
important drugs in highly active treatment in this patient group. In the present study, a dose
8 / 16
Fig 3. Percentage Ki67 positive cells in BE(2)-C tumour following doxo treatment in the PSCT model. Fraction of
cells (%; mean±SD) expressing Ki67 96 hours after indicated treatments. Mock = diluent (1xPBS). Data based on 5
PSCT per group. For statistical analysis, see text.
9 / 16
Fraction of cells (%; mean±SE) expressing indicated markers 96 hours following indicated treatments. Mock = diluent (1xPBS). Data based on 5 PSCTs per group.
Statistical comparisons between the mock- or doxo-treated group: NS = p>0.05
; = p<0.01.
dependent cytotoxic effect following doxo treatment was observed in the human embryonic
PSCT-model, reiterating known characteristics of sensitive and multi-resistant phenotypes of
two metastatic high-risk NB. Repeated doxo treatment was consistently more effective
compared to an equivalent dose administered only once. This observation is in line with
longstanding clinical experience of enhanced anti-tumour effects from repeated chemotherapy cycles
Tumour-selective effects were indicated from the applied doxo regimens. Assessments of
non-malignant highly proliferative tissues (developing embryonic neural epithelium, cartilage
and muscle, located in the proximity of the studied NB-growth) revealed no increase in
cytotoxic effects after doxo treatment compared to mock treatment. This finding is important for
the analysis of anti-tumour effects in that it decreases, but not eliminates, the risk of
confounding doxo-induced toxicity in the local microenvironment.
A somewhat unexpected finding in the PSCT model was that post doxo treatment the BE
(2)-C tumour growth presented an increased Ki67-index (from 43% to 64%; p<0.01). Ki67 is a
protein absent from non-cycling cells, strictly associated with cell proliferation and detectable
in all active phases of the cell cycle [
], and thus the observation indicated a shift towards
cycling cells induced by the exposure to doxo. This was dependent on repeated doxo
treatment, possibly reflecting gradual tissue absorption of the drug [
]. Support for the notion that
quiescent (G0) tumour cells entering cell cycle after doxo treatment was obtained also in vitro.
The Ki67-index was investigated following exposure of cultured BE(2)-C cells to a
pharmacologically relevant dose of doxo (1μM), allowing a uniform bioavailability of doxo in the culture
medium. This resulted in a massive increase of Ki67 positive cells (97%; p<0.0001), >3-fold
increase of PH3/EdU double positive cells (19%; p<0.001), however no cell death (0.2%
TUNEL). Notably, also a reduction of EdU positive cells was observed (from 99% to 79%;
p<0.0001) along with the appearance of EdU negative cells in G2/M-phase (9.6%; p<0.001),
together indicating induction of cellular arrest, partly in G2/M-phase.
These findings are partly in line with previous reports that MYCN-amplified NB avoids
arrest in G1- and/or S-phase, favouring a G2/M-phase enrichment and reduced cell death [36±
41]. The results are consistent with findings in human hepatocellular carcinoma in which
doxo was shown to accelerate cell cycle transition, at first allowing cell cycle continuation, but
ultimately leading to cell cycle arrest [
]. Here it is also of potential interest that cells lacking a
functional p53/p21 pathway have been shown to arrest in G2/M-phase through down
regulation of cdk1 kinase activity by p14ARF [
Further investigations may elucidate whether the doxo-induced G2/M-arrested state in BE
(2)-C is permanent or reversible. There might be a putative gain for the multi-resistant
BE(2)10 / 16
Fig 4. The effects of doxo on PSCT non-malignant embryonic tissues. Immunohistochemistry staining of
formalinfixed paraffin-embedded PSCT histological slides, following intra peritoneal injection of the host mouse with 8mg/kg
doxo (A-D), or 4+4mg/kg doxo (E-H). High frequencies of positive staining for Ki67 in tissues compatible with neural
epithelium, muscle and cartilage can be seen, indicative of extensive proliferation (A,C,E,G). Low frequencies of
positive staining for cleaved caspase 3 can be seen in NE, muscle and cartilage, indicative of low frequencies of
apoptosis (B,D,F,H). Size bars: 50μm.
C cells to accumulate in G2/M-phase arrest. It has been shown that stable tetraploid clones are
more resistant to chemotherapy-induced apoptosis than diploid counterparts, due to increased
DNA repair and anti-apoptotic factors [
11 / 16
A small number of studies have suggested enhanced tumour re-growth following
]. Tumour growth/re-growth has been suggested to be stimulated through
apoptosis-induced proliferation, and diverse model systems have shown that apoptotic cells can
secrete mitogens to directly stimulate cell proliferation. For example, in vitro studies have
shown IGFII-like factor secretion from BE(2)-C cells into the culture medium, resulting in an
autocrine/paracrine stimulation of DNA replication and cell growth [
]. In another type of
study, long-term drug selection of BE(2)-C MYCN amplified NB cells with doxorubicin was
shown to enrich for a cancer-stem-cell-like subpopulation [
]. Calcagno and colleagues
reported a similar conclusion from in vitro studies of breast cancer [
]. Kurtova and
colleagues showed that chemotherapy induces prostaglandin PGE2 in neighbouring cells,
triggering cell division of putative cancer stem cell populations in bladder cancer [
Fraction of cells (%; mean±SE) expressing indicated markers following treatment at the indicated time points (48 or 96 hours). Data based on three independent
experiments. Statistical comparisons between the mock- or doxo-treated group: NS = p>0.05
= p< 0.0001.
12 / 16
However, a proliferative boost was not observed of IMR-32 tumours after doxo-therapy,
neither in vivo nor in vitro. Exposure in vitro to 1μM doxo revealed instead extensive cell death
(68% TUNEL positive). The combined in vivo/in vitro analysis thus indicated presence of a
functionally active p53 pathway. Evidently, numerous factors, genetic and epigenetic, may
relate to this difference but IMR-32 cells have been previously shown to express low levels of
the p53 downstream target p21 and cells with low levels of p21 are more likely to enter
A repeated doxo exposure in vitro (1+1μM) further enhanced the differences between BE
(2)-C and IMR-32 with regard to survival, possibly reflecting their difference in p53 status.
The vast majority of BE(2)-C cells survived a second exposure (TUNEL 0.6%) and maintained
positive staining for Ki67 (91%). However, most cells went into growth arrest (98% EdU
negative), mainly in late G2/M-phase (55% PH3 positive). IMR-32 on the other hand exhibited
high levels of growth arrest (88% EdU negative) but here leading to late apoptosis/cell death
(TUNEL 88%). Only a small fraction of IMR-32 was arrested in late G2/M-phase following the
repeated doxo-exposure (2.7% PH3 positive).
Doxorubicin-induced death has been reported to be independent of caspase in N-type NB
cells (e.g. IMR-32) [
]. In our study a single doxo in vivo or in vitro exposure of IMR-32 did
not alter the frequencies of c-casp3 positive cells. However, assessments of double exposures (4
+4mg/kg in vivo and 1+1μM in vitro) resulted in a significant increase of cleaved caspase-3
(p<0.01 in both cases). The reason for this discrepancy needs to be further explored.
In summary, we have demonstrated the use of the human embryonic microenvironment in
the PSCT model for in vivo evaluation of chemotherapy response in high-risk NB. The results
are encouraging for the further development of clinically relevant studies of intra tumour
heterogeneity and asynchronous tumour response to therapy in NB and other tumours
originating early in life. Notably, a phenomenon of compensatory re-growth of resistant cells/clones
following CT was detected for the multi-resistant p53 mutated NB tumour line BE(2)-C, but
not for the drug-sensitive p53 wild type NB line IMR-32. Further investigations are needed to
study the molecular regulation of arrest after recurrent treatment. Chemotherapy is long
known as a double-edged sword and the new findings next need to be evaluated for generality
and potential clinical relevance.
We thank Lotta Elfman and Inger Bodin for technical help, Nikolas Herold, Jan Mulder and
Malin WickstroÈm for scientific advice.
Conceptualization: Isabell Hultman, Lars AÈ hrlund-Richter.
Investigation: Isabell Hultman, Linnea Haeggblom, Ingvild Rognmo, Josefin Jansson Edqvist,
Evelina Blomberg, Lottie Phillips, Shahrzad Shirazi Fard.
Methodology: Isabell Hultman, Shahrzad Shirazi Fard.
Resources: Rouknuddin Ali.
Software: Josefin Jansson Edqvist.
Supervision: Per Kogner, Lars AÈ hrlund-Richter.
13 / 16
Validation: Isabell Hultman, Bengt Sandstedt, Shahrzad Shirazi Fard, Lars AÈ hrlund-Richter.
Writing ± original draft: Isabell Hultman, Linnea Haeggblom, Bengt Sandstedt, Shahrzad
Shirazi Fard, Lars AÈ hrlund-Richter.
14 / 16
(12):3555±65. Epub 2013/04/12. https://doi.org/10.1158/0008-5472.CAN-12-2845 PMID: 23576551;
PubMed Central PMCID: PMCPMC3686886.
15 / 16
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