From www.bloodjournal.org by guest on July 14, 2017. For personal use only.
Cytokine Therapy With Gene-Transfected Cells: Single Injection of
Irradiated Granulocyte-Macrophage Colony-Stimulating
Factor-Transduced Cells Accelerates Hematopoietic
Recovery After Cytotoxic Chemotherapy in Mice
By Felicia M. Rosenthal, Reinhard Fruh, Reinhard Henschler, Hendrik Veelken, Peter Kulmburg, Andreas Mackensen,
Bernd Gansbacher, Roland Mertelsmann, and Albrecht Lindemann
Development of cell-based delivery systems that can release
therapeutic levelsof hematopoietic growth factors into the
systemic circulation would facilitate treatment of patients
requiring cytokine therapy. In this study, we have investi-
gated the potential of granulocyte-macrophage colony-stim-
ulating factor (GM-CSF)-secreting,irradiatedsyngeneic mu-
rine cells to accelerate hematopoietic recovery after
cytotoxic chemotherapy. As a model, CMS-5 fibrosarcoma
cells, were transduced with a retroviral vector containing
the murine GM-CSF cDNA. Transduced cells secreted 38 ng
GM-CSFI10 cells in 24 hours. After irradiation,in vitro GM-
CSF production initially increasedup to fivefold and was
measurable for about 2 weeks. One and 2 days after injection
of irradiated, GM-CSF-secreting CMS-5 cells (NPICMVGM-
H EMATOPOIETIC GROWTH factors have been shown
to accelerate bone marrow (BM) recovery after che-
motherapy and radiation therapy in mice.7 Inhumans, gran-
ulocyte colony-stimulating factor (G-CSF) and granulocyte-
macrophage colony-stimulating factor (GM-CSF) are widely
used to shorten the phase of neutropenia after cytotoxic ther-
apy and after BM transplantation.82However, serum half-
life of these cytokines is relatively short (eg, 35 minutes for
GM-CSF after interperitoneal injection in mice) and continu-
ous parenteral administration, eg, by the use of Alza minios-
motic pumps, or repetitive injections, has been used to main-
tain biologically effective serum concentrations.I5Methods
to provide sustained cytokine serum levels for several days
without the need for daily injections or surgical implantation
of delivery devices would be desirable. Autologous or allo-
geneic cells, genetically modified to constitutively secrete
the cytokine of interest, could fulfill these criteria. Several
investigators have already described the successful introduc-
tion of genes, coding for enzymes like al-antitrypsin or
hormones like insulin, into murine fibroblasts or myoblasts,
and the therapeutic application of these cells in animal defi-
ciency models.z4 Other groups have introduced cytokine
Fromthe Departments of Medicine I (Huematology/Oncology)
and Surgery, University Medical Center Freiburg, Feiburg, Ger-
many; and the Department of Medicine, Memorial Sloan-Kettering
Cancer Center, New York, NY.
Submitted March 16, 1994; accepted July 5, 1994.
Supported by a grant from the Deutsche Forschungsgemeinschafi
(Sonderforschungsbereich 364).
Address reprint requests to Feliciu Rosenthal, MD, University
Medical Center Freiburg, Department of Internal Medicine l, Hae-
matology/Oncology, Hugstetter Strasse 55, 79106 Freiburg, Ger-
many.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8409-0023$3.00/0
2960
CSFICMS5# 6 cells)into mice, GM-CSF serum levels of 405f
58 pgImLand 1832 36 pgImL were measured, respectively.
Serum levels were comparable with levels detected 3 hours
afterinjection of100 ng recombinantmurineGM-CSF
(rmGM-CSF) subcutaneously (90 pg/mL). Injectionof N21
CMVGM-CSFICMS5 # 6 cells in cyclophosphamide-treated
mice was as effective in accelerating neutrophil recovery as
twice dailysubcutaneousinjectionsofrmGM-CSF.These
data suggest that irradiated hematopoietic growth factor-
secreting cellsmight offer an alternative to parenteral injec-
tions of recombinant cytokines in the treatment of neutro-
penic patients.
0 1994 by The American Society of Hematology.
genes into tumor cells to induce antitumor immune responses
or into hematopoietic cells to study the controls modulating
self-renewal and differentiation of hematopoietic ~ e l l s . ~ ~ - ~
Neutropenia after cytotoxic therapy is only transient, and
thus, requires cytokine substitution for a limited time period
only. Tani et alZ8have designed a subcutaneous diffusion
chamber apparatus for cytokine-producing fibroblasts that
allows regulation of cytokine secretion either by reimplanta-
tion of additional cells into the chamber or by removing
cells through ethanol treatment of the chamber. Ideally, gene
expression would be regulated at the transcriptional level,
eg, by the use of inducible promoters. Here we propose the
use of irradiated cytokine-gene-transduced cells that have
lost the capability to proliferate in vivo, but have retained
the ability to secrete biologically active levels of cytokines
for several days to weeks. Moreover, irradiation would be
a precaution when cells are injected in vivo, as malignant
transformation of genetically engineered fibroblasts has been
described after in vivo implantation in mice.
In this report, we show acceleration of hematopoietic re-
covery after injection of irradiated GM-CSF-transduced
murine fibrosarcoma cells in cyclophosphamide-treated
mice.
MATERIAL AND METHODS
Retroviral vector design and conversion of vectors into viruses.
Cloning of retroviral vectors NZICMVGM-CSF and DCIADIRIGM-
CSF has been described p r e v i ~ u s l y Retroviral
.~~ vector constructs
were converted to the corresponding virus by electroporating vector
DNA into the helper-free, ecotropic packaging cell line (GP+E-
86)? Colonies were isolated by G418 selection (0.7 m@& of
Geneticin; GIBCO Laboratories, Grand Island, NY), expanded to
producer cell lines and cell free supernatant was tested for viral titer.
Cell line and infection of tumor cells. CMS-5 is a methylcholan-
trene-induced, nonmetastatic fibrosarcoma cell line of BALBIc ori-
gin. Cells were cultured in Dulbeccos modified Eagles medium
supplemented with 10% fetal calf serum, 100 U/mL of penicillin,
100 pg/mL of streptomycin and 2 mmoVL L-glutamine at 37C in
5% CO2 atmosphere. Virus-producer cell lines secreting high titer
of virus (1 to 5 X IO5) were usedto infect CMS-5 cells. Bulk-
Blood, Vol 84, No 9 (November l ) , 1994: pp 2960-2965
 From www.bloodjournal.org by guest on July 14, 2017. For personal use only.
THERAPY WITH GENE-TRANSFECTEDCELLS
infected cells were isolated by G41 8 selection, expanded to cell lines
and used for further analysis.
Cyrokine assay. Secretion of GM-CSF into supernatants by ret-
rovirally infected tumor cells and GM-CSF serum levels were deter-
mined using an appropriate bioassay and confirmed by enzyme-
linked immunosorbent assay (ELISA; Endogen, Boston, MA).
Supernatant from semiconfluent irradiated or unirradiated parental
or cytokine-secreting CMS-5 cells was collected after 24 hours, and
assayed for GM-CSF production after determining the number of
viable cells. Irradiation was performed at room temperature with a
'37Cs-irradiationsource at a dose-rate of 3 Gy/min. Irradiated (35,
50, and 100 Cy) CMS-5 andN2/CMVGM-CSF/CMS5 # 6 cells
(lo6) were plated in 25-mL tissue-culture flasks and the amount of
GM-CSF in a 24-hour culture supernatant l , 3,6,9, or 12 days after
irradiation was determined. GM-CSF biologic activity was measured
by testing the ability of GM-CSF-containing preparations to induce
'H-thymidine incorporation into DNA of GM-CSF-dependent mu-
rine 3 2 x 1 3 cells as previously de~cribed.2~
Treament of mice. Female BALB/c mice were purchased from
the Zentralinstitut f i r Versuchstierzucht in Hannover, Germany and
used at 9 to 11 weeks old. On day 0 and day 2, all mice received
150 mg/kg of cyclophosphamide interperitoneally. One group of
mice did not receive further treatment, another group was injected
subcutaneously with 100 ngof recombinant murine GM-CSF
(rmGM-CSF; Boehringer Mannheim, Germany) twice daily on days
3 through 10. This dose of rmGM-CSF has previously been shown
to accelerate hematopoietic recovery after chemotherapy in mice.'
The last group of mice was injected subcutaneously on day 3 with
a total of lo7irradiated N2lCMVGM-CSFlCMS5 # 6 cells, split into
two injection sites. From day 4, blood was drawn daily from lateral
tail veins into EDTA-coated microvette tubes (Sarstedt, Heidelberg,
Germany). After lysis of erythrocytes with Turk solution, absolute
leukocyte counts were determined in a Neubauer Chamber. Differen-
tial counts were performed every other day. GM-CSF serum levels
were determined on days 1 through 4 after injection of GM-CSF-
producing cells.
Statistical analyses. Mean ? SE was calculated. Differences
between groups were examined for statistical significance by a two-
tailed, two-sample t-test. A P value s .05 was considered to indicate
statistical significance.
RESULTS
Infection ofjibrosarcoma cells. The structures of the ret-
roviral vectors used to transduce the murine fibrosarcoma
cell line CMS-5 have been described previou~ly?~ Virus-
containing, cell-free supernatant of infected packaging cell
clones was used to transduce CMS-5 cells. G418-resistant
CMS-5 bulk-infected cells were screened for GM-CSF ex-
pression by analyzing GM-CSF release into the culture su-
pernatant. Parental CMS-5 cells did not secrete any detect-
able GM-CSF in either assay. G418-resistant cytokine gene
transduced cells differed in their ability to synthesize and
secrete GM-CSF, depending on the retroviral vector con-
struct and the virus-producer clone used to transduce target
cells (Table 1). GM-CSF secretion of bulk-infected fibro-
sarcoma cells transduced with the GM-CSF vector ranged
from 0 to 128 ng/106 cells in 24 hours. GM-CSF production
of cells was found to decrease after extended periods of
culture without G418 selection. Bulk-infected NUCMVGM-
CSF/CMSS # 6 cells that were used for the in vivo studies
described here, secreted 38 ng/106 cells in 24 hours unless
irradiated (see below).
296 1
Table 1. GM-CSF Secretion by Transduced CMSS Bulk Culturesaa
Daerminod by ELBA
Virus Clone GM-CSF Ing/lO' cells and 24 h)
~ _ _ _ _ _ ~
N2lCMVGM-CSFE86 # 6 128 t- 6.4
N21CMVGM-CSFE86 # 10 80 2 1.4
NZlCMVGM-CSFE86 # l ? 126 t- 1.8
DC/ADlRlGM-CSFE86 # 4 16 t- 2.5
DC/AD/RlGM-CSFiE86 # 15 8 2 3.4
DCIADIRIGM-CSFlE86 # 17 25 t- 0.1
DCIADIRIGM-CSFIE86# 18 21 2 3.6
Data are represented as means of duplicate determinations t- SE.
GM-CSF secretion after irradiation of cytokine-gene-
transducedjibrosarcoma cells. To prevent proliferation of
cytokine producing cells in vivo, cells were irradiated before
injection. To study how long cytokine secretion would con-
tinue after irradiation, N2/CMVGM-CSF/CMS5 # 6 cells
were irradiated with 35, 50, and 100 Gy, respectively, and
plated at a density of lo6cells/25-mL tissue-culture medium.
GM-CSF levels in 24-hour culture supernatants were deter-
mined 1, 3, 6, 9, and 12 days after irradiation. Unirradiated
N2/CMVGM-CSF/CMS5 # 6 cells produced 38 ng/106 cells
in 24 hours (Fig 1, day -1 before irradiation). After irradia-
tion, cytokine secretion initially increased threefold to five-
fold depending on the dose of irradiation applied. GM-CSF
levels decreased to levels similar to those observed before
irradiation by day12.As the number of viable cells de-
creased continuously, these data suggest that biologically
active GM-CSF is also released by radiation-damaged cells
for several days.
GM-CSF serum levels after injection of GM-CSF-secre-
tingjibrosarcoma cells in BALB/c mice. To obtain informa-
tion about the time course of GM-CSF serum concentrations,
blood was drawn at different time points after subcutaneous
injection of irradiated N2/CMVGM-CSF/CMS5 # 6 cells or
3 and 10 hours after subcutaneous injection of rmGM-CSF.
GM-CSF serum levels were determined in a bioassay and
confirmed by ELISA. In Fig 2A, GM-CSF serum levels of
mice injected subcutaneously with 100 ng rmGM-CSF are
shown 3 and 10 hours after injection. Three hours after injec-
tion of rmGM-CSF, a serum level of 93 t 1 1 pg/mL was
measured, whereas at 10 hours, serum GM-CSF levels were
below the limit of detection. Figure 2B shows GM-CSF
serum levels 1 to 4 days after injection of lo7irradiated N2/
CMVGM-CSFfCMS5 # 6 cells, which is 3 to 6 days after
initiation of cytotoxic therapy. One and 2 days after injection
of GM-CSF-secreting cells, GM-CSF serum levels of 405
t 58 pg/mL and 183 2 36 pg/mL were measured, respec-
tively. On days 3 and 4, GM-CSF serum levels were 88 2
24 pg/mL and 82 t 26 pg/mL, respectively, comparable
with the level detected 3 hours after subcutaneous injection
of 100 ng rmGM-CSF. NoGM-CSF was found in the serum
of mice injected with the same number of parental, nontrans-
duced CMS-5 cells.
Hematologic changes in cyclophosphamide-treated mice,
after injection of GM-CSF-secreting fibrosarcoma cells or
twice daily injections of recombinant GM-CSF. Nine-to
1l-week-old female BALB/c mice were injected intraperito-
 From www.bloodjournal.org by guest on July 14, 2017. For personal use only.

ROSENTHAL ET AL

1 T

t 1

-1 1 3 6 B 1 2 -1 1 3 6 B 1 2 -1 1 3 6 0 1 2

Dlya Pl0.r ImdMkn

Fig 1. GM-CSF production by W/CMVGM-CSF/CMSL # 6 CMS-5 cells and cell survival efter irradiation. One million cells were irradiated
on day 0 with 35 (A). 50 (B). or 100Gy (C), respectively, and platedin 25-mL tissue-cutture flasks. GM-CSF levels in 24-hour culture supernantants
were determined by bioassay on days -1 (before irradiation) anddays 1,3,6,9, and 12 after irradiation and after supernatant collection cells
were trypsinized andcounted. Values are means k SE of six (GM-CSF) or two (cell number) determinations.

neally on days 0 and 2 with l50 mgkg cyclophosphamide,
the control group didnot receive further treatment. One
group of mice was injected subcutaneously with IO' irradi-
ated N2/CMVGM-CSF/CMSS # 6 cells on day 3, another
group was injected twice daily (d3-d10) subcutaneously with
100 ng of rmCM-CSF. Figure 3 shows hematologic changes
in BALB/c mice after therapy. Baseline leukocyte counts in
BALB/c mice were between 9,000 and lO,OOO/pL. Three
days after initiation of cyclophosphamide therapy leukocyte
counts dropped to nadir levels of 1,000 to 1,5OO/yLin all
mice, persisting until day 6. Starting from day 7, a difference
in absolute leukocyte counts between GM-CSF-treated mice
and control mice injected with cyclophosphamide only was
noted. On day 8, absolute white blood cell (WBC) counts

I n IWd. CM86 RC.

400- 4a!

a- sm

m- a00
Fig 2. GM-CSF serum levels in BALBlc miceafter
injection ofGM-CSF-secreting CMS-5 cells, parental
CMS-5 cells or recombinant murine GM-CSF. (A) GM-
CSF serum levels 3 and 10 hours aftersubcutaneous
rm 1m
injection of 100 n g rmGM-CSF. (B} GM-CSF serum
levels 1.2.3, and 4 days after injection of IO' irradi-
ated (35 Gyl WICMVGM-CSFICMS5 # 6 cells or un-
transduced CMS-5 cells. Values are means 2 SE of
0 0 fourmicefrom two different experiments (days 1
a 10 i 2 a 4
and 2, and 10 hours) or 2 mice (days 3 and 4, and 3
Dsys pon InjsUlon hours). nd, not done; >, below limit of detection.
 From www.bloodjournal.org by guest on July 14, 2017. For personal use only.

THERAPY WITH GENE-TRANSFECTEDCELLS 2963

Fig 3. Peripheral WBC countsincyclophospha-

mide-treated BALBlcmice, after subcutaneous injec-
tion of rmGM-CSF or irradiated GM-CSF-secreting
*
fibrosarcoma cells. Rwulta represent means SE
of five mice from two dlfferent experiments (days3
through 81 and two mice (days9 and 10). Subcutane-
ous injmtiona of rmGM-CSF or NZ/CMVGM-CSF/
CMS5 I 6 cella are illustrated by arrows. ( P values
for day 8 cydophosphemide v GM-CSF subcutane-
ously: P = .010; cyclophosphemide v NZICMVGM-
CSFlCMS5 m P = ,036).

in mice injected subcutaneously twice daily with rmGM-
CSF as well as in mice treated with a single injection of
irradiated GM-CSF-secreting syngeneic cells were signifi-
cantly higher as compared with control mice (cyclophospha-
mide v GM-CSF subcutaneously: P = .010,cyclophospha-
mide v NUCMVGM-CSFICMSS#6: P = .036). Average
WBC counts on day 7 were 4,38O/pL in the cytokine treat-
ment group compared with 3,005/pL in control mice. On
day 8,WBC count of the cytokine treatment group had in-
creased to 9,26O/pL, and in controls to 6,34O/pL. In mice
treated with rmGM-CSF subcutaneously or with GM-CSF-
secreting cells, leukocyte counts continued to increase until
day 9 and returned toward baseline levels on day 10. After
recovery of hematopoiesis (eg, after day 9) no siginficant
difference in absolute leukocyte counts is e ~ p e c t e d . ~
Table 2 shows differential W C counts of treatment
groups as absolute values of leukocyte subpopulations on
days 7 and 8 after initiation of treatment. Untreated BALB/
c mice had a differential count of about 400 monocytes,
1,800 granulocytes and 7,100 lymphocytes. On day 3
through 5 , ie, 1 to 3 days after cytotoxic therapy, blood
smear examinations showed absolute neutropenia (<100/
pL) in all treatment groups (data not shown). Onday 7,
mice treated with subcutaneous rmGM-CSF or GM-CSF-
secreting cells showed almost twice the absolute leukocyte
levels of control mice, accounted for by a higher proportion
of myeloid cells. Baseline myeloid counts were reached in
both GM-CSF-treated populations 7 days after the first cy-
clophosphamide injection or 4 days after initiation of cyto-
kine treatment, respectively, whereas control mice had only
reached approximately half-normal values at that time. No
significantdifferencesin absolute lymphocyte counts were
noted between different treatment groups. On8,day the differ-
ence in absolute myeloid cells between cyclophosphamide-
only-treated mice and those with addition of hematopoietic
growth factor treatment was even more pronounced (cyclo-
phosphamide v GM-CSF subcutaneously: P = .019, cyclo-
phosphamide v NUCMVGM-CSFICMS5 M: P = .W).
DISCUSSION
The use of recombinant hematopoietic growth factors has
reduced morbidity of cytotoxic chemotherapy in tumor pa-
 From www.bloodjournal.org by guest on July 14, 2017. For personal use only.
2964
Table 2. Leukocyte Subpopulations
Control
Day 7
Lymphocytes 7,100 t 350
Myeloid cells 2,200 i 480
Day 8
Lymphocytes 7,100 t 350
Myeloid cells 2,200 +- 480
Results are shown for untreated control animals, and for days 7 and 8 after cyclophosphamide administration and subcutaneous injection of
rmGM-CSF or irradiated GM-CSF-secreting syngeneic NP/CMVGM-CSF/CMSB #6 cells (GM/CMS #6).Values are means SE of five mice from
two different experiments. (Pvalues for day 8, myeloid cells: cyclophosphamide v subcutaneous GM-CSF: P = .019, cyclophosphamide v N2/
CMVGM-CSF/CMS5#6 P = ,044).
tients by amelioration of neutropenia and its associated infec-
tious complications.82Currently, growth factors are applied
by daily subcutaneous injections or continuous intravenous
infusion. In this study, we show that a single injection of
irradiated GM-CSF-transduced murine fibrosarcoma cells
is as effective in accelerating hematopoietic recovery after
cyclophosphamide chemotherapy as twice daily subcutane-
ous injections of recombinant GM-CSF.
Because long-term expression of GM-CSF is not desirable
for the treatment of chemotherapy-induced neutropenia, non-
proliferating cells with a finite life span should be used.
Irradiation of transfected cells is one option and has the
additional advantage of preventing outgrowth of possibly
transformed cells in vivo.28Here we show that after irradia-
tion of GM-CSF gene-transduced cells with up to 100 Gy,
cytokine production persisted for more than 12 days in vitro
(Fig 1). After irradiation, GM-CSF secretion initially in-
creased. This effect could possibly be caused by cytokine
leakage from radiation-damaged cells or by radiation-in-
duced activation of transcription factors.*, Irradiated cells
were incapable of further proliferation in vitro, as shown by
decreasing cell numbers over time. There was no evidence
of tumor growth or other side effects in mice observed for
up to 2 months after treatment (data not shown).
After injection of lo7 irradiated GM-CSF-secreting syn-
geneic cells, circulating GM-CSF was measurable for 4 days,
reaching peak levels of 405 ? 58 pg/mL on the first day
after injection and decreasing slowly thereafter (Fig 2). GM-
CSF serum level 3 hours after subcutaneous injection of 100
ng rmGM-CSF was 93 2 11 pg/mL, comparable with that
found 3 and 4 days after injection of irradiated GM-CSF-
secreting cells. Ten hours after subcutaneous injection, GM-
CSF was no longer detectable in serum. These data show
that after injection of cells that continuously secrete GM-
CSF, sustained serum levels are achieved as opposed to only
transient elevation found after subcutaneous injection.
Accelerated recovery of WBCs and total myeloid cells
after twice daily subcutaneous GM-CSF treatment after cy-
clophosphamide therapy has been previously shown by
Gamba-Vitalo et al.3 Here we confirm the effects of twice
daily subcutaneous rmGM-CSF injections on WBC and my-
eloid recoveryandshow that injection of irradiated GM-
CSF-secreting syngeneic cells is at least as efficacious (Fig
3 and Table 2). Although implanted devices as described by
ROSENTHAL ET AL
Cyclophosphamide + GM-CSF + GM/CMS5 U6
1,674 t 102 1,869 i 261 1,465 t 304
1,331 t 415 2.771 i 438 2,655 t_ 508
3,410 t 316 4,078 i 651 2.977 i 638
3,657 t 163 5,304 2 310 6,713 f 802
Tani et alZ8or miniosmotic pumps are likely to be equally
effective, both approaches require surgical interventions.l4,l5
With current technology, it is unlikelythat cytokine-gene-
transfected cells would replace administration of recombi-
nant growth factors in transient, chemotherapy-induced neu-
tropenias except in special circumstances when poor patient
compliance or other factors make daily administration diffi-
cult or impossible. However, successful treatment of chronic
neutropenic disorders like Kostmanns syndrome (congenital
neutropenia) and cyclic or idiopathic neutropenias with ge-
netically engineered cells or slow-release formulations of G-
CSF might be possible in the nearfut~re.~. In this situation,
the application of G-CSF gene-transfected unirradiated, non-
proliferating autologous cells, possibly in conjunction with
a suicide gene allowing interruption of cytokine secretion in
case of detrimental effects or other clinical indications, ap-
pears to bea promising strategy, and is currently under inves-
tigation in our laboratory.zz~23~36
ACKNOWLEDGMENT
We thank Carolin Heifer for excellent technical assistance and
Claudia Schmoor for help with statistical analyses.
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 From www.bloodjournal.org by guest on July 14, 2017. For personal use only.

1994 84: 2960-2965

Cytokine therapy with gene-transfected cells: single injection of

irradiated granulocyte-macrophage colony-stimulating
factor-transduced cells accelerates hematopoietic recovery after
cytotoxic chemotherapy in mice
FM Rosenthal, R Fruh, R Henschler, H Veelken, P Kulmburg, A Mackensen, B Gansbacher, R
Mertelsmann and A Lindemann

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