Group Title: Journal of Translational Medicine 2008, 6:37
Title: Exosomes as a tumor immune escape mechanism: possible therapeutic implications
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Title: Exosomes as a tumor immune escape mechanism: possible therapeutic implications
Series Title: Journal of Translational Medicine 2008, 6:37
Physical Description: Archival
Creator: Ichim TE
Zhong Z
Kaushal S
Zheng X
Ren X
Hao X
Joyce JA
Hanley HH
Riordan NH
Koropatnick J
Bogin V
Minev BR
Min WP
Tullis RH
Publication Date: 39651
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: Open Access: http://www.biomedcentral.com/info/about/openaccess/

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Journal o Translational Medicine
Journal of Translational Medicine g.o.ed Central


Research

Exosomes as a tumor immune escape mechanism: possible


Open Access


therapeutic implications
Thomas E Ichim*1,2,9, Zhaohui Zhongt3, Shalesh Kaushalt4, Xiufen Zheng2,
Xiubao Ren5, Xishan Hao5, James A Joyce6, Harold H Hanley6,
Neil H Riordan', James Koropatnick2, Vladimir Bogin', Boris R Minev7'8,
Wei-Ping Min2 and Richard H Tullis6


Address: 'Medistem Laboratories Inc, San Diego, USA, 2Department of Surgery, Pathology, Oncology, Microbiology and Immunology, University
of Western Ontario, London, Canada, 3Department of Surgery, the Second Xiangya Hospital of Central South University, Changsha, PR China,
4Department of Ophthalmology, University of Florida, Gainesville, USA, 5Department of Surgery, Tianjin Medical University, Tianjin, PR China,
6Aethlon Medical, San Diego, California, USA, 7Moores UCSD Cancer Centre, San Diego, USA, 8Division of Neurosurgery, University of California
San Diego, San Diego, USA and 9Medistem Laboratories, 9255 Towne Centre Drive, Suite 450, San Diego, California, 92122, USA
Email: Thomas E Ichim* thomas.ichim@gmail.com; Zhaohui Zhong jzhonguro@gmail.com; Shalesh Kaushal skaushal@eye.ufl.edu;
Xiufen Zheng xzheng26@uwo.ca; Xiubao Ren weiping.min@uwo.ca; Xishan Hao weiping.min@uwo.ca;
James A Joyce jj@aethlonmedical.com; Harold H Hanley h3@aethlonmedical.com; Neil H Riordan nhriordan@gmail.com;
James Koropatnick jkoropat@uwo.ca; Vladimir Bogin vbogin@cromospharma.com; Boris R Minev bminev@ucsd.edu; Wei-
Ping Min weiping.min@uwo.ca; Richard H Tullis rhtullis@aethlonmedical.com
* Corresponding author tEqual contributors



Published: 22 July 2008 Received: 17 March 2008
journal of Translational Medicine 2008, 6:37 doi: 10. 186/1479-5876-6-37 Accepted: 22 July 2008
This article is available from: http://www.translational-medicine.com/content/6/1/37
2008 Ichim et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Advances in cancer therapy have been substantial in terms of molecular understanding of disease
mechanisms, however these advances have not translated into increased survival in the majority of
cancer types. One unsolved problem in current cancer therapeutics is the substantial immune
suppression seen in patients. Conventionally, investigations in this area have focused on antigen-
nonspecific immune suppressive molecules such as cytokines and T cell apoptosis inducing
molecules such as Fas ligand. More recently, studies have demonstrated nanovesicle particles
termed exosomes are involved not only in stimulation but also inhibition of immunity in
physiological conditions. Interestingly, exosomes secreted by cancer cells have been demonstrated
to express tumor antigens, as well as immune suppressive molecules such as PD-IL and FasL.
Concentrations of exosomes from plasma of cancer patients have been associated with
spontaneous T cell apoptosis, which is associated in some situations with shortened survival. In this
paper we place the "exosome-immune suppression" concept in perspective of other tumor
immune evasion mechanisms. We conclude by discussing a novel therapeutic approach to cancer
immune suppression by extracorporeal removal of exosomes using hollow fiber filtration
technology








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Journal of Translational Medicine 2008, 6:37


Cancer is Recognized by the Immune System
The concept of whether cancer is recognized by the
immune system has been a topic of intense discussions
and experimentation for more than a century. Philosoph-
ically speaking, the argument revolves around one central
aspect: cancer originates from "self" tissue, therefore why
should the immune system attack it? The traditional con-
cept of immunology which teaches that the main purpose
of the immune system is to distinguish between "self" and
"non-self" suggests that since cancer is "self" there should
be no immune response against it. Current-day immuno-
logical advances, however, have struck down this notion.
However, before going into these advances in detail, we
will first overview the history of cancer immunotherapy in
order to provide a background for our discussion.

In the late 1800s a physician at Sloan Kettering Cancer
Center, William Coley, made the empirical observation
that certain types of tumors would go into remission sub-
sequent to bacterial infections. One of the first patients he
saw in his career was a young girl who died of a rapidly
progressing sarcoma originating in her arm. A different
patient with a similar type of sarcoma in the neck had
lived for seven years after diagnosis with no detectable
signs of cancer. The only noteworthy difference between
the two patients, in his mind was that the latter patient
had repeated encounters with bacterial infections. This
prompted Coley to search the medical literature, where he
found that sarcoma remission had previously been docu-
mented to be associated with erysipelas, a streptococcal
infection of the skin. This prompted Coley to begin pur-
posely inoculating patients with various bacterial extracts
with the aim of stimulating an immune response that
would somehow "cross-over" and lead to cancer regres-
sion. He reported that the first patient purposely inocu-
lated suffered from an inoperable late stage neck sarcoma.
This patient was administered extracts of a streptococcal
broth that was generated from another patient. According
to the published description, this treatment led to a signif-
icant remission of a "hen egg"-sized tumour within ten
days, and resulted in patient survival for eight years, after
which he died of tumour relapse [1,2]. Eventually, due to
the uncontrollable effects of unstandardized bacterial
mixtures, Coley generated a combination of heat-killed
serratia marcescens and heat-killed streptococci which
were eventually named "Coley's Toxins" and sold in the
United States by Parke-Davis from 1923 to 1963 [3]. The
advent of chemotherapy, as well as unpredictable reac-
tions that patients would have to Coley's Toxin contrib-
uted, at least in part, to the discontinuation of this
therapy. Nevertheless, William Coley is considered by
many the father of modern day cancer immunotherapy
[4]. Interestingly, a standardized version of Coley Vaccine
is currently being developed by the Canadian biotechnol-
ogy company MBVax.


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Despite the suggestion that immune response to bacterial
infections may somehow "re-awaken" the immune sys-
tem to kill cancer, scientifically, there could have been
other explanations for the effect of Coley's Toxins. For
example, it may be possible that compounds inside the
bacterial extracts had ability to directly kill cancer cells [5],
or to preferentially inhibit tumor angiogenesis [6].
Accordingly, in our discussion of whether the immune
response actually inhibits cancer or not, we will turn to
animal models.

The era of molecular biology has allowed for gene-specific
deletion in animals. This means that genes associated
with immune responses can be "knocked-out" of animals
so as to study the importance of the specific gene in an in
vivo setting. Speaking in very general terms, there are two
pathways that the immune system can take when it is acti-
vated. The first type is called "Thl", which is involved in
destroying cells of the body that are infected from the
inside, such as virally infected cells. The second type of
immune response is called "Th2", which is responsible for
killing targets that reside outside of the cells of the body,
such as parasites and certain bacteria [7,8]. Since cancer
consists of cells of the body that have distinguishing prop-
erties from the other cells (ie high proliferation, ability to
metastasize, etc), it may be possible to rationalize the Thl
path of the immune response would be the one responsi-
ble for control of cancer, if the immune response is
involved at all. Indeed, Coley's toxin (and its constitu-
ents) were identified decades later to be potent inducers of
the Thl cytokine TNF-alpha, as well as activators of this
general immune response pathway [9,10]. The discovery
of transcription factors that induce Thl or Th2 immunity
has allowed experimental assessment of the roles of these
types of immune responses in control of cancer. Tran-
scription factors controlling Thl immunity include T-bet
[11], and STAT4 [12], and those controlling Th2 include
GATA-3 [13 and STAT6 [14,15 ]. When various tumors are
administered to STAT6 knockout animals (therefore hav-
ing a Thl predisposition since the Th2 pathway is
ablated), these tumors are either spontaneously rejected
[16], or immunity to them is achieved with much higher
potency compared to wild-type animals [17]. Further-
more, in STAT6 knockout animals, immunologic resist-
ance to metastasis formation is observed [18]. On the
other hand, STAT4 knockout mice lack Thl capability and
therefore have only upregulated Th2 immunity. Such ani-
mals allow accelerated cancer formation after treatment
with chemical carcinogens [19].

While the above suggest the importance of the Thl
immune response in controlling tumors, in many cases
animal data does not translate efficiently to human dis-
ease. Accordingly, we turn our attention to situations
where immune suppression is induced either by genetic


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Journal of Translational Medicine 2008, 6:37


abnormality or in response to a medical condition. In
general, natural killer (NK) cell activity is associated with
Thi immune responses and tumor immunity [20].
Patients with the congenital abnormality Chediak-
Higashi Syndrome, are characterized by absent or severely
diminished NK function. In this population, the overall
incidence of malignant tumors is 200-300 times greater
than that in the general population [21]. Another example
of an inborn trait associated with immune deviation is
patients born with a specific polymorphism of the IL-4
receptor gene that is known to be associated with aug-
mented Th2 responses. Multivariate regression analysis
showed that this polymorphism was an independent
prognostic factor for shorter cancer survival and more
advanced histopathological grade [22]. In addition to
inborn genetic abnormalities, the immune suppressive
regimens used to prevent transplant rejection are associ-
ated with a selective inhibition of Thl responses [23-25].
In support of the concept that suppression of Thl immu-
nity is associated with cancer onset, the incidence of can-
cer in the post-transplant population is markedly
increased in comparison to controls living under similar
environmental conditions [26-31]. In terms of disease
associated immune suppression, HIV infected patients
also have a marked predisposition to a variety of tumors,
especially, but not limited to lymphomas, as a result of
immunodeficiency [32].

The above examples support a relation between immune
suppression (or Th2 deviation) and cancer proliferation,
the opposite circumstance, of immune stimulation result-
ing in anticancer response is also documented. Numerous
clinical trials using antigen specific approaches such as
vaccination with either tumor antigens alone [33,34],
tumor antigens bound to immunogens [35,36], tumor
antigens delivered alone [37] or in combination with cos-
timulatory molecules by viral methods [38], tumor anti-
gens loaded on dendritic cells ex vivo [39-41], or
administration of in vitro generated tumor-reactive T cells
[42], have all demonstrated some, albeit modest clinical
effects. It is documented that inappropriate immune
responses (broadly speaking Th2 responses) can actually
stimulate tumor growth [43,44]. Accordingly, these data
all support the presumed recognition of cancer by the
immune system and the notion that the immune system,
if stimulated properly, may induce cancer regression.

The philosophical question posed at the beginning of this
discussion; how can the immune system recognize cancer
when cancer is part of self, is answered in the following
manner. The immune system is not responsible for seeing
only "self" versus "non-self" but actually seeing and
responding to different variations of "self". The tumor, in
its quest for proliferative advantage, ability to metastasize,
and need for formation of new blood supply, actually


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expresses new molecules at levels that are recognized by
the immune system. Immunological recognition of mole-
cules needed for the tumor to have the "cancer pheno-
type" has been well-documented. We will not provide an
overview of these data here but will provide some exam-
ples. Specifically, the proliferative advantage of tumors is
associated with growth factor receptor upregulation,
accordingly immune responses to various such receptors
are known to exist naturally or to be inducible [45,46].
The same is true for matrix metalloproteases involved in
tumor extravasation and metastasis [47,48], as well as for
angiogenic factors involved in formation of new blood
vessels [49,50]. The question, of course remains, if the
immune system can see cancer, why does it not eradicate
it, and why has the clinical implementation of cancer
immunotherapy yielded such poor results at the bedside?

Cancer Suppresses the Immune System
The development of successful immune responses to can-
cer is hindered by numerous factors, including primarily,
that ability of the tumor to cause suppression of a produc-
tive host immune response to cancer. The interaction
between the tumor and the immune system has been lik-
ened to pregnancy, in which an allogeneic graft (the fetus)
rapidly develops without rejection by an immunogically
competent host. The ability of the fetus to evade the
maternal immune response of the mother is not due to
anatomical barriers, since maternal immune cells have
been demonstrated to cross the placenta and actually
enter the fetus [51]. What seems to occur in pregnancy is
similar to the cancer patient in that there occurs a selective
depletion of immune components, while other immuno-
logical parameters are left intact. Both in pregnancy and
cancer a specific depletion of certain T cells occurs via
numerous common mechanisms such as FasL [52-54].
Before elaborating on specific mechanisms by which FasL
kills immune system cells, we will first discuss some of the
historical work that led us to the notion that cancer sup-
presses the immune system.

Experiments in the 1970s demonstrated the existence of
immunological "blocking factors", then-unidentified
components of plasma found in cancer patients and preg-
nant women that inhibited antigen-specific lymphocyte
responses. Some of this early work involved culturing
autologous lymphocytes with autologous tumor cells in
the presence of third party healthy serum. This culture
resulted in an inhibition of growth of the autologous
tumor as a result of the lymphocytes. Third party lym-
phocytes did not inhibit the growth of the tumor. Interest-
ingly when autologous serum (i.e. from the cancer
patient) was added to the cultures, the lymphocyte-medi-
ated inhibition of tumor growth was not observed. These
experiments gave rise to the concept of antigen-specific
"blocking factors" found in the body of cancer patients


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that incapacitate successful tumor immunity [55-57]. This
work stimulated the more recent demonstration of tumor-
suppression of immune function in experiments showing
that T cell function is suppressed in terms of inability to
secrete interferon gamma due to a cleavage of the critical
T cell receptor transduction component, the TCR-zeta
chain [58]. Originally, zeta chain cleavage was identified
in T cells prone to undergo apoptosis [59]. Although a
wide variety of explanations have been put forth for the
cleavage of the zeta chain, one particular cause was postu-
lated to be tumor-secreted microvesicles [60]. Since the
immune suppressive effects of cancer are systemic, the
ability of microvesicles secreted by tumor cells to specifi-
cally induce T cell modulation through circulating
through-out the body has attracted considerable atten-
tion. While there are several known mechanisms of cancer
to suppress the immune system that do not use microves-
icles, their sheer number in the cancer patient, their ability
to systemically influence numerous immune parameters,
as well as the fact that administration of cancer microves-
icles stimulates cancer progression, all point to their
important role in cancer evasions of the immune
response.

Cancer Secreted Microvesicles: A Mechanism of
Escaping from the Immune System
In the 1980's Dr. Douglas Taylor described microvesicles
secreted by tumor cells [61]. They were estimated to be
between 50-200 nanometers in diameter and associated
with a variety of immune inhibitory effects. Specifically, it
was demonstrated that such microvesicles could not only
induce T cell apoptosis, but also block various aspects of
T cell signaling, proliferation, cytokine production, and
cytotoxicity [62-64]. Although much interest arose in the
biology of microvesicles, few therapeutic applications
developed since microvesicles were uncharacterized at a
molecular level.

Other research identified another type of microvesicular-
like structures, which were termed "exosomes". Originally
defined as small 80-200 nanometers in diameter, exo-
somes were observed initially in maturing reticulocytes
[65,66]. Subsequently it was discovered that exosomes are
a potent method of dendritic cell communication with
other antigen presenting cells. Exosomes secreted by den-
dritic cells were observed to contain extremely high levels
of MHC I, MHC II, costimulatory molecules, and various
adhesion molecules [67]. In addition, dendritic cell exo-
somes contain antigens that said dendritic cell had previ-
ously engulfed [68]. The ability of exosomes to act as
"mini-antigen presenting cells" has stimulated cancer
researchers to pulse dendritic cells with tumor antigens,
collect exosomes secreted by the tumor antigen-pulsed
dendritic cell, and use these exosomes for immuno-
therapy. Such exosomes were seen to be capable of eradi-


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rating established tumors when administered in various
murine models [69,70]. The ability of dendritic exosomes
to potently prime the immune system brought about the
question if exosomes may also possess a tolerance induc-
ing or immune suppressive role. Since it is established that
the exosome has a high concentration of tumor antigens,
the question arose whether exosomes may induce an
abortive T cell activation process leading to energy [71].
Specifically, it is known that numerous tumor cells, and
exosomes derived therefrom, express the T cell apoptosis-
inducing molecule FasL [71-73].

FasL is an integral type II membrane protein belonging to
the TNF family whose expression is observed in a variety
of tissues and cells such as activated lymphocytes and the
anterior chamber in the eye. FasL induces apoptotic cell
death in various types of cells target cells via its corre-
sponding receptor, CD95/APO1. FasL not only plays
important roles in the homeostasis of activated lym-
phocytes, but it has also been implicated in establishing
immune-privileged status in the testis and eye, as well as a
mechanisms by which tumors escape immune mediated
killing. Accordingly, given the expression of Fas ligand on
a variety of tumors, we and others have sought, and suc-
cessfully demonstrated that FasL is expressed on exosomes
secreted by tumor cells [71]. Due to the ability of exo-
somes to mediate a variety of immunological signals, it
was proposed that at the beginning of the neoplastic proc-
ess, tumor secreted exosomes selectively induce antigen-
specific T cell apoptosis, through activating the T cell
receptor, which in turn upregulates expression of Fas on
the T cell, subsequently, the FasL molecule on the exo-
some induces apoptosis. This process may be occurring by
a direct interaction between the tumor exosome and the T
cell, or it may be occurring indirectly by tumor exosomes
binding dendritic cells, then subsequently when T cells
bind dendritic cells in lymphatic areas, the exosome actu-
ally is bound by the dendritic cell and uses dendritic cell
adhesion/costimulatory molecules to form a stable inter-
action with the T cell and induce apoptosis. In the context
of more advanced cancer patients, where exosomes reach
higher concentrations systemically, the induction ofT cell
apoptosis occurs in an antigen-nonspecific, but FasL,
MHC I-dependent manner.

The recent recognition that tumor secreted exosomes are
identical to the tumor secreted microvesicles described in
the 1980s [74], has stimulated a wide variety of research
into the immune suppressive ability of said microvesicles.
Specifically, immunesuppressive microvesicles were iden-
tified not only in cancer patients [60,63], but also in preg-
nancy [75-77], transplant tolerance [78,79], and oral
tolerance induction [80,81]. Accordingly, one ideal
method of stimulating the immune response of a cancer
patient would be the removal of these immunosuppres-


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sive microvesicles from circulation through the use of an
extracorporeal filter.

Microvesicle Removal by Extracorporeal
Filtration: The HemopurifierTM
Previous efforts have been made to remove cancer associ-
ated immune suppressive factors through extracorporeal
means. One most prominent example of this is a report by
Lentz's group in which 16 patients with metastatic cancers
were treated with ultrapheresis, a procedure that removes
certain fractions of blood associated with immune sup-
pression. Treatment was associated with a marked
increase in lymphocytic infiltration of tumor and tumor
necrosis as seen by repeat biopsy. In some patients, immu-
nological energy was reversed and Karnofsky status
improved. Six of the 16 patients had reduction of the sum
of mean cross-sectional diameters of measureable lesions
by 50% or more [82]. Although this study demonstrated
the proof-of-principle that stimulation of cancer immune
responses can be clinically performed by extracorporeal
removal of immune suppressants, this approach has sev-
eral limitations: a) not-selective for specific inhibitors; b)
theoretically would result in loss of immune stimulatory
cytokines; and c) is not applicable on a wide scale.

The San Diego biotechnology company, Aethlon Medical,
has developed a novel hollow-fiber cartridge (Hemopuri-
fier'T) that is compatible with standard dialysis machines.
Recently completed clinical trials have demonstrated its
safety in hepatitis patients. Effective removal of HIV parti-
cles, which have a similar size to exosomes, was demon-
strated using a variety of settings [83-85].

The Hemopurifierf utilizes an affinity substrate a propri-
etary lectin-based resin, which possesses high affinity for
heavily glycosylated viral surface proteins, which can
selectively deplete circulating virus with resultant accumu-
lation of virus in the cartridge. Tumor cell membranes and
shed immunosuppressive microvesicles are also highly
glycosylated and thus possess a preferential affinity to
lectins in comparison to non-malignant cells [86-88].
Cancer patients have a much higher level of circulating
microvesicles. The level of microvesicles in cancer patients
is 2,000-5,000 ug/ml protein/ml of blood versus 0-0.5
ug in healthy volunteers [62,76]. Given the high concen-
tration of circulating microvesicles, as well as the potential
for selected depletion due to glycosylation differences, the
Hemopurifier" in its current state is an attractive candi-
date for removal of cancer-secreted immunosuppressive
microvesicles.

The ability to attach different molecules and antibodies to
the resin of the Hemopurifier" cartridge, increases the
potential selective exosome removal targets. For example,
clinically used antibodies such as Herceptin, could be


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attached to the Hemopurifier" resin in order to deplete
microvesicles expressing HER2 protein. Additionally,
numerous proteins exist that are found on cancer-secreted
microvesicles such as FasL, MHC I, MHC II, CD44, placen-
tal alkaline phosphatase, TSG-101, MHC I-peptide com-
plexes, MHC II-peptide complexes. Antibodies to these
proteins are commonly available and can be incorporated
into the Hemopurifier' cartridge with little effort.

Conclusion
The Hemopurifier" cartridge can enter the cancer thera-
peutics arena with relative ease due to several factors.
Firstly, the cartridge is already produced under Good
Manufacturing Practices with clinical safety data available,
albeit for different indications. Secondly, the cartridge is
compatible with standard dialysis systems which are
present at every major medical institution. Thirdly, the
patient population whose treatment is proposed with this
novel approach has few or no treatment alternatives.
Fourthly, the Hemopurifier" cartridge can be used not
only as a monotherapy, but also as an adjuvant to cur-
rently used immunotherapeutic approaches. The com-
mercialization of therapies as adjuvants to existing
therapies is well-accepted in the industry, as seen in the
approval of Herceptin and Avastin for use with specific
chemotherapeutic agents.

In conclusion, the use of the Hemopurifier" as a means of
de-repressing immune functions in cancer patients is a
novel, easily implemented approach to cancer therapeu-
tics that will not only provide an alternative to the cur-
rently ineffective approaches, but also provide a
framework for development of strategies that may benefit
cancer patients in a multidimensional manner.

Authors' contributions
TEI, ZZ, SK, XZ, XR, XH, JAJ, HHH, NHR, JK, VB, MRM,
WP, and RHT were involved in conceiving the paper and
reducing it to practice.


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