Journal of Cancer Therapy, 2011, 2, 384-393
doi:10.4236/jct.2011.23053 Published Online August 2011 (http://www.SciRP.org/journal/jct)
Construction and Evaluation of a Combined
Cyclophosphamide/Nanoparticle Anticancer
Vaccine
Kurt Andrew Yaeger, Robert Anthony Kurt
Department of Biology, Lafayette College, Easton, USA.
Received June 9th, 2011; revised July 8th, 2011; accepted July 16th, 2011.
ABSTRACT
Tumor immunotherapy is a rapidly emerging form of cancer treatment. In the current study, a nanoparticle-based vac-
cine was constructed and the effi cacy w as assessed t hrough anal ysi s of i mmune cel l popu l at i ons , tumor growth rates, and
metastasis. The vaccine was fabricated through encapsulation of plasmid DNA encoding the tumor-associated antigen
Mage-b, and the TLR9 agonist CpG oligodeoxynucleotides by a biodegradable polymer, poly(L,D-lactic-co-
glycolic acid) (PLGA). The size and shape of the nanoparticles suggested that they were an appropriate size for uptake
by professional antigen presenting cells; dendritic cells. Furthermore, effects of the immunopotentiating drug cyclo-
phosphamide was included to decrease systemic populations of regulatory T cells (Treg); immune system sentinels that
down-regulate immune responses. The vaccine was assessed using the 4T1 murine mammary carcinoma model which is
a model for stage IV breast cancer. The combined cyclophosphamide/nanoparticle vaccine was shown to significantly
reduce 4T1 tumor growth rates and lung metastasis in female BALB/c mice.
Keywords: Nanoparticle Vaccine, Breast Cancer, Metastasis, 4T1
1. Introduction
In theory, an adaptive immune response could efficiently
control a neoplastic growth provided specific tumor-
associated antigens (TAA) exists within the malignant
cell population to prompt the response [1]. Unlike con-
ventional cancer treatments, this therapeutic modality
would be specific and systemic, able to target single
cancerous cells as well as distant metastases without cy-
totoxic side effects on healthy cells [2]. For instance, the
TAA Mage-b is a member of the melanoma antigen
(MAGE) family of TAA which is overexpressed by
many different tumors and exhibits low levels of expres-
sion by most normal adult tissues [3]. Indeed vaccination
with Mage-b was able to influence growth and metastasis
of the aggressive murine mammary carcinoma model
4T1 [4,5]. However, there are several factors that prevent
complete tumor rejection by host immune function.
Examples of these factors include: (1) inadequate antigen
presentation by immune cells such as macrophages and
dendritic cells (DC) [6], (2) poor distinction between
TAA and normal self epitopes [7], and (3) the accrual of
immunotolerance towards TAA [8]. Recently, methods
of overcoming such barriers have been established, pav-
ing the way for more effective cancer vaccines.
DC are antigen-presenting cells (APC) that promote
stimulation of naïve T lymphocytes through antigen en-
gulfment and subsequent presentation of the antigen by
major histocompatibility complex (MHC) proteins [9].
Upon encountering an antigen, peripheral DC mature and
migrate to the nearest lymph nodes, presenting epitopes
to T cells to trigger cell mediated immunity [10]. Unfor-
tunately, TAA do not often elicit an immune response
sufficient enough for tumor rejection. However, nanopar-
ticles may be able to selectively deliver TAA to DC since
peptide or DNA-based antigens encapsulated by PLGA
nanoparticles are efficiently taken up by DC [11,12]. The
spherical shape and size range of these nanoparticles
allow for efficient phagocytosis and antigen presentation
by DC. Also, due to extended delivery of nanoparticle
encapsulated antigens there exists prolonged antigen ex-
posure to DC, a characteristic that generates a more po-
tent immune response.
Although a nanoparticle-based vaccine can be effec-
tive, many tumor antigens are indistinct from normal
self-proteins and therefore may be passed over by DC

Construction and Evaluation of a Combined Cyclophosphamide/Nanoparticle Anticancer Vaccine385
immunosurveillance [13]. In these situations, toll-like
receptor (TLR) activation may be of benefit [14]. TLR
detect a wide spectrum of pathogen associated molecular
patterns (PAMPs), from unique bacterial products such
as lipoproteins (TLR2) and flagellin (TLR5), to nucleic
acid motifs intrinsic to bacterial or viral sources (TLR3,
7-9), and initiate expression of inflammatory mediators
that regulate inflammation. Moreover, TLR activation of
DC promotes the transport of peptide/MHC complexes to
the plasma membrane enhancing antigen presentation
[15]. This process underscores the vital role TLR play in
regulating both local and systemic inflammation, via the
activation of DC. Although TLR deal primarily with dis-
tinctly microbial antigens, their activation greatly poten-
tiates an immune response allowing TLR agonists to be
used as adjuvants. Recently, the activity of TLR agonists
in solid tumors has been elucidated. In the presence of
TAA, DC activation by the TLR9 agonist CpG oligode-
oxynucleotides (CpG ODN) initiates the capture, proc-
essing, and presentation of TAA by DC [16]. A TAA
vaccine delivered with CpG ODN as an adjuvant is
therefore more immunogenic than the vaccine alone [17].
Similar results were observed in two studies after DC
stimulation by nanoparticle encapsulated TAA with
TLR3 and TLR4 agonists [18,19]. In both cases,
nanoparticle/adjuvant delivery led to DC maturation and
migration to the lymph nodes for antigen presentation.
The potency of such vaccines, however, may be hin-
dered by immunotolerance towards TAA. Immunotoler-
ance, a state induced by regulatory (CD25+CD4+FOXP3+)
T cells (Treg), normally follows a period of infection and
is defined by the cessation of an adaptive immune re-
sponse in order to prevent chronic inflammation [20]. In
many cancers, persistent antigen presentation can lead to
tolerance, a result of specific Treg cell accumulation for
certain TAA [21]. Tumor-associated Tregs can suppresses
natural killer (NK) cell function and inhibit activity of
APC and T cells through the steric obstruction of MHC
molecules, or release of interleukin-10 (IL-10) and trans-
forming growth factor—(TGF-
 ). While immunotoler-
ance seems to be a natural tendency of the inflammatory
process, much progress has been made in artificially ar-
resting Treg cell mediated immune suppression, allowing
a persistent, unabated immune response [22]. Cyclophos-
phamide, originally an anti-tumor chemotherapeutic agent,
has been shown on several occasions to inhibit Treg cell
activity and potentiate CTL responses when given in com-
bination with a DC vaccine [23-27]. Furthermore, cyclo-
phosphamide treatment of tumor-bearing mice elevates
CD3+/CD4+, and CD3+/CD8+ T cells within tumors [28].
Thus, priming the tumor microenvironment with cyclo-
phosphamide prior to treatment with a cancer vaccine
may enhance anti-tumor immunity.
The present study examined the combined effect of
cyclophosphamide treatment and nanoparticle vaccination
in tumor-bearing mice. Prior to any immunostimulation,
BALB/c mice with 4T1 tumors were treated with con-
secutive low doses of cyclophosphamide to deplete Treg
cells [26]. Then, using nanoparticles containing a vector
encoding Mage-b as well as CpG ODN, as TAA and TLR
ligand respectively, mice were vaccinated and subse-
quently followed for tumor growth and metastasis. The
results show that this three-pronged strategy significantly
influenced tumor growth and lung metastasis of the ag-
gressive murine mammary carcinoma model 4T1. Thus,
depleting Tregs, in combination with boosting innate as
well as adaptive anti-tumor immunity using a nanoparti-
cle-based vaccine holds promise as a therapeutic vaccine
approach.
2. Material and Methods
2.1. Mice and Cell Lines
4T1 tumor cells used for this study were maintained in
complete RPMI (cRPMI) (RPMI 1640, Lonza, Walkers-
ville, MD) supplemented with 10% heat-inactivated fetal
bovine serum (Lonza), glutamine (2 mM, Lonza), penicil-
lin (100 U/mL, Lonza), streptomycin (100 ug/mL, Lonza),
nonessential amino acids (Sigma, St. Lois, MO), 2-mer-
captoethanol (5 × 10–5 M, Sigma), and sodium pyruvate
(1mM, Lonza). Balb/c mice were bred on site and were
housed in a thoren caging system (Thoren Caging Sys-
tems Inc., Hazelton, PA). Food and water were provided
ad libitum. All mice were used in accordance with an
Institutional Animal Care and Use Committee approved
protocol that followed the guide-lines for ethical conduct
in care and use of animals.
2.2. DNA Preparation
The vector encoding Mage-b for use in the vaccine was
generously provided by Dr. Claudia Gravekamp (De-
partment of Cellular and Structural Biology, University
of Texas Health Science Center). One Shot Chemically
Competent E. coli (Invitrogen, Carlsbad, CA) were
transformed with the vector. Bacteria were incubated on
ice for 30 minutes and heat shocked for 30 seconds at
42˚C. After immediate transfer to ice, 250 µl of room
temperature SOC media (Invitrogen) was added. The
reaction was shaken horizontally at 200 rpm (Innova
4300, New Brunswick Scientific, Edison NJ) and 37˚C
for 1 hour, plated on Luria-Bertani media containing am-
picillin (50 ug/ml, LB/amp) plates and incubated over-
night at 37˚C.
Due to the large amount of DNA required for produc-
tion of the vaccine (~2 mg), purification was carried out
using a QIAfilter MAXI filtration kit (QIAGEN, Valen-

Construction and Evaluation of a Combined Cyclophosphamide/Nanoparticle Anticancer Vaccine 386
cia, CA). One isolated colony from a LB/amp plate was
added to 5 ml LB/amp broth and incubated overnight at
37˚C, shaking at 150 rpm (Innova 4300). Next, 200 µl of
this starter culture was diluted in 100 ml LB/amp me-
dium and incubated at 37˚C for 16 hours. The culture
was split equally between four 50 ml tubes and centri-
fuged at 4˚C and 6370× g for 15 minutes. Cell pellets
were resuspended in 2.5 ml Buffer P1, and separate tubes
were combined. To this, 10 ml Buffer P2 was added, and
the solution was incubated at room temperature for 5
minutes. Ten ml chilled Buffer P3 was added, and after
room temperature incubation for 10 minutes, the lysate
was passed through a QIAGEN-tip 500 filter, which had
been equilibrated by gravity filtration of Buffer QBT.
The QIAGEN-tip 500 was washed twice with 30 ml
Buffer QC, the DNA was eluted with 15 ml Buffer QF
into a 50 ml tube, and precipitated by adding 10.5 ml
isopropanol. The reaction was mixed and centrifuged at
5000× g for 60 minutes. After decanting the supernatant,
the DNA pellet was washed with 5 ml 70% ethanol and
centrifuged at 5000× g for 60 minutes. The pellet was
dried in air and DNA yield was quantified by absorbance
at 260 nm ([DNA] = A260 nm × dilution × 50 ng/ul)
after resuspension in water.
2.3. Vaccine Preparation
The PLGA nanoparticle vaccine, loaded with the Mage-b
vector and CpG ODN was prepared by the double emul-
sion solvent evaporation method [29]. For this purpose
the vector encoding Mage-b DNA (~2.0 mg) was diluted
in 300 µl CpG ODN (Invivogen, San Diego, CA) solu-
tion (50 µg/ml) and 200 µl 1% (w/v) PVA in water. For
the first emulsion, 200 mg of the PLGA polymer
(Sigma-Aldrich, St. Louis, MO) was dissolved in 2 ml
dichloromethane (DCM), and 100 µl of the Mage-b/CpG
solution was added. Using a microtip probe sonicator
(Sonic Dismembrator Model 100, Fischer Scientific,
Pittsburgh, PA), the reaction was pulsed at level 2 for 20
seconds. This emulsion was immediately added to 100
ml 1% (w/v) Polyvinyl Alcohol (PVA) in water, forming
the second emulsion. The reaction was rapidly stirred
overnight at room temperature to evaporate the DCM.
The product was washed six times with 50 ml sterile dis-
tilled water and frozen overnight at –80˚C. Nanoparticles
were then lyophilized and stored until use in a desiccator
at room temperature.
2.4. Nanoparticle Analysis
A particle size distribution was obtained using a Zeta-
sizer (Zetasizer Nano, Malvern Instruments Ltd, Wor-
chestershire, UK). The procedure assesses the Brownian
motion of a sample of particles, and correlates this ran-
dom diffusion to particle radius via the Stokes-Einstein
equation. The Zetasizer detects dynamic light scattering
of a population of particles, which is a function of diffu-
sion. Nanoparticles were visualized by scanning electron
microscopy (Department of Colloids, Max Planck Insti-
tute for Colloids and Interfaces). A release assay was
performed to quantify the hydrolysis and release of DNA
from the nanoparticles with respect to time. For this pur-
pose, nanoparticles were resuspended in 5 ml sterile dis-
tilled water and incubated with shaking at 37˚C for fif-
teen days. Three samples were taken each day and ana-
lyzed for absorbance at 260 nm to quantify DNA con-
centration.
2.5. Vaccination and Tumor Growth
For each experiment 30 mice received 5 × 104 4T1 tumor
cells in 100 µl HBSS. Ten mice in each group were
maintained as a positive control. The 20 remaining indi-
viduals were injected intraperitoneally with 20 mg/kg
cyclophosphamide 4, 3, and 2 days prior to nanoparticle
vaccination. The cyclophosphamide protocol was devel-
oped by Barbon et al. [26] and shown to effectively de-
crease Treg cell activity. Ten of the cyclophospha-
mide-treated mice received 100 µl of the nanoparticle
vaccine in HBSS (3 mg/ml), injected into the left tibialis
muscle. Figure 1 summarizes the timeline for treatment
of the mice. Tumor growth rates were determined begin-
ning 7 days after nanoparticle delivery using vernier
calipers to measure tumor dimensions and calculating
tumor volume = L × W2/2.
2.6. Analysis of Metastasis
Following sacrifice, lungs were harvested from each
mouse to analyze metastases. The tissues were minced
and digested in enzyme cocktails containing 1 mg/mL
collagenase type IV (Worthintgon Biochemical Corp.,
Lakewood, NJ) and 0.1 mg/mL elastase (Worthington) at
room temperature in spinner flasks for 1 hour. The cells
were then washed and resuspended in 10 ml cRPMI.
Two dilutions per organ were made (9/10 and 1/10) and
Figure 1. Treatment regimen. For each experiment 30 mice
were given tumors on day one. Twenty of the mice received
cyclophosphamide (CY) treatment on days 4, 5, and 6. Ten
of the CY treated mice received the nanoparticle vaccine on
day 8. The experiment was completed three separate times.

Construction and Evaluation of a Combined Cyclophosphamide/Nanoparticle Anticancer Vaccine 387
microscopy (SEM) images indicated consistencies in plated on tissue culture dishes with 10 μM thioguanine
(Sigma). Samples were incubated at 37˚C and 5% CO2.
Fourteen days later the cells were fixed with methanol
(Fisher), stained with 0.03% methylene blue (Sigma),
and colonies counted.
shape of the nanoparticles (Figure 2(a)), while a size
distribution obtained using a zetasizer revealed the nano-
particles ranged in diameter from approximately 50 nm
to 900 nm, and the distribution centered at approximately
350 nm (Figure 2(b)). To assess release of the encapsu-
lated DNA the nanoparticles were resuspended in water
and samples were taken every 24 hours to assess DNA
concentration. The DNA release began within 24 hours
and plateaued after 12 days (Figure 3). These data re-
vealed the successful generation of nanoparticles the
proper size for DC uptake, and that the nanoparticles
were capable of releasing the encapsulated DNA.
2.7. Flow Cytometry
Vaccine draining (inguinal) lymph nodes and splenocytes
were harvested eight days after vaccination and prepared
for flow cytometry. Lymphocytes were removed from
the organs by pressing the organs with the flat end of a
syringe plunger. For staining, 1 ml of cells at 5 × 105
cells/ml in cRPMI was added to 15 ml tubes and 1 μg of
each antibody was added. To trace the profile of helper T
cells, cytotoxic T cells, and regulatory T cells antibodies
specific for CD3, CD4, CD8, CD25, and isotype controls
were used (BD Biosciences, San Jose, CA). After incu-
bation on ice for 30 minutes, cells were resuspended in 1
ml 3.7% formaldehyde, incubated again on ice for 10
minutes, and washed with 10 ml HBSS. Cells were re-
suspended in 1 ml HBSS and sent to Pennsylvania State
University Hershey (Hershey, PA) for analysis.
3.2. T Cell Subsets Are Not Altered by
Nanoparticle Vaccination
In an attempt to gauge whether the vaccine caused an
expansion of effector cells we assessed vaccine draining
lymph nodes and splenocytes 8 days after vaccination.
Within the lymph nodes CD3+/CD4+ (TH), CD3+/CD8+
(CTL), and CD4+/CD25+ (Treg) cells made up approxi-
mately 50%, 20%, and 3% of the cells respectively for all
experimental groups (Figure 4). Although the lack of an
increase in TH or CTL in the lymph nodes was surprising
3. Results
3.1. Nanoparticle Analysis since 8 days following vaccination there should be an-
ongoing immune response relative to control mice, it was
not surprising that the Treg cell numbers were normal
Before vaccinating mice with the nanoparticles we
wanted to determine whether the particles were the de-
since the cells were assessed 10 days following the last
sired size and whether the encapsulated DNA would be
released in an aqueous environment. Scanning electron cyclophosphamide treatment. Analysis of splenocytes
(a)
(b)
Figure 2. Nanoparticle shape and size. (a) Scanning electron micrographs of the PLGA nanoparticle vaccine. (b) Nanoparticle
size distribution as determined by Zetasizer. The distribution was centered at approximately 350nm. The experiment was run
three times, one of which yielded this representative distribution. The size (d = diameter) is measured in nanometers.

Construction and Evaluation of a Combined Cyclophosphamide/Nanoparticle Anticancer Vaccine 388
Figure 3. DNA release from the nanoparticles over time. A sample of the nanoparticle vaccine was resuspended in water and
maintained at 37˚C in a shaking incubator. The amount of DNA in solution plateaued after 12 days, indicating complete
nanoparticle hydrolysis. The plot contains the average +/– standard deviation of the DNA concentration of the three separate
samples taken each day.
also revealed no significant differences between treat-
ment groups. Although there were fewer TH, CTL and
Treg cells in the spleens from cyclophosphamide treated
and nanoparticle vaccinated mice the differences were
not significant (Figure 4). It is possible that the small
decrease in these populations could be attributed to cells
exiting the spleen and localizing to the tumor site. Re-
gardless, collectively these data reveal that 8 days fol-
lowing vaccination with the nanoparticles there was no
obvious expansion of effector T cells in the spleens or
vaccine draining lymph nodes.
3.3. Tumor Growth and Metastasis Are
Decreased by Nanoparticle Vaccination
To evaluate whether the nanoparticle vaccine influenced
tumor progression we monitored tumor growth over time
as well as lung metastasis. All tumors, regardless of
treatment, followed a relatively exponential growth rate
(Figure 5). In the early stages, before the 7th day
post-vaccination, tumors in control mice grew faster than
tumors in mice treated only with cyclophosphamide and
tumors in nanoparticle vaccinated mice. After this point,
however, tumors in the cyclophosphamide only treated
group began to grow at a rate similar to the controls,
whereas mice that received the nanoparticle vaccine con-
tinued to exhibit a slower growth rate. Although mice
treated with cyclophosphamide alone and mice that re-
ceived the nanoparticle vaccine both exhibited signifi-
cantly slower tumor growth rates than control mice, the
tumors were much smaller in the mice that received the
nanoparticle vaccine. Following 24 days of analysis the
tumors in control mice averaged 2753 +/– 386 mm3,
whereas tumors in the cyclophosphamide only treated
mice averaged 2213 +/– 216 mm3, and tumors in the
mice that received cyclophosphamide and the nanoparti-
cle vaccine averaged 1107 +/– 161 mm3 (Figure 5).
These data suggest that cyclophosphamide alone has a
benefit in leading to a reduction in tumor growth rate,
although this reduction was not as significant as when the
mice also received the nanoparticle vaccine.
We chose the 4T1 murine mammary carcinoma model
for this study because of its aggressive nature and ability
to spontaneously metastasize. Since metastasis is often a
major contributor to death of patients with cancer we
wanted to know whether the nanoparticle vaccine also
had any effect on lung metastasis. For this reason, the
metastatic ability of primary 4T1 tumors from the dif-
ferent treatment groups was assessed by quantifying
colonies that grew from the resected and digested lungs.
Lung tissue from control mice exhibited the greatest
number of metastatic colonies (2306), while cyclophos-
phamide only treated mice exhibited significantly fewer
colonies (1777), and lungs from mice that received the
nanoparticle vaccine exhibited the fewest metastatic
colonies (1528) (Figure 6). These data suggest that
cyclophosphamide alone has a benefit in reducing me-
tastasis, although this benefit was not as significant as
when the mice also received the nanoparticle vaccine.
Thus, mice that received cyclophosphamide and the
nanoparticle vaccine exhibited the greatest reduction in
tumor growth rate and lung metastasis.
4. Discussion
The size distribution of the nanoparticles, which centered
around 350 nm, and spherical shape assortment, indicated
by zetasizer analysis and SEM imaging respectively,
revealed a nanoparticle vaccine that should be phagocyto-
sed by APC; an advantage of the nanoparticle approach.

Construction and Evaluation of a Combined Cyclophosphamide/Nanoparticle Anticancer Vaccine 389
(a)
(b)
Figure 4. Analysis of lymphocytes from lymph nodes and spleens. The number of helper T cells (Th), cytotoxic T cells (CTL),
and regulatory T cells (Treg) in inguinal lymph nodes (a) and spleens (b) were assessed 8 days after vaccination by flow cy-
tometry. The data represent the average +/– standard deviation of all three experiments.
Figure 5. Tumor growth rates. Growth rates of 4T1 tumors from untreated mice (control), mice treated with cyclophos-
phamide alone (CY), and mice treated with CY and the nanoparticle vaccine (CY + nanoparticles) are shown. Tumors were
measured daily using vernier calipers. The data represent the average +/– standard deviation of all three experiments. Where
indicated (*) p < 0.001 using Student’s t-Test relative to the control.

Construction and Evaluation of a Combined Cyclophosphamide/Nanoparticle Anticancer Vaccine 390
Figure 6. Number of metastatic colonies recovered from the lungs of tumor-bearing mice. Lungs were harvested 24 days after
vaccination, processed, and incubated for 14 days before assessing colony outgrowth. The data represent the number of me-
tastatic colonies from each individual mouse (x), and the average (□) of all mice from two separate experiments. Where indi-
cated (*) p < 0.001 using Student’s t-Test relative to the control.
For nanoparticles with cytotoxic drugs internalized, this
would also limit systemic cytotoxicity. The nanoparticle
itself is biodegradable and nontoxic, deteriorating rapidly
in solution to an assortment of water-soluble substances,
such as lactic and glycolic acids, which are simply ex-
creted [30]. Analysis of release from the nanoparticles
indicated a rapid hydrolysis of the particles with DNA
release between 4 and 10 days, and complete discharge
of all encapsulated DNA after the 12th day. The steady
release of antigen over a period of a few days is espe-
cially important in eliciting a potent immune response, as
it helps maintain antigen concentration.
In this study, at early stages of tumor growth (1 to 5
days post-vaccination), tumors of the two groups treated
with cyclophosphamide had similar growth rates. Ini-
tially, cyclophosphamide action would have decreased
Treg cells, leading to an increased capacity for an im-
mune response. However, by day 8, when lymph nodes
and spleens were harvested, the effects of the cyclo-
phosphamide treatment seem to have passed as suggested
by a similar number of Treg cells in the different ex-
perimental groups. These data are in agreement with
Barbon et al. [26], who determined that the impact of
cyclophosphamide lasts between 6 to 8 days. Around this
same time growth rates of tumors in mice treated only
with cyclophosphamide began to more closely parallel
growth rates in control mice. This may have been caused
by tumor-induced immunosuppression as a result of an
up-regulation of Treg cells, which inhibits a tumor-specific
T cell response [31].
In theory, there are two mechanisms by which the
nanoparticles deliver antigen: through extracellular
nanoparticle hydrolysis and antigen release, leading to
free antigen uptake by APCs, or through endocytosis of
nanoparticles and intracellular antigen release. Although
we have not explored which mechanism predominates in
this study, elements of both may occur in vivo. Nonethe-
less, intracellular antigen release is likely an efficient
method, and steps could be taken in future studies to
emphasize this route. For instance, recent studies have
indicated that covalent modifications to the surface of
nanoparticles are possible, and indeed allow greater
specificity for accumulation and endocytosis [12,19].
Thus, modifying the surface of the nanoparticles with
antibodies or TLR ligands may increase effectiveness of
the vaccine.
However, when the nanoparticle vaccine was used in
conjunction with cyclophosphamide treatment tumor
growth rates were significantly lower throughout the ex-
periment. This may be attributed to a primed immune
environment formed through initial cyclophosphamide
depletion of Treg cells and followed by an efficient T
cell response initiated by uptake of the nanoparticle vac-
cine containing DNA encoding the Mage-b TAA to
stimulate an immune response, and CpG ODN to stimu-
late innate immunity. In theory, this anti-tumor immune

Construction and Evaluation of a Combined Cyclophosphamide/Nanoparticle Anticancer Vaccine391
response could have been sustained longer, but in the
absence of booster vaccinations, this response was even-
tually restricted. In many instances, vaccine boosters
have been used to maintain the anti-tumor immune re-
sponse [10,18,19]. Although beyond the scope of the
current study, it would be interesting to determine
whether additional cyclophosphamide treatments and
nanoparticle vaccinations could further impact the tumor
growth rate. It would also be worthwhile to explore the
impact of the nanoparticle vaccine without cyclophos-
phamide treatment in order to determine the extent to
which Treg depletion was important for efficacy of the
vaccine. Another interesting area to investigate would be
to look at prophylactic vaccination with the vaccine
rather than therapeutic vaccination as studied here. With
many tumor models prophylactic vaccination often re-
sults in a more significant impact on tumor progression
than therapeutic vaccination.
Interestingly, we did not find evidence of T cell ex-
pansion following vaccination, and we did not verify
whether an antigen-specific immune response was elicited
by the vaccine. Subsequent studies are undoubtedly nec-
essary and warranted to delineate how the vaccine works.
For this purpose we are interested in looking at further
subsets of T cells (Th1, Th2, memory cells, antigen spe-
cific tetramer positive cells), as well as additional lym-
phocyte populations such as B cells, NK cells and
 TCR+ T cells. Initial studies in nude or SCID mice
would help delineate whether an antigen specific immune
response is important for vaccine efficacy and subsequent
studies such as ELISPOT, cytokine release, cytotoxicity,
and proliferation assays would be extremely useful in
determining how the vaccine works to decrease tumor
growth and metastasis.
In this study, the extent of lung metastasis also corre-
lated with tumor growth inhibition. The lung metastasis
in mice treated with cyclophosphamide alone and mice
treated with the nanoparticle vaccine were significantly
less than the lung metastasis in control mice. However,
although there were fewer lung metastasis in nanoparticle
vaccinated mice, lung metastasis in mice treated only
with cyclophosphamide, and mice treated with cyclo-
phosphamide in combination with the nanoparticle vac-
cine were not significantly different. These data under-
score the importance of Treg cell depletion in limiting
metastatic growth, and that coupled with a nanoparticle
vaccine the effect is even greater. As with tumor growth,
it would be interesting to look at the effect of additional
cyclophosphamide treatments and booster vaccinations
on lung metastasis in future studies.
Collectively, the data presented here indicate a com-
bined cyclophosphamide/nanoparticle vaccine was suc-
cessful in both significantly slowing primary tumor
growth rates and lung metastasis in mice with 4T1 tu-
mors. The data suggest that through a multi-stage
mechanism of action, the immune response is augmented
upon depletion of Treg cells with cyclophosphamide, and
is triggered to respond to the tumor cells with the
nanoparticle vaccine containing DNA encoding a TAA
and CpG ODN to stimulate innate immunity. However,
inhibition of tumor growth was only temporary; without
additional vaccine boosters, the tumors began to grow at
an unrestricted rate. The effectiveness of booster vacci-
nations and additional cyclophosphamide treatments
should be evaluated in future studies. In theory, contin-
ued treatments could prolong a more extensive anti-tu-
mor immune response. Nonetheless, the results obtained
using a therapeutic vaccine approach for this highly ag-
gressive murine mammary carcinoma model highlights
the potential for nanoparticle vaccines in eliciting anti-
tumor immunity.
5. Acknowledgements
We would like to thank the Department of Biology at
Lafayette College for support of this work. We would
also like to thank Drs. James Ferri (Department of
Chemical and Biomolecular Engineering), David Husic
(Department of Chemistry), and Charles Nutaitis (De-
partment of Chemistry) for all of their advice and assis-
tance with the generation of the nanoparticle vaccine.
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