**9. Membrane vesicles as therapeutic agents**

Secreted microvesicles, known as exosomes, provide a form of cell to cell signaling in which the recipient cell can be modified by the contents of the delivered exosomes. The contents (cytosolic proteins, lipids, siRNA, miRNA, DNA, etc) (Tan *et al.*, 2010; Khan *et al.*, 2011) are protected and stably delivered unlike those secreted into the extracellular matrix (Pap *et al.*, 2010); thus securing the bioactivity of the delivered contents. Exosomes could be an attractive tool as an immunotherapeutic as they maintain much of the anti-tumor activity of a dendritic cell with the advantage of a cell free vehicle. Understanding that exosomes are involved in many levels of tumorigenesis, and potential strategies against this form of cellular communication has led to the creation of potential therapeutics against exosomes.

TEXs are a protective measure for tumor cells that aids in survival, growth, invasion, metastasis, and evasion of the immune system (Clayton *et al.*, 2007; Valenti *et al.*, 2007; Anderson *et al.*, 2010; Khan *et al.*, 2011). Taxol and vinca alkaloids are well known chemotherapeutic agents, but they also limit exosome release via the microtubule formation. Exosome formation has also been shown to be blocked by decreasing acidity of the microcellular environment (Iero *et al.*, 2008; Pap *et al.*, 2010). Prevention of exosome formation involves a broad variety of proteins and is essential for life in all cells. Therefore any inhibition of exosomes within the body could lead to unwarranted side effects (Pap *et al.*, 2010). It was thought that inhibition or treatment focused against TEXs and its ability to aid tumor cells could bear significant benefit to patients, but that was overshadowed by the realization that exosomes could potentially be employed as biomarkers and diagnostic tools of malignancy in blood (Anderson *et al.*, 2010) and urine (Nilsson *et al.*, 2009). One such study analyzed the correlation between serum PSA and urinary-exosomes, but none was observed. However, the data suggests that healthy donor urinary-exosomes were negative for PSA, PSMA, and 5T4 (Mitchell *et al.*, 2009).

Realizing that exosomes are secreted/released by a wide variety of cell types (Skokos *et al.*, 2001; Wolfers *et al.*, 2001; Chaput *et al.*, 2004b; Tan *et al.*, 2010; Khan *et al.*, 2011) including

The Application of Membrane Vesicles for Cancer Therapy 43

attachment and fusion, or through transcytosis. Physiologically, these extracellular vesicles have been shown to play a role in inter-cellular communication, disposal of defective or weakened proteins, formation of morphogen gradients for tissue patterning during development and for antigen presentation to T cells. Pathologically, these vesicles have been shown to aid in the transmission of viruses, prions, -amyloid in Alzheimer's disease, and specifically to this chapter, tumor pathogenesis. Although additional investigation is still needed to fully exploit these extracellular vesicles for therapy, there is increasing evidence that in better understanding them and the molecules that they transport, determinations of diagnosis and prognosis and the prediction of response to treatment may be possible. However, it is also the hope that these vesicles may also act at therapeutic targets and

Conventional treatments for cancer include the use of chemotherapeutic drugs, radiation therapy and interventional surgery if the tumors are operable. Recent data suggests that the application of dendritic cell-derived, tumor cell-derived and even ascitic cell-derived exosomes could be developed as novel treatments for cancer. In fact, is has already been established that exosomes can be safely administered to patients, though the number of patients in these trials have been small and thus argue for larger studies. Furthermore, the goal of exosome therapy is to increase the biological magnitude of the immune response and as such exosomal immunotherapy is heavily reliant on the immune system. It will therefore be important to address issues such as patient immunosuppression due to therapeutic treatment, as the efficacy of the immunotherapy will be reliant upon the capability of the immune system. In order to increase the biological magnitude of the immune response researchers are artificially coating and engineering exosomes with tumor antigens to make them more recognizable to the immune system. The affects of heat shocking exosomes is also being investigated as heat shocked tumor exosomes are more effective than those not receiving heat treatment. Heating of the exosome confers a greater immunogenicity and thus elicits a greater immune response. Another possible application for exosomes is in vaccine delivery as exosome-producing cells could be engineered to produce a miRNA or specific genetic component or toxin that could be loaded into a exosome with the proper surface molecules for a cell specific uptake and response. Finally, the application of exosomes as immunotherapeutics represents a new chapter in cancer treatment development. However, much more research must go into elucidation of mechanism and

The authors would like to thank the sponsors of their work on exosomes over the past few years: NCMHD Project EXPORT Program 5P20MD001632/Project 3 (NRW), also a generous start up package by Loma Linda University (NRW) and a National Merit Test Bed (NMTB) award sponsored by the Department of the Army under cooperative agreement number

Abusamra, A.J., Zhong, Z., Zheng, X., Li, M., Ichim, T.E., Chin, J.L. & Min, W.P. (2005a)

Tumor exosomes expressing Fas ligand mediate CD8+ T-cell apoptosis. *Blood Cells,* 

perhaps even replacement therapies.

targeting in order to improve potency.

*Molecules & Diseases*, 35, 169-173.

**11. Acknowledgment** 

DAMD17-97-2-7016 (NRW).

**12. References** 

immune cells and tumor cells, many studies set out to manipulate exosomes for cancer therapy, specifically immunotherapy. Ever since B lymphocytes were described to contain MHC class II molecules and stimulate CD4+ T cells (Raposo *et al.*, 1996), studies have suggested exosomes may have an immunogenic role. Currently, DEXs from pulsed dendritic cells are under investigation for their ability to enhance and prime the immune system against tumor cells. Dendritic cells activate T cell responses through direct dendritic to T cell contacts. However, exosomes secreted by dendritic cells are also able to stimulate T cells (Zitvogel *et al*., 1998).

#### **9.1 Anti-cancer therapy**

The importance of exosomes in cell to cell communication can be seen in their antigen presenting ability. Seminal work during the late 1990's showed DC's pulsed/loaded with cancer antigens or tumor peptides could yield DEXs which elicited better immune responses towards tumor cells (Zitvogel *et al.*, 1998; Wolfers *et al.*, 2001; Couzin, 2005; Chaput *et al.*, 2006; Tan *et al.*, 2010). The immune response was due to DEXs displaying the appropriate MHC class I molecules and tumor peptides. In fact, DC can be pulsed with both MHC class I and II, therefore effectively priming T cells against tumors (Thery *et al.*, 2002; Andre *et al.*, 2004; Chaput *et al.*, 2004a; Mignot *et al.*, 2006) as well as stimulating the activation and proliferation of NK cells (Clayton *et al.*, 2007; Viaud *et al.*, 2009). Current literature suggests that MHC class I and II containing exosomes are potential cell-free cancer vaccines (Zitvogel *et al.*, 1998; Andre *et al.*, 2004; Chaput *et al.*, 2004a; Tan *et al.*, 2010). Unlike current clinical cancer vaccines, exosomes are not technically vaccines as they are preventative and not therapeutic. The prophylactic vaccines are only effective against oncogenic viruses and do not treat the cancer directly, not to mention too expensive for the average patient (Tan *et al.*, 2010). Treatment of tumors with DEXs has had beneficial results such as initiation of immune response (Couzin, 2005), tumor growth suppression (Zitvogel *et al.*, 1998), tumor shrinking (Viaud *et al.*, 2009), and tumor rejection (Wolfers *et al.*, 2001; Mignot *et al.*, 2006). Exosomes are a better alternative than DC in terms of therapy because its composition can be identified, measured, is stable for storage, and has predictable behavior after administration (Thery *et al.*, 2002; Chaput *et al.*, 2004b).

The utilization of exosomes for cancer immunotherapy is a very new but very rapidly growing field. Most studies were conducted *in vitro*, but it is still unclear how oncologists will be able to translate the literature's data into the realm that will be most beneficial for the patient. One such complication is how the DEXs will be collected and implemented for patient treatment, and will the collected DEXs be sufficient enough? One potential strategy involves isolating DEXs through filtration of the patient's blood which will then be returned and employed to stimulate the immune system (Couzin, 2005). This would certainly allow for personalized therapy, but the question still remains: would there be enough DEXs and if not could adjuvant augmentation increase their numbers. Although the mode of exosome action in vivo is not clear yet, DEXs are a very interesting and potential substitute for dendritic cells in tumor vaccination therapy.

### **10. Conclusion**

Various secreted extracellular vesicles have been found in blood, saliva, breast milk, bronchoalveolar lavage fluid, urine and amniotic fluid. Cellular uptake mechanisms have been shown to range from ligand/receptor interactions, integrin/cell adhesion molecule

immune cells and tumor cells, many studies set out to manipulate exosomes for cancer therapy, specifically immunotherapy. Ever since B lymphocytes were described to contain MHC class II molecules and stimulate CD4+ T cells (Raposo *et al.*, 1996), studies have suggested exosomes may have an immunogenic role. Currently, DEXs from pulsed dendritic cells are under investigation for their ability to enhance and prime the immune system against tumor cells. Dendritic cells activate T cell responses through direct dendritic to T cell contacts. However, exosomes secreted by dendritic cells are also able to stimulate T

The importance of exosomes in cell to cell communication can be seen in their antigen presenting ability. Seminal work during the late 1990's showed DC's pulsed/loaded with cancer antigens or tumor peptides could yield DEXs which elicited better immune responses towards tumor cells (Zitvogel *et al.*, 1998; Wolfers *et al.*, 2001; Couzin, 2005; Chaput *et al.*, 2006; Tan *et al.*, 2010). The immune response was due to DEXs displaying the appropriate MHC class I molecules and tumor peptides. In fact, DC can be pulsed with both MHC class I and II, therefore effectively priming T cells against tumors (Thery *et al.*, 2002; Andre *et al.*, 2004; Chaput *et al.*, 2004a; Mignot *et al.*, 2006) as well as stimulating the activation and proliferation of NK cells (Clayton *et al.*, 2007; Viaud *et al.*, 2009). Current literature suggests that MHC class I and II containing exosomes are potential cell-free cancer vaccines (Zitvogel *et al.*, 1998; Andre *et al.*, 2004; Chaput *et al.*, 2004a; Tan *et al.*, 2010). Unlike current clinical cancer vaccines, exosomes are not technically vaccines as they are preventative and not therapeutic. The prophylactic vaccines are only effective against oncogenic viruses and do not treat the cancer directly, not to mention too expensive for the average patient (Tan *et al.*, 2010). Treatment of tumors with DEXs has had beneficial results such as initiation of immune response (Couzin, 2005), tumor growth suppression (Zitvogel *et al.*, 1998), tumor shrinking (Viaud *et al.*, 2009), and tumor rejection (Wolfers *et al.*, 2001; Mignot *et al.*, 2006). Exosomes are a better alternative than DC in terms of therapy because its composition can be identified, measured, is stable for storage, and has predictable behavior after

The utilization of exosomes for cancer immunotherapy is a very new but very rapidly growing field. Most studies were conducted *in vitro*, but it is still unclear how oncologists will be able to translate the literature's data into the realm that will be most beneficial for the patient. One such complication is how the DEXs will be collected and implemented for patient treatment, and will the collected DEXs be sufficient enough? One potential strategy involves isolating DEXs through filtration of the patient's blood which will then be returned and employed to stimulate the immune system (Couzin, 2005). This would certainly allow for personalized therapy, but the question still remains: would there be enough DEXs and if not could adjuvant augmentation increase their numbers. Although the mode of exosome action in vivo is not clear yet, DEXs are a very interesting and potential substitute for

Various secreted extracellular vesicles have been found in blood, saliva, breast milk, bronchoalveolar lavage fluid, urine and amniotic fluid. Cellular uptake mechanisms have been shown to range from ligand/receptor interactions, integrin/cell adhesion molecule

cells (Zitvogel *et al*., 1998).

**9.1 Anti-cancer therapy** 

administration (Thery *et al.*, 2002; Chaput *et al.*, 2004b).

dendritic cells in tumor vaccination therapy.

**10. Conclusion** 

attachment and fusion, or through transcytosis. Physiologically, these extracellular vesicles have been shown to play a role in inter-cellular communication, disposal of defective or weakened proteins, formation of morphogen gradients for tissue patterning during development and for antigen presentation to T cells. Pathologically, these vesicles have been shown to aid in the transmission of viruses, prions, -amyloid in Alzheimer's disease, and specifically to this chapter, tumor pathogenesis. Although additional investigation is still needed to fully exploit these extracellular vesicles for therapy, there is increasing evidence that in better understanding them and the molecules that they transport, determinations of diagnosis and prognosis and the prediction of response to treatment may be possible. However, it is also the hope that these vesicles may also act at therapeutic targets and perhaps even replacement therapies.

Conventional treatments for cancer include the use of chemotherapeutic drugs, radiation therapy and interventional surgery if the tumors are operable. Recent data suggests that the application of dendritic cell-derived, tumor cell-derived and even ascitic cell-derived exosomes could be developed as novel treatments for cancer. In fact, is has already been established that exosomes can be safely administered to patients, though the number of patients in these trials have been small and thus argue for larger studies. Furthermore, the goal of exosome therapy is to increase the biological magnitude of the immune response and as such exosomal immunotherapy is heavily reliant on the immune system. It will therefore be important to address issues such as patient immunosuppression due to therapeutic treatment, as the efficacy of the immunotherapy will be reliant upon the capability of the immune system. In order to increase the biological magnitude of the immune response researchers are artificially coating and engineering exosomes with tumor antigens to make them more recognizable to the immune system. The affects of heat shocking exosomes is also being investigated as heat shocked tumor exosomes are more effective than those not receiving heat treatment. Heating of the exosome confers a greater immunogenicity and thus elicits a greater immune response. Another possible application for exosomes is in vaccine delivery as exosome-producing cells could be engineered to produce a miRNA or specific genetic component or toxin that could be loaded into a exosome with the proper surface molecules for a cell specific uptake and response. Finally, the application of exosomes as immunotherapeutics represents a new chapter in cancer treatment development. However, much more research must go into elucidation of mechanism and targeting in order to improve potency.
