Dendritic cells loaded with exosomes derived from cancer stem cell‐enriched spheroids as a potential immunotherapeutic option

Abstract Cancer stem cells (CSCs) are responsible for therapeutic resistance and recurrence in colorectal cancer. Despite advances in immunotherapy, the inability to specifically eradicate CSCs has led to treatment failure. Hence, identification of appropriate antigen sources is a major challenge in designing dendritic cell (DC)‐based therapeutic strategies against CSCs. Here, in an in vitro model using the HT‐29 colon cancer cell line, we explored the efficacy of DCs loaded with exosomes derived from CSC‐enriched colonospheres (CSCenr‐EXOs) as an antigen source in activating CSC‐specific T‐cell responses. HT‐29 lysate, HT‐29‐EXOs and CSCenr lysate were independently assessed as separate antigen sources. Having confirmed CSCs enrichment in spheroids, CSCenr‐EXOs were purified and characterized, and their impact on DC maturation was investigated. Finally, the impact of the antigen‐pulsed DCs on the proliferation rate and also spheroid destructive capacity of autologous T cells was assessed. CSCenr‐EXOs similar to other antigen groups had no suppressive/negative impacts on phenotypic maturation of DCs as judged by the expression level of costimulatory molecules. Notably, similar to CSCenr lysate, CSCenr‐EXOs significantly increased the IL‐12/IL‐10 ratio in supernatants of mature DCs. CSCenr‐EXO‐loaded DCs effectively promoted T‐cell proliferation. Importantly, T cells stimulated with CSCenr‐EXOs disrupted spheroids' structure. Thus, CSCenr‐EXOs present a novel and promising antigen source that in combination with conventional tumour bulk‐derived antigens should be further explored in pre‐clinical immunotherapeutic settings for the efficacy in hampering recurrence and metastatic spread.


| INTRODUC TI ON
Colorectal cancer (CRC) is one of the most frequent malignancies and the leading cause of cancer death worldwide. 1,2 Despite efficient clinical interventions in early stages of CRC including surgery, chemotherapy and radiotherapy, at later stages they frequently are palliative and improve patients' life quality, but survival-influencing tumour recurrence and metastasis still account for a high mortality rate. 3,4 Immune responses are well known as the vanguard of inherent anti-cancer strategies, and progress in immunotherapeutic approaches has improved the efficacy of cancer treatment over the past decades. 5,6 However, the overall outcome is limited and unsatisfactory due to the inability of treatment strategies to target cancer stem cells (CSCs). Accumulating evidences suggest that tumour mass-resident CSCs, a rare population of heterogeneous tumour cells, which display tumour initiating and self-renewal capacity, and account for drug resistance, impose treatment failure. [7][8][9] This issue precludes curative cancer treatment through tumour recurrence following therapy. Therefore new therapeutic strategies selectively targeting this particular stem-like population are warranted.
A variety of immunological modalities have been utilized for CSCs eradication; these include targeting of CSC-specific antigens (Ags) and niche, adoptive CSC-primed T-cell therapy, stimulation of innate immune responses and CSC lysate vaccines. [10][11][12][13][14] In this respect, dendritic cells (DCs) as specialized antigen-presenting cells (APCs) and effective initiators of adaptive immune responses, have played a pivotal role in priming and boosting anti-tumour immune responses and developing cancer vaccines. 15,16 Emerging studies have demonstrated that CSC-based DC (CSC-DC) vaccines can target CSCs by promoting the induction of cytotoxic T cells (CTLs), leading to inhibition of tumour growth and relapse rate reduction. In addition, these vaccination modalities harnessed lung metastases, reduced tumour size and prolonged survival rates in animal tumour models through induction of interferon (IFN)γ production and activation of humoural and cellular immune responses against CSCs with no major adverse effects such as autoimmune reactivity. [17][18][19][20][21][22][23] DC targeting of CSCs has been demonstrated to be advantageous overutilization of DC vaccines pulsed with either tumour bulk or parent cell lysate. [19][20][21] These findings suggest the potential capacity of the immune system and in particular of DC-based vaccines in eradicating CSCs.
Due to the limited and inadequate response rates induced by CSC lysates, identification of proper antigen sources for CSCs targeting is warranted. One option to improve cancer immunotherapy may rely on DC loading with tumour-derived exosomes (TEXs). TEX, membrane nanovesicles (30-140 nm), are released by tumour cells and were described to prepare the tumour microenvironment and pre-metastatic niche in favour of tumour progression, metastasis and immune escape. [24][25][26][27][28] In spite of their role in immune suppression, TEXs are enriched in both tumour antigens and costimulatory molecules and can induce anti-cancer immunity, [29][30][31] particularly when presented by DC. TEXs are taken up by DCs, induce their maturation, and capacity to stimulate antigen-specific CTLs and IFNγ delivery. [32][33][34] It is important to note that uptaken antigens are digested and exclusively loaded into newly generated MHCII molecules including the phenomenon of cross-priming. Accordingly, DC-TEXs vaccination inhibited tumour growth and improved the survival rate in several protective and therapeutic tumour vaccination models. 32,[35][36][37] TEXs-loaded DCs have been shown to elicit superior antitumour immune responses compared to cell lysate-loaded DC in vivo and in vitro. 32,[38][39][40][41][42] However, the efficacy of DC-based therapy using CRC-derived TEXs vs CSCs-derived exosomes (CSC-EXOs) remains to be determined.
As the first step towards developing an effective CSC-DC vaccine in CRC, we searched for an optimal antigen source in an in vitro model. TEXs are enriched in tumour antigens. Thus, we speculated that exosomes derived from CSC-enriched populations may also be enriched in CSC-selective antigens and thereby become particular therapeutic targets and most valuable immunogenic sources for DC loading. To our knowledge, thus far, there is no published report investigating the immunogenic potency of CSC enr -EXOs in the context of anti-tumour responses; here, we explored whether DCs loaded with exosomes derived from CSC enr -EXOs could promote in vitro stimulation of T lymphocytes against colorectal cancer stem cells.
DCs were also loaded with CSC-enriched spheroid lysate, HT-29 lysate and HT-29-EXOs as other potential Ag sources. Our preliminary results approved that CSC-enriched spheroid-derived EXOs do not interfere with phenotypic and functional maturation of DCs.
Importantly, DCs loaded with CSC enr -EXOs as a novel Ag source stimulated T-cell proliferation and CSC-directed cytotoxicity.

| RNA isolation and quantitative PCR
To analyse the expression of KLF4, SOX2, NANOG and OCT4 key stemness genes, the HT-29 parental and spheroid cells were washed thrice with cold PBS and total RNAs were isolated using RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's instruction. To remove genomic DNA contamination, RNA samples were treated with DNase I. RNA quantity and integrity was determined by Nanodrop (ThermoFisher Scientific, USA) and an agarose gel. cDNA was generated using cDNA synthesis kit (GeneAll, Korea). Real-time polymerase chain reaction (RT-qPCR) was performed with the SYBR Premix Ex Taq II real-time PCR kit (TaKaRa, Japan) on the Rotor-Gene Q LightCycler (Qiagene, Germany). The house-keeping gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference gene. Primers are listed in Table 1.

| Exosome purification and cell lysate preparation
HT-29 cells were grown up to 70% confluence in complete medium (DMEM/High glucose + 10% FBS). The medium was discarded and the cells were washed three times with PBS and cultured in DMEM supplemented with 10% Gibco™ exosome-depleted FBS for 48 hours, when the culture supernatant was collected. Due to the serum-free culture condition of spheroids, the conditioned medium was harvested after 10 days of culture. After centrifugation (300 g for 10 minutes) to remove cellular debris, the conditioned mediums were concentrated by a 100-kD molecular weight cut-off (MWCO) Amicon ultra capsule filter (Millipore, USA). Exosome purification was performed through precipitation by size exclusion chromatography using the Exo-spin™ kit (EXO1-8, Cell Guidance Systems, UK) according to the manufacturer's instruction. Purified exosomes were pooled. For cell lysate preparation, HT-29 and CSC-enriched spheroid cells were harvested and washed three times with PBS. The cell lysates were obtained by ten freeze-thaw cycles using liquid nitrogen and a 37°C water bath and then were centrifuged at 20 000 g for 20 minutes to remove the cellular debris. The total protein concentration of isolated exosomes and cell lysates was determined by BCA protein assay kit (Takara, Japan) and stored at −80°C until use.
To prevent rapid evaporation of the fixative, slides were put on top of petri dishes filled with PBS. Finally, samples were coated with gold-palladium and observed by a scanning electron microscope (SEM, Seron Technology, AIS-2100, Korea).

| Western blotting
To characterize the exosomes, purified exosomes and cell extracts were lysed in the RIPA lysis buffer (150 mmol/L NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 1% SDS, 50 mmol/L Tris (pH = 8)) and a protease inhibitor cocktail (Sigma, USA). After lysis, 15 µg of each sample was re-suspended in reducing sodium dodecyl sulphate (SDS) loading buffer and incubated for 5 minutes at 95°C. Thereafter the samples were subjected to 12% SDS-polyacrylamide gel electrophoresis. Following electrotransfer of separated proteins onto a polyvinylidene fluoride (PVDF) transfer membrane and a blocking step, blot was subsequently developed using a chemiluminescent HRP substrate and chemiluminescence was detected using a LAS3000 instrument (Fujifilm, Japan). The primary antibody was omitted in the control group and rabbit IgG was used as isotype control.

| Generation of monocyte-derived DCs
Human peripheral blood mononuclear cells (PBMCs) were separated  (Table 2). After 4 hours, 50 ng/mL lipopolysaccharide (LPS, Sigma, USA) was added to induce complete DC maturation. In some wells immature DCs were only treated with LPS as the positive control group (LPS-alone), and also in some wells immature DCs were treated with PBS instead of Ag or LPS (negative control). After a 48 hour-incubation at 37°C, mature exosome-or lysate-loaded DCs were harvested (day 8) and used as APCs.

| Phenotypic characterization of CSCs and DCs
DCs

| Cytokine release from DCs
After the 8-day cultures for generation of mature DCs, supernatants from all groups were collected and analysed for cytokine concentration. The level of IL-12p40 and IL-10 in the supernatants was determined by enzyme-linked immunosorbent assay (ELISA, R&D system, USA) according to the manufacturer's protocol.

| Measurement of spheroid destruction in coculture with activated T cells
Co-culturing of spheroids with activated T cells was performed as previously described. 43 Briefly, T cells were stimulated twice on days zero and eight of co-culture using DCs at the ratio of 10:1 (T:DC); Thereafter, the functional capacity of activated T cells was investigated via running co-cultures between T cells and HT-29-derived spheroids for 24 hours. The rate of destruction was examined via measuring the diameter of spheroids using Image J software (IJ 1.46r version, NIH, USA) on the phase-contrast microscope pictures. More than 20 independent microscopic fields from spheroids of each group were counted.

| Statistical analysis
All experiments were performed at least three times and data are

| Preparation of CSC-enriched spheroids
Colon cancer spheroids were generated from the HT-29 cell line in serum-free and non-adherent condition ( Figure 1A-C). Enrichment of cancer stem-like cells was confirmed by investigating the expression of SOX2, KLF4, NANOG and OCT4 key stemness genes using real-time PCR. Spheroids showed a significant increase in OCT4, KLF4 and SOX2 gene expression compared to parental HT-29 cells ( Figure 1D). To further verify CSC enrichment, the CRC-CSC surface markers CD44, CD133, DCLK1 and CD166 were assessed by flow cytometry. As shown in Figure 1E and Table 3, single cell suspension from spheroids revealed all 4 markers being expressed at a higher percentage, but only the increase in CD133 + and CD166 + cells was significantly higher than in HT-29 cells.

| Characterization of exosomes derived from HT-29 and CSC-enriched spheroids
After confirming CSCs enrichment in spheroids, exosomes were  Figure 2C). Nonetheless, these results confirmed the nature and purity of the isolated exosomes.

| DC maturation status in the presence of exosomes and cell lysates of HT-29 cells and CSCenriched spheroids
Loading of immature DCs with tumour lysates or EXOs promotes DC maturation. 31,44 To investigate the potential impact of CSC enr -EXOs on the maturation status of DCs, immature DCs were loaded with CSC enr -EXOs and in separate groups with CSC enr lysate, HT-29 lysate and HT-29-EXOs (Table 2) (Table 2) and the LPS-alone groups ( Figure 3A). However, the HT-29-pulsed DCs showed a significantly higher expression of CD40 compared to the CSC enr -EXO group. As related to CD83, both HT-29 lysate-and HT-29-EXO groups showed a significantly lower expression compared to the typical maturation group (LPS-alone), whereas the CSC enr lysate-and CSC enr -EXO-pulsed DCs did not significantly differ from the LPS-alone group ( Figure 3A).
A different picture emerged evaluating IL-12 and IL-10 secretion. All antigen groups supported IL-12 secretion with no significant difference to the LPS group, but HT-29 lysates being slightly more efficient than CSC enr lysates. Instead, with the exception of HT-29-EXOs, IL-10 secretion was suppressed compared to the LPS group ( Figure 3B,C). Accordingly, the IL-12 to IL-10 ratios of CSC enr -EXO-, CSC enr lysate-and HT-29 lysate-loaded DCs significantly exceeded that of the LPS group, the ratio being highest for HT-29 lysate-loaded DCs. With the exception of HT-29 lysates, differences in the IL-12 to IL-10 ratios between the three remaining antigen-pulsed DCs were not or only borderline significant ( Figure 3D). Note: The data were reported as mean ± SD% of three independent flow cytometry experiments.
There was a significant increase in the expression of CD133 and CD166 CSCs markers in spheroids when compared to parental cells, (*P < .05).

| CSC enr -EXOs-pulsed DCs induce autologous T-cell proliferation
To examine the capacity of CSC enr -EXOs-treated DCs in induction of T-cell proliferation and comparing it to the results of other groups, T cells were stimulated for 8 days by co-culture with antigen-loaded mDCs from the four antigen groups ( Table 2). The primed T cells were labelled with CFSE and DC-promoted T-cell proliferation was evaluated by CFSE dilution of restimulated T cells via a second stimulation with accordingly loaded DCs for 5 days. IL-2 treated T cells served as the positive control. As shown in Figure 4A,

| Cytotoxicity of CSC-EXOs-pulsed DCs activated T cells
To further monitor the functional consequence of DC-based T-cell priming in the context of different antigen groups (Table 2)

Furthermore, cytotoxic activities of T cells stimulated by DCs from
CSC enr -EXOs slightly exceeded that of the remaining three groups and that of HT-29-EXO exhibited the weakest advantage compared to T cells stimulated in the absence of antigen. However, the latter disadvantage, which may be linked to the up-regulation of IL-10, was minor ( Figure 5B). Thus, co-culture with antigen-loaded DC promotes cytotoxic T-cell activation. Though differences between the four antigen groups did not reach a significant level, T cells primed with CSC enr -EXO-loaded DCs showed the strongest effect.

| D ISCUSS I ON
Great progress in immune response induction by tools as in vitromatured Ag-pulsed DCs has opened promising paths for tumour immunotherapy, which was hampered for a long time by weak immunogenicity of most tumour-associated antigens and immunosuppressive features of tumour cells. 45,46 However, tumours con- the anti-tumour immune responses were not HLA-restricted. [76][77][78] The vaccine efficacy could be improved by providing additional F I G U R E 4 The proliferation of primed T cells in co-culture with mature DCs. Human CFSE-labelled T cells were primed using mature DCs from CSC enr -EXOs and other antigen groups; following co-culture with the same DCs for 5 d, the proliferation of CFSE-labelled T cells was immunostimulatory signals through alloreactive T cells secreting high levels of T-cell activating cytokines, helping to induce allo-and auto-antibodies, enhancing the cross-priming of antigen-presenting cells (APCs) for CD8+ T cells responding to tumour antigens. [78][79][80][81][82][83] The presence of allo-MHC class II molecules may also be beneficial for Th activation. 84 Those studies also would unravel overlapping and distinct CSC and differentiated tumour cell antigens. Whether a repetition with CRC-CSC or differentiated CRC cell lysates adds valuable information cannot, at present, be judged on and may for a vaccination approach not be necessary. Despite these missing informations, our data unequivocally demonstrate that vaccination with CSC enr -and HT-29-EXO-loaded DC suffices for the activation of CRC-specific CTL with a slight advantage of CSC enr -EXO. Thus, CSC enr -EXOs are a promising antigen sources to initiate a cytotoxic immune response against this subpopulation as well as terminally differentiated tumour cells.
Taken together, this first report on the immunogenic potential of sphere-derived exosomes (CSC enr -EXOs) unravelled that CSC-EXOs support DC maturation and contain immunogenic antigens promoting anti-tumour responses. In fact, CSC-EXOs exert no suppressive/inhibitory effects on DC maturation, and even shift the IL-12/IL-10 ratio in favour of immunostimulation, accompanied by significant autologous T-cell proliferation and spheroid-directed cytotoxic T-cell activation, the immune response induction efficacy of CSC-EXOs being comparable or slightly superior to that of CSC enr lysate. Furthermore, no disadvantages of CSC enr -compared to HT-29-EXOs/lysates were noted in DC maturation and T-cell stimulation by DC. It remains to be proven, but appears most likely that the antigen profiles of CSC enr -EXOs contain CSC-and differentiated cancer cell-specific antigens. As far as this is not the case or the ratio is very imbalanced, a combination strategy making use of CSC enrand HT-29-EXOs or CSC and HT-29 lysates could be advantageous.
We suggest that the explicit engagement of CSC in vaccination protocols provides a major breakthrough in cancer immunotherapy.
With this in mind, CSC-EXOs deserve extensive in vitro and in vivo investigations to unravel their content and their immunogenicity as a weapon against the tumour mass and the deleterious CSCs.

ACK N OWLED G EM ENTS
We acknowledge the grant by Deputy for Research and Technology, Ministry of Health and Medical Education, IR Iran (Grant no: 700/169). This study also was supported by a grant from Iran University of Medical Sciences (Grant no: 30098).

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.