Therapy-induced chemoexosomes: Sinister small extracellular vesicles that support tumor survival and progression

Abstract

Chemotherapy involves the use of multiple cytotoxic or cytostatic drugs acting by various mechanisms to kill or arrest the growth of cancer cells. Chemotherapy remains the most utilized approach for controlling cancer. Emerging evidence indicates that cancer cells activate various pro-survival mechanisms to cope with chemotherapeutic stress. These mechanisms persist during treatment and often help orchestrate tumor regrowth and patient relapse. Exosomes due to their nature of carrying and transferring multiple biologically active components have emerged as key players in cancer pathogenesis. Recent data demonstrates that chemotherapeutic stress enhances the secretion and alters the cargo carried by exosomes. These altered exosomes, which we refer to as chemoexosomes, are capable of transferring cargo to target tumor cells that can enhance their chemoresistance, increase their metastatic behavior and in certain cases even aid in endowing tumor cells with cancer stem cell-like properties. This mini-review summarizes the recent developments in our understanding of the impact chemoexosomes have on tumor survival and progression.

1. Introduction

In spite of advancement in novel technologies and therapies for diagnosis and treatment, cancer remains a major challenge to health professionals [1, 2] Cancer treatment options include surgery, radiotherapy, immunotherapy, chemotherapy, bone marrow transplant and targeted therapy [3, 4]. Currently, much of the research in cancer is focused on precision medicine and immunotherapy, with the hope that these treatment approaches will transform cancer care [5-9]. Precision medicine involves the tailor made individualized treatment of patients by taking their genetic makeup, medical history, test results and distinctive characteristics into account. Immunotherapy is specifically intended to manipulate and improve the patient’s own immune system to fight cancer. Chemotherapy is aimed at attacking and killing cancer cells directly and is still the most widely used form of anti-cancer therapy [10-12]. Chemotherapy utilizes drugs to inhibit the growth of rapidly growing cancer cells either by killing them or by stopping them from dividing. Depending upon the cancer type, specific drugs are used either alone or in combination for treatment. Chemotherapeutic drugs are classified into different types on the basis of mechanism of action which includes alkylating agents, antimetabolites, topoisomerase inhibitors, mitotic inhibitors, antibiotics, proteasome inhibitors and tyrosine kinase inhibitors [13]. Novel cytotoxic and cytostatic drugs are routinely being incorporated into cancer treatment regimens [14]. In spite of usage of more potent novel drugs and administration of multiple rounds of chemotherapeutic drugs alone or in combination, cancer cells often persist after chemotherapy and regrow leading to patient relapse. Emerging discoveries indicate that chemotherapeutic stress reprograms cancer cells, causing them to release soluble factors that are delivered to other cancer cells thereby enhancing their resistant to therapy. The resulting therapy resistant cancer cells having an altered genetic and proteome content are endowed with the potential to efficiently regrow and drive aggressive tumor progression [15]. This negative side effect of chemotherapy and the mechanisms in play are not well understood.

In addition to soluble factors, cancer cells undergoing chemotherapy induced stress also release large amounts of extracellular vesicles. This refers to a heterogeneous group of vesicles that includes exosomes, microvesicles and apoptotic bodies [16-18]. Because of overlapping size and functions, accurate classification of extracellular vesicles is still a matter of debate, but in general is related to their mechanism of secretion. Exosomes are 30 to 150 nm vesicles formed by inward budding of endosomal membranes with their subsequent release into the extracellular space. Due to the lack of definitive biomarkers to identify exosomes, it has been recommended that particles within this size range be referred to as small extracellular vesicles [18]. However, because most of the studies to date use the term exosome to identify small extracellular vesicles, we continue to refer to exosomes in this review, but acknowledge that in most publications the identity of these particles is based on size not on proof that they originate from endosomal compartments. Microvesicles are 150 to 1000 nm vesicles formed by outward budding and fission of plasma membrane. Both exosomes and microvesicles are secreted from live cells but apoptotic bodies are formed during the execution phase of apoptosis, their size ranges from 500 to 2000 nm. Irrespective of their origin and size, both exosomes and microvesicles share overlapping functions which involve carrying and delivering lipids, proteins and genetic content to other cells, thereby driving multiple physiological and pathological processes [19, 20]. Confined by a double-layered lipid membrane exosome cargo is protected from degradation and can be transported long distances. Importantly, exosomes secreted by tumor cells have a cargo signature reflective of the malignant cell. Upon internalization by target cells exosome cargo can reprogram the target tumor cell to promote aggressive behavior or to evade therapy [21]. Cancer cells are surrounded by non-malignant stromal cells that support cancer growth. Stromal cells include, but are not limited to, fibroblasts, macrophages, pericytes and endothelial cells [22, 23]. Recent studies indicate that stromal cells also communicate with tumor cells via exosomes that support tumor growth, metastasis, invasion and resistance to therapy.

While exosomes secreted by tumor cells play an important role in mediating tumor-tumor and tumor-host cross talk, less is known about the impact of chemotherapy on exosome secretion and function. When chemotherapeutic drugs are administered to cancer cells, studies have revealed that secretion of exosomes is substantially enhanced [24-27]. Moreover, exosomes secreted following chemotherapy can also have altered composition. We recently introduced the term chemoexosome to identify these exosomes, and to distinguish them from exosomes secreted prior to exposing the tumor cells to chemotherapy [24]. The finding that the secretion and composition of exosomes is altered by drugs delivered during anti-cancer therapy has led to the investigation of the impact of these chemoexosomes on cancer progression. This review focuses on chemoexosomes and their potential for conferring chemoresistance, metastasis and stemness properties on tumor cells.

2. Chemoexosomes promote chemoresistance

Chemoresistance is defined as the ability of tumor cells to evade chemotherapeutic treatment and survive. This is often followed by proliferation, invasion and metastasis of tumor thereby leading to disease progression associated with morbidity and mortality [28-30]. Chemotherapeutic resistance can be intrinsic due to genetic and phenotypic changes within the tumor or it can be extrinsic due to the mutual communication between the tumor supporting host cells and tumor cells present within the tumor microenvironment. When tumor cells are exposed to chemotherapy, soluble factors released from dying tumor cells communicate in a paracrine fashion to impart changes in surviving tumor cells that enhances their resistance to therapy. Host cells present within the tumor microenvironment also are exposed to chemotherapy triggering release of soluble pro survival factors. Together these factors secreted by tumor and host cells result in genetic, molecular and phenotypic changes of tumor cells that promote chemoresistance [31].

By carrying multiple biologically active components such as proteins, lipids, DNA, RNA and miRNA in tiny, compact vesicular structures, chemoexosomes are likely players in tumor chemoresistance [30, 32-37]. Chemoexosomes released from chemoresistant cancer cells can confer chemoresistance in chemosensitive tumor cells [38]. In addition, miRNA’s delivered by exosomes or exosome mediated enhancement of miRNA’s in cancer cells regulates expression of genes that play a vital role in chemoresistance. Emerging evidence indicates that chemotherapy alters the cargo within exosomes and enriches them with multiple components capable of conferring chemoresistance in sensitive cells. This enables tumor cells to evade therapy and grow aggressively.

2.1. Effect of chemotherapy on chemoexosomes production by tumor cells

Paclitaxel

Paclitaxel is a frontline chemotherapeutic drug that stabilizes microtubule formation and is used in combination with other drugs for treating breast cancer [39]. MDA-MB-231 breast cancer cells treated with a physiologically relevant dose of paclitaxel dramatically enhanced chemoexosome secretion by 1.5-fold compared to vehicle treated cells [26]. In addition, exosome composition was also altered, including an increase in the protein survivin. Survivin is upregulated in many cancers leading to inhibition of apoptosis and a decrease in rates of cell death. It is also known to be a key player in conferring resistance to therapy and promoting aggressive tumor behavior [40]. Although paclitaxel did not enhance cellular expression of survivin, it altered survivin localization and increased its concentration in chemoexosomes by 30 fold when compared to vehicle treated cells [26]. Nocodazole, another class of chemotherapeutic drug that disrupts microtubule assembly also enhanced survivin levels in the chemoexosomes [26]. Other chemotherapeutic drugs that impact cell survival, growth and proliferation did not enhance survivin levels in chemoexosomes demonstrating that survivin enrichment in the chemoexosomes is not due to a general phenomenon resulting from chemotherapeutic stress, but it appears to be specific to the chemotherapeutic drugs that disrupt normal function of microtubules. When incubated with SKBR3 breast cancer cells, chemoexosomes delivered their survivin that enhanced tumor cell resistance to paclitaxel[26]. These studies indicate that drugs impacting microtubules can impact survivin levels thereby enhancing behaviors that drive tumor progression.

Cisplatin

Another drug used to treat various types of solid organ cancers including lung cancer, head and neck cancer, and ovarian cancer is cisplatin [41]. Cisplatin is an alkylating agent that binds to DNA and interferes with DNA repair mechanisms eventually leading to cancer cell death. Extracellular vesicles from cisplatin treated ovarian cancer (A2780) cells incubated with bystander tumor cells induced cisplatin resistance [42]. This effect was reduced when extracellular vesicles were added in the presence of heparin, a known inhibitor of extracellular vesicle uptake by cells [43, 44]. Compared to controls, exposure of cells to cisplatin triggered release of extracellular vesicles having enhanced phosphorylated p38 alpha, JNK2, JNK-Pan and p53. It has been well established that p38 and JNK signaling pathways promote chemoresistance thereby indicating a potential molecular mechanism for extracellular vesicle mediated cisplatin resistance [42]A549 NSCLC¹ cells treated with cisplatin enhanced chemoexosome secretion in a concentration dependent manner [45]. Short term or long term treatment of NSCLC cells with cisplatin specifically enhanced miR-425-3p levels in the chemoexosomes compared to exosomes that are secreted from NSCLC cells treated with vehicle (control exosomes). Chemoexosomes that are secreted after cisplatin therapy were readily internalized by NSCLC cells. When chemoexosomes enriched with miR-425-3p were incubated with cisplatin sensitive NSCLC cells, it enhanced cisplatin resistance in those cells. This enhanced resistance was diminished when the cisplatin sensitive NSCLC cells were transfected with miR-425-3p inhibitor prior to incubation with the chemoexosomes. This indicates a direct role of exosomal mir-425-3p in cisplatin resistance in NSLC cells. Further analysis revealed that the chemoexosome mediated delivery of miR-425-3p enhanced autophagic activity in the recipient cells thereby conferring cisplatin resistance. This was confirmed by incubating cells with the autophagy inhibitor BafA1 which diminished cisplatin resistance. MiR-425-3p levels were shown to be increased in exosomes isolated from NSCLC patients serum after therapy compared to exosomes isolated from patients before therapy thus providing a strong in vivo correlation with the above in vitro findings [45].

Gemcitabine

Gemcitabine (Gem) belongs to a family of anti-metabolite drugs that attack cells at very specific phases in the cell cycle eventually leading to cell death [46]. It is an important chemotherapeutic agent in gastrointestinal malignancies including pancreatic cancer where it is used alone or in combination with other drugs. Patel et al., demonstrated that the chemoexosomes released from gemcitabine treated pancreatic cancer cell lines, MiaPaCa and Colo357, induced chemoresistance in naive pancreatic cancer cells in comparison to cells incubated with exosomes released by cells treated with vehicle (control exosomes) [47]. Mechanistically, these gemcitabine-induced chemoexosomes stimulate upregulation of the reactive oxygen species (ROS) detoxifying enzymes super oxide dismutase (SOD) and catalase (CAT)) and down regulation of gemcitabine metabolizing enzyme deoxy cytidine kinase, two independent but cohesive mechanisms that enhance chemoresistance. Gemcitabine treatment significantly enhances SOD and CAT transcript levels in chemoexosomes compared to control exosomes. These chemoexosomes delivered SOD and CAT transcripts to naive pancreatic cancer cells, significantly enhancing SOD and CAT protein levels. The amount of miR-155, a miRNA that targets dCK is significantly enhanced in chemoexosomes compared to control exosomes. Functional inhibition of miR-155 by anti-miR-155 abrogated chemoexosome mediates acquired chemoresistance confirming this mechanism of exosome mediated chemoresistance [47]. These studies indicate that although gemcitabine is widely used as an anti-cancer drug, it can have the unwanted effect of promoting chemoresistance. Therapeutic interference of the exosomes produced by cells treated with gemcitabine holds potential to improve therapy.

Proteasome inhibitors

Bortezomib and carfilzomib are two proteasome inhibitors widely used in frontline anti-myeloma therapy [48]. Proteosomes comprise a proteolytic complex that eliminates misfolded, used and non-functional proteins by degradation. Degraded proteins are then recycled to facilitate synthesis of new proteins in the cell. Inhibition of proteasomes by proteasome inhibitors results in the buildup of ubiquitinated proteins in the cells leading to cell death. We have previously shown that myeloma cells treated with proteasome inhibitors bortezomib or carfilzomib dramatically enhances the secretion of exosomes [24]. In addition, myeloma cells treated with the genotoxic agent melphalan, also enhances exosome secretion. These anti-myeloma therapies activate the NF-kB pathway thereby triggering heparanase expression in myeloma cells [49]. Heparanase is an endoglycosidase that trims heparan sulfate chains of proteoglycans, leading to alteration in gene expression, shedding of the syndecan-1 proteoglycan from the cell surface and activation of a mechanism that drives tumor metastasis and angiogenesis thereby promoting aggressive tumor behavior in myeloma [50-55]. In addition to these functions, heparanase also regulates exosome biogenesis. Myeloma and breast cancer cells expressing high heparanase secrete a greater number of exosomes compared to myeloma cells expressing low heparanase [56]. Subsequent studies revealed that heparanase, by trimming heparan sulfate chains of the syndecan-1 proteoglycan, activated the syndecansyntenin-ALIX pathway to stimulate exosome biogenesis [57]. In addition to its impact on exosome biogenesis, we found that heparanase alters the composition of cargo inside the exosomes to favor myeloma growth and progression [56]. These findings indicate that the chemotherapy-mediated increase in heparanase is responsible for enhanced chemoexosome secretion in myeloma cells. Interestingly, chemoexosomes released by the myeloma cells contained high levels of heparanase and when these chemoexosomes contacted myeloma cells they delivered heparanase to the cells and enhanced ERK signaling. This is important because ERK signaling increases myeloma chemoresistance [49]. Together these studies reveal that proteasome therapy, a widely utilized approach to control myeloma, can have the unexpected effect of promoting chemoresistance. Thus, development of therapies that would block exosome function during proteasome therapy could lead to enhanced patient outcomes.

2.2. Effect of chemotherapy on chemoexosomes production by stromal cells

When chemotherapeutic drugs are administered to cancer patients, stromal cells within the tumor microenvironment are also exposed, and react to, chemotherapeutic drugs. Many reports have demonstrated that cancer associated fibroblasts (CAF) enhance chemoresistance in various cancer types including pancreatic cancer. When exposed to gemcitabine, CAF’s secrete more chemoexosomes compared to cells exposed to vehicle [58]. Gemcitabine treatment remarkably enhanced mRNA levels of Snail and miRNA146a in the chemoexosomes secreted by CAFs. Snail is a transcription factor known to be involved in epithelial to mesenchymal transition and chemoresistance. In addition, snail directly regulates miR146a. Incubation of Snail and miR146a containing chemoexosomes with pancreatic ductal adenocarcinoma cells (PDAC) delivered the Snail and miR146a to these cells and increased their chemoresistance. Chemoresistance was abrogated when PDAC cells were co-cultured with CAF’s pretreated with Gemcitabine and the exosome secretion inhibitor Gw4689, thereby demonstrating the direct role of the exosomes from gemcitabine treated CAF in chemoresistance. These findings were further confirmed in vivo by implanting CAF’s and pancreatic cancer cells subcutaneously in NOD/SCID mice. These mice were treated with saline control, gemcitabine or GW4869 (exosome inhibitor) + gemcitabine for two weeks. Tumor size increased over time in PBS treated and gemcitabinetreated mice whereas tumor size did not increase in mice treated with gemcitabine and GW4869 [58].

Resistance to therapy contributes to poor outcomes in cancer patients. The studies mentioned above support the emerging concept that chemoexosomes secreted by tumor and host cells may play an important role in driving chemoresistance and patient relapse. . These previously unappreciated side effects of chemotherapy require further investigation with the goal of introducing interventional drugs that block chemoexosome secretion or function thereby diminishing chemoresistance.

3. Chemoexosomes promote metastasis

Metastasis is the spreading of cancer to distant sites, a process seen in many cancers in advanced stages. Patients with metastatic disease often have a poor response to chemotherapy and a dismal outcome. The process of metastasis involves multiple key steps including, but not limited to, cancer cell polarity changes, invasion into the blood stream, immune cell evasion, seeding at a distant site, extracellular matrix degeneration, and neovascularization [59, 60]. This entire process involves cancer – host interactions at each stage through secretion of multiple cytokines including interleukins, growth factors, and proteinases like matrix metalloproteinases that facilitate breaching the extracellular matrix barriers [59, 60]. Not surprisingly, chemotherapy with its often harsh effect on the homeostasis of host cells, can have the negative impact of promoting metastasis of tumor cells that survive an initial chemotherapeutic assault [61]. Exosomes secreted by cancers act in an autocrine and paracrine manner, leading to alteration in biology of the cancer cells promoting dedifferentiation, escape from immune surveillance and ultimately metastasis [30, 62-64]. Given the fact that tumor cells produce relatively high levels of exosomes that have been shown to have biological function, it is reasonable to expect that when tumor cells are exposed to chemotherapeutic drugs, the secreted chemoexosomes differ in function from those secreted by cells not exposed to drug. In fact, as mentioned previously, exosome cargo is often altered after tumor exposure to chemotherapy and likely due in part to tumor cell response to stress [24]. Understanding how chemoexosomes contribute to regulating the biology of surviving tumor cells post-therapy is crucial in understanding chemoresistance, tumor relapse and pro-metastasis alterations.

At this time, relatively little is known regarding the role of chemoexosomes in enhancing pro-metastatic potential of cancers. Breast cancer cells (MDA-MB-231) treated with paclitaxel resulted in loss of cell polarity [26]. When cancer cells lose polarity, there is enhanced propensity for epithelial-mesenchymal transition, an important early process in metastasis [65-68]. It has been reported that when mice with breast cancer were treated with paclitaxel and doxorubicin, there were higher levels of annexin A6 in the exosomes post-chemotherapeutic treatment [35]. These chemoexosomes also transfer annexin A6 to lung endothelial cells, resulting in activation of the nuclear factor kappa-B signaling pathways. Consequentially, there was an upregulation of C-C motif chemokine ligand 2 on the endothelial cells which acts as a ligand for the monocytes and tumor cells which possess the C-C chemokine receptor, resulting in tumor cell colonization in the lungs. Annexin A6 is a highly conserved protein which is found on the plasma membrane and has been linked to cell survival, proliferation, differentiation and inflammation [69]. It has been associated with invasiveness in cancer [70-72] and its downregulation sensitizes the cancer cells to targeted chemotherapy [73]. Examination of circulating exosomes following therapy for breast cancer revealed an increase in the level of annexin A6. This was most noticeable in patients who had no response to the chemotherapy [35]. Together these studies point to increases in exosomal annexin A6 as an important contributor to resistance, increased invasiveness and tumor progression in breast cancer.

As mentioned above, exposure of myeloma cells to proteasome inhibitors dramatically enhances the amount of heparanase present in the exosomes. Interestingly, heparanase was localized to the exosome surface where it was capable of degrading heparan sulfate within an extracellular matrix [24]. Recent data has revealed that extracellular vesicles are abundant within the extracellular matrix where they can actively participate in proteolytic tissue remodeling [74, 75]. Thus, it is reasonable to speculate that the chemoexosomes bearing heparanase play an important role in matrix degradation during tumor cell invasion and metastasis. Additionally, because heparanase delivered by exosomes can stimulate syndecan-1 shedding [24], this can lead to formation of a complex with α4β1 integrin and VEGFR2 and the resulting increase in myeloma metastasis and endothelial migration [55]. Importantly, myeloma chemoexosomes, via their heparanase cargo, also can stimulate macrophage migration, an event that may further facilitate tumor invasion and metastasis [24].

In an in vitro study, ovarian cancer cell lines, A2870 and IGROV-1 were separately treated with cisplatin, a frontline chemotherapy in ovarian cancer, and chemoexosomes were isolated. When these ovarian cancer cells were treated with chemoexosomes and their migration ability was assessed using a matrigel transwell system, there was 5-6 fold increase in the number of cells that became invasive when treated with chemoexosomes compared to cells treated with exosomes harvested under baseline conditions [42]. Although this is an in vitro study and does not directly address metastasis, it does reveal that chemoexosomes can impact invasion, a key step in the metastatic process.

As indicated above, relatively little is known regarding the role of chemoexosomes in enhancing pro-metastatic potential of cancers. However, there is mounting evidence that chemoexosomes can participate in regulating key steps in the metastatic process. Clearly there is need for further studies that will enhance our understanding of the role of chemoexosomes in promoting relapse and metastatic spread of cancer.

4. Chemoexosomes promote stemness

Cancer stem cells (CSCs), also known as tumor initiating cells, are a small subpopulation of cells that constitute <1-8% of cells within a tumor [76]. They have the ability to self-renew and differentiate into heterogeneous cell populations that contribute to aggressive tumor growth. CSCs have been reported in multiple types of cancers ranging from leukemia to solid tumors [77-79]. CSCs play a vital role in tumor initiation, progression, chemoresistance and relapse. Chemotherapy induces the expression of stemness related genes in cancer cells by various mechanisms such as activation of hypoxia inducible factors and calcium release in cancer cells [80]. Recent studies have provided evidence that chemoexosomes can impact stemness thereby presenting yet another way that chemoexosomes regulate tumor growth and progression[81].

Shen et al. [80] report that breast cancer cells treated with a sub-lethal dose of chemotherapeutic agents docetaxel or doxorubicin induced secretion of extracellular vesicles that transferred their cargo and induced a cancer stem cell like phenotype in breast cancer cells. These extracellular vesicles, when introduced to breast cancer cells, enhanced their ability to form spheres, one of the characteristic features of cancer stem cells. When breast cancer cells were pretreated with these extracellular vesicles followed by injection into mammary fat pads of NSG mice, it enhanced tumorigenicity compared to breast cancer cells that were treated with extracellular vesicles from cells not exposed to chemotherapy. In addition, the post-therapy extracellular vesicles also enhanced the expression of stemness associated genes such as NANOG, SOX9, NOTCH1 and OCT4 in breast cancer cells. Characterization of the vesicle cargo revealed enhanced levels of miRNA’s including miR-9-5-p, miR-203a-3p and miR-195-5p. Chemotherapy treatment did not elevate the cellular expression of these miRNA’s but it selectively enhanced their packaging into extracellular vesicles. Further, the vesicle mediated transfer of miR-9-5-p, miR-203a-3p and miR-195-5p suppressed the expression of the ONECUT2 transcription factor leading to enhanced stem cell like phenotype Enhanced expression of ONECUT2 has been reported in various cancer types and inhibition of this transcription factor was shown to enhance tumor cell proliferation and migration. In a mouse xenograft mammary tumor model, docetaxel treatment caused elevation of miR-9-5p, miR-195-5p, and miR-203a-3p in circulating extracellular vesicles, an increased level of stemnessassociated genes and decreased ONECUT2 expression [80]. These effects were diminished in tumors deficient in exosome secretion demonstrating that the vesicles responsible for the above mentioned effects were chemoexosomes and that they played a direct role in promoting cancer stemness.

As discussed earlier in this review, anti-myeloma therapy enhances the secretion of chemoexosomes having a high level of heparanase. When these chemoexosomes carrying high heparanase were incubated with myeloma cells under spheroid forming conditions, the myeloma cells formed more and larger spheroids than incubated with control exosomes. Moreover, and expression of stem cell markers ALDAH1, GLI1 and SOX2 increased dramatically when chemoexosomes were present [81]. In addition, chemoexosomes isolated from the serum of a myeloma patient after chemotherapy also enhanced the stemness of myeloma cells.

The stemness inducing ability of myeloma chemoexosomes was diminished when chemoexosomes were added together with the heparanase inhibitors OGT2115 or Roneparstat, or when heparanase expression was knocked down in cells secreting the exosomes. These data indicate a direct role of chemoexosome mediated transfer of heparanase in enhancing the stem cell phenotype of myeloma cells. Moreover, when the heparanase deficient cells that lacked stemness properties were injected into mice they formed tumors poorly compared to myeloma cells expressing a higher level of heparanase [81]. Thus, treatment of myeloma cells with standard chemotherapy drugs can lead to secretion of chemoexosomes high in heparanase cargo that can enhance tumor stemness, eventual tumor regrowth and patient relapse.

Treatment of ovarian cancer cell spheroids with cisplatin caused release of small extracellular vesicles (i.e., chemoexosomes) that enhanced the ability of mesenchymal stem cells to promote tumor growth and progression [82]. The bone marrow derived MSCs used in the study when exposed to the chemoexosomes enhanced expression of metalloproteinases MMP1, MMP2 and MMP3 compared to cells treated with control exosomes. Secretion by the mesenchymal stem cells of interleukin-6, interleukin-8 (IL-8), and vascular endothelial growth factor A (VEGFA) [82] was also increased, all of which may play a role in promoting tumor progression.

5. Chemoexosomes as biomarkers to monitor therapy response

Exosome cargo is specific to the cell type from which they are produced and it reflects the condition of that particular cell. Because they are composed of a range of biological molecules including proteins, lipids, nucleic acids, miRNA’s and long non-coding RNA’s they hold great potential for as carriers of prognostic and diagnostic biomarkers [27]. In colorectal carcinoma (CRC) it has been shown that exosomes isolated from CRC patient’s serum are enriched with multiple miRNAs that are significantly decreased after surgical resection indicating that these miRNA’s can serve as potential diagnostic markers for CRC . In patients with pancreatic cancer, exosomes isolated from the serum are enriched with high levels of glypican-1 that are now being utilized for pancreatic cancer diagnosis [83]. Long non-coding RNA ZFAS1 is enriched in exosomes isolated from gastric cancer patients serum and is associated with lymphatic metastasis and tumor stage [84].

As mentioned earlier, chemotherapy not only enhances the production of exosomes, it also alters exosome cargo. During the course of chemotherapy these chemoexosomes with altered cargo are released into the body fluids [27]. It is reasonable to speculate that this cargo reflects characteristics of the tumor and thus may provide clues to tumor response to therapy and eventual patient outcome. Multiple techniques are available for exosome characterization including proteomic analysis, lipidomic analysis, deep sequencing and next generation sequencing for long non-coding RNA’s and miRNA’s all of which can be utilized to gain information on the tumor of origin. However, relatively few studies have probed the potential of chemoexosomes for identification of biomarkers. Profiling of serum exosomal miRNA from breast cancer patients before, during and after neoadjuvant treatment revealed enhanced expression of miRNA-21 in metastatic patients and it was directly correlated with the size of tumor [85]. Annexin A3 was also found in breast cancer cell derived exosomes and levels of annexin A3 are increased after treatment with drugs [86]. In myeloma, heparanase is present on the surface of myeloma cell derived exosomes and its levels are significantly enhanced following chemotherapy [24]. Because heparanase is a potent promotor of myeloma tumors, its presence in high levels in chemoexosomes may prove useful as a tool to predict eventual patient relapse. Thus, although there is a paucity of studies in which chemoexosomes have been probed for biomarkers, the initial findings indicate that they hold important information that may be useful clinically.

6. Conclusions and future perspectives

The last decade has seen an explosion in our knowledge related to the importance of extracellular vesicles in regulating cell behaviors in cancer. Evidence is mounting that treatment of tumor cells with chemotherapeutic drugs can alter the biogenesis and composition of exosomes leading to deleterious effects. As discussed in this review, release of chemoexosomes can lead to enhanced tumor cell chemoresistance thereby contributing to patient relapse and poor outcome (Figure 1). This enhanced chemoresistance may be in part due to these vesicles enhancing a stem cell phenotype in some tumor cells. Moreover, the contribution of chemoexosomes to promoting tumor metastasis may explain why chemotherapy in some cases is known to increase metastasis. There is also a possibility that these chemoexosomes released during chemotherapy act on immune cells to suppress anti-tumor immune response so that the tumors can evade immune response and develop chemoresistance and eventually tumor relapse. Exploring chemoexosomes cargo provides the opportunity to better understand the way both tumor and host cells respond to chemotherapy. One of the main challenge to this approach is difficulty in separating tumor cell derived exosomes from the exosomes that are derived from host cells. Studies aimed at exploring markers that can differentiate between tumor cell derived exosomes and host exosomes is an important area of research that needs immediate attention.

Fig. 1.

Role of chemoexosomes in tumor survival and proliferation

1- Exposure of tumor cells to chemotherapeutic drugs stimulates exosome biogenesis and secretion and alters exosome composition (e.g. increased level of survivin and heparanase). 2 – Once released by cells, chemoexosomes dock with tumor cells and deliver their cargo and enhance chemoresistance. Some cells exhibit elevated levels of transcription factors (e.g. SOX9, NANOG, GLI1) consistent with their obtaining cancer stem cell-like properties (CSC). 3 - These stem-like cancer cells divide and proliferate leading to tumor relapse. 4 - Chemoexosomes that enter bone marrow can stimulate release of pro- angiogenic cells that migrate to tumor where they initiate angiogenesis further enhancing tumor proliferation. 5 - Chemoexosomes released by tumor cells dock with mesenchymal stem cells and enhance their ability to promote tumor growth and progression. Chemotherapeutic drugs stimulate cancer associated fibroblasts to secrete chemoexosomes having a high level of Snail, a transcription factor known to stimulate epithelial to mesenchymal transition (EMT) and promote metastasis. Those chemoexosomes also increase chemo-resistance of the tumor cells.

Although the many negative side effects that chemotherapy has on patients are well documented, our understanding of the impact of chemoexosomes is in its infancy. It is now critical to determine specific mechanisms that lead to drug-induced changes in exosome biogenesis and function. In addition, understanding the mechanisms by which chemoexosomes can impact tumor, specifically those mechanisms that contribute to chemoresistance, should be a high priority for research. Lastly, discovery of ways to interfere directly with chemoexosome secretion and/or function have potential to enhance the effectiveness of chemotherapy, lessen tumor relapse and improve patient outcome.