Intratumoral Administration Immunogenic Exosomes can Modify Tumor Immune Microenvironment

doi:10.21203/rs.3.rs-3891975/v1

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Intratumoral Administration Immunogenic Exosomes can Modify Tumor Immune Microenvironment

https://doi.org/10.21203/rs.3.rs-3891975/v1

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It has been observed that external stress or stimuli can initiate apoptosis and produce extracellular vesicles known as exosomes. Recent studies suggest that exosomes can trigger an anti-tumor immune response. In the current study, exosomes secreted by the 4T1 triple-negative breast cancer (TNBC) cell line under stress conditions (Dox, X-ray irradiation, and cold plasma treatments) were studied. The stress-induced exosomes were harvested, differing in their ability to present some DAMP proteins such as HSP70 and HMGB1. These exosomes can enhance the expression of pro-inflammatory molecules by immune cells at different levels in different treatments. Additionally, intratumoral administration of these exosomes has been shown to modify the tumor immune microenvironment (TIME) in a TNBC murine model differently. We have concluded that exosomes secreted by the 4T1 cell line under Dox treatment can significantly reduce tumor volume and modify the tumor microenvironment. However, other treatment methods produce immunogenic exosomes that are neither effective nor appropriate. Nevertheless, many studies report that these methods have significant therapeutic effects when used directly.

Biological sciences/Cancer

Biological sciences/Immunology

The tumor microenvironment (TME) is comprised of diverse cell types interacting with each other in a hypoxic and acidic environment. The tumor immune microenvironment (TIME), consisting of immune cells and their mediators, is one of the most significant components of TME ¹. Both TIME and TME play a vital role in the development, progression, and treatment of cancer; therefore, modifying them is necessary for combating the disease ^2–4. There are various cancer treatment methods, including targeted therapy, radiotherapy, and chemotherapy, which can be used to tackle these environments ^{5 39}. These interventions can eliminate cancer cells and release specific substances that may activate the immune system, and enhance potential immune responses ^3,6–9.

Immune cells can detect internal or external danger signals in their environment ³. Endogenous danger signals released by cells in response to cell death and stress are called damage-associated molecular patterns (DAMPs) ^10,11. In contrast, pathogen-associated molecular patterns (PAMPs) are external molecules with conserved motifs like bacterial DNA or formyl peptides ^3,11. DAMPs and PAMPs can stimulate the immune system by binding to pattern recognition receptors (PRRs) on immune cells. ¹².

The death of cells causes the release of various DAMPs, such as adenosine triphosphate (ATP) ,High Mobility Group Box 1 (HMGB1), heat shock proteins (HSPs), and uric acid ¹³.Other molecules such as CALR, HSPs, ANXA1, and ATP are also released from the endoplasmic reticulum of dying cells. DAMPs may be released through extracellular vesicles (EVs) and bind to cell surface receptors such as the TLR family ^14,15. Exosomes are a type of extracellular vesicles that are nanosized and contain various bioactive molecules, including nucleic acids and proteins ^3,16. They can easily travel throughout the body and interact with distant cells ^3,17. Exosomes are key players in cell-to-cell communication by facilitating the transfer of biologically active molecules to recipient cells ^3,18. Physiological challenges, such as injury, inflammation, infection, disease, or exposure to external stress like heat or radiation, can modify the composition and also function of exosomes ¹⁹.

It has been suggested that stress-modified exosome cargos can be an effective platform for activating an antitumor immunogenic effect ^19–21. As the release of DAMPs during therapy or cell death can stimulate an inflammatory response by means of the TLR/NF-kB signaling pathway, TME can change from cold to hot ^3,21. Our research showed that radiation, chemo DOX, and cold plasma can increase the release of immune-stimulating exosomes by 4T1 cell lines. These exosomes contain a molecular profile including an abundance of molecules, such as DAMPs, which are significant for stimulating immune cells. Furthermore, peripheral blood mononuclear cells (PBMCs) treated by these exosomes increase the expression of proinflammatory interleukins. Intratumoral administration of exosomes has been shown to be effective in modulating immune responses within the tumor microenvironment. In the present study, 4T1 cells were treated with doxorubicin, x-ray, and cold plasma, and then their exosomes named EXO^dox, EXO^x−ray, and EXO^{cold−plasma} ,respectively, were isolated.. Afterwards, we used exosomes to determine their immunogenic properties in in vitro and therapeutic properties against the breast cancer tumors in in vivo studies as shown in Fig. 1.

Isolation and characterization of 4T1 cells derived exosomes:

The 4T1 cells were treated with doxorubicin, X-ray, and cold plasma. Then, exosomes isolated from cells using the same protocols. The study confirmed the successful isolation of pure and intact exosomes. The size and shape of the exosomes were determined using DLS and TEM, respectively, revealing an average size of 50 nm (as shown in Figs. 2A and B). The Malvern Zetasizer showed that the exosomes had a surface charge of 0 mV. Moreover, UV absorbance at 530 nm (the absorption region of doxorubicin) demonstrated that doxorubicin was not present in the vesicles, meaning that doxorubicin can activate the innate immune system, which can cause the release of inflammatory cytokines, leading to inflammation in non-targeted organs ²².

Exosomes are a type of vesicle that can be distinguished from other similarly sized vesicles by the expression of specific markers of the endosomal pathway. In this study, the presence of calnexin, CD63, Hsp70, and Hsp90 proteins were analyzed from different treatment groups using western blot analysis (as shown in Figs. 3A-D). The results confirmed that the purified vesicles were indeed exosomes.

Immunogenicity assay of exosomes:

Immunostimulating agents, including HMGB1, uric acid, ATP, and heat shock proteins (HSP), are released when cells die. These agents can be found in extracellular vesicles, including exosomes and micro-vesicles ³. Exosome markers were detected through immunoblot analysis (Fig. 3A-C), and HMGB1 was found to act as a DAMP immunostimulant in treatment groups (Fig. 3D). To assess their performance, blood mononuclear cells from Balb/c mice were isolated and incubated with EXO^dox, EXO^x−ray EXO^{cold−plasma} for 24 hours. The cells were then analyzed for IL-1β, IL-6, IL-12, and IFN-β generation. The groups receiving EXO^dox produced significant amounts of IL12 and high levels of IL6 and IL1β, while the groups receiving EXO^x−ray and EXO^{cold−plasma} did not produce these interleukins at the same level. The effectiveness of the therapy was demonstrated by strong stimulation in the EXO^dox, followed by EXO^x−ray, and to a lesser extent, EXO^{cold−plasma} (Fig. 4A-D).

Intratumoral treatment the 4T1 model:

In a mouse model with established tumors, exosomes treatment was administered five times at three-day intervals, starting 10 days after 4T1 cell injection. The control group mice were injected with PBS on the same schedule. The results showed that the treatment schedule notably delayed tumor growth (volume) compared to the untreated control (p < 0.05) (refer to Fig. 5A).

Immunohistochemical staining and optical density (OD) values showed that all treatments significantly increased IL1β, IL6, IL12, and IFNβ expression in tumor tissue compared to the control group (p ≤ 0.05) (Fig. 6A-E).

To assess the impact of leukocyte cell infiltration, a specific segment of the tumor was used for immunohistochemistry analysis, and the findings were quantified. It was found that there was an increase in CD4 and CD8 T-cell subsets, as well as CD56, a marker for NK cells, on the tumor following intratumoral administration of exosomes (as shown in Fig. 7A-C).

When cells die, they release DAMPs, including HMGB1, uric acid, ATP, and HSPs ³. These are the most frequently observed DAMPs in cells undergoing stress ¹¹. By binding to the immune cell receptors, DAMPs can induce inflammation and releasing cytokines, such as IFN-γ ³. In addition to the TME, DAMPs are also found in exosomes of tumor cells stimulated by treatments ^3,23. DAMPs found in exosomes may affect the inflammatory balance in tumor sites, and some studies show that tumor-derived exosomes can mediate toll-like receptor (TLR) signaling pathway ^24,25. The study's findings align with prior research indicating that therapies enhance exosome release, a process contingent on the specific treatment². For example, exposure to hyperthermia-treated cancer cells exosomes increases macrophage activation markers and exosomal Hsp70 content, suggesting a potential immunogenic role for exosomes. The results revealed that EXO^dox, EXO^x−ray and EXO^{cold−plasma} significantly possess a molecular profile rich in HMGB1 in a different range and proteins, including Calnexin and HSPs, linked to exosomes ¹⁹. Exosomes produced by treated cells showed a molecular profile rich in HMGB1 and protein molecules Calnexin and HSPs associated with exosomes (Fig. 3). In vitro assay on PBMCs reveals that the incubation by EXO^dox, EXO^x−ray, and EXO^{cold−plasma} leads to an increase in cytokine protein levels of IL-6, INF-β, IL-1β, and IL-12, possibly due to the presence of DAMPs in exosomes (Fig. 4). Of note, HMGB1 protein was specifically increased in exosomes secreted by treated cells. HMGB1 can bind to immune cell receptors, such as TLR2 and TLR4 ²⁶, triggering inflammation and releasing cytokines, such as TNF-α and IFN-γ ^3,23. Studies demonstrate that HMGB1 protein is found in various types of exosomes from various cancer cells, including breast, bronchial, chronic, colorectal, and glioblastoma ^3,27,28. The treatment method may also affect the presence of HMGB1 in exosomes. Cold Atmospheric Plasma (CAP) is a partially ionized gas triggering the production of reactive oxygen and nitrogen species (RONS)^29,30. The investigation of the protein profile of exosomes revealed that EXO^{cold−plasma} had reduced levels of HMGB1. In vitro studies demonstrated that these exosomes had a diminished ability to induce an immune response. Similar studies on the anti-cancer effect of cold plasma showed that the highest amount of DAMPs was Calreticulin and extracellular ATP, inducing strong inflammatory immune responses when directly treated. Surprisingly, the exosomes. Although the exosomes derived from this treatment did not have as much ability to change the tumor microenvironment as direct treatment, they increased inflammatory cytokines at the tumor site. Meanwhile, this treatment did not lead to a decrease in the volume of the tumor at the end of the treatment period. Other factors were likely trapped inside the vesicles, preventing them from functioning effectively. Therefore, further studies are needed to explore this phenomenon.

We observed in our study that treatment with EXO^x−ray, although very similar in protein content to EXO^dox (both of them had a lot of HMGB1), mice treated with these exosomes died after receiving two doses of these exosomes and did not complete the course of treatment. Exposure to X-ray radiation during radiation therapy increases the intracellular level of reactive oxygen species (ROS), leading to cell cycle arrest in the G0/G1 phase. This mechanism is influenced by several factors, including DNA damage, hypoxia, mitochondrial impact, and increased ROS production³¹. The reason for the unexpected death of the mice that received these exosomes is unclear. It is highly questionable why, in studies using X-ray radiation directly, immunogenic cell death in cancer cells was evident despite the production of high levels of ROS³². In the present study, treatment with EXO^x−ray did not demonstrate the desired effect, necessitating further research and consideration.

HSPs induce a pro-inflammatory response in mouse and human monocytes, leading to the activation of T cytotoxic cells, natural killer cells, macrophages, and DCs upon interaction with them ^3,33. The expression of HSP70 on exosomes increased under stressful conditions, as shown in Fig. 3C. Previous studies have demonstrated that the expression of HSP60, HSP70, and HSP90 in exosomes derived from HepG2 cells was different after treatment with chemotherapeutic drugs³⁴. A similar study suggested that HSP70-positive exosomes from melanoma cells were able to enhance NK cells' cytolytic activity against YAC-1 cells ³⁵. Another study demonstrated that exosomes containing HSP70 promote anti-tumor immunity by enhancing the maturation of DCs and Th1 cells ³⁶. Like previous studies, our in vitro data show that exosomes released from cancer cells are able to modulate immune responses and modify tumor microenvironment at different levels in different treatments^21,37. To assess the immunological state of the exosomes in the tumor microenvironment, treated cells-derived exosomes were intratumorally injected. The results showed that the intratumoral injection of exosomes produced by all types of treatments led to tumor growth inhibition. However, a better response was obtained by EXO^dox. It is shown that EXO^dox can effectively induce an immune system response in murine breast cancer models, resulting in an increase in pro-inflammatory cytokines, such as IFN-β, IL-12, IL-1β, IL-6, and CD8 + T cells. -(Figs. 6 and 7). Recently, it has been demonstrated that exosomes generated via the photothermal technique exhibit elevated levels of immune-activating molecules compared to those obtained through hyperthermia. Upon administration of these exosomes into animal models, they bolster the capacity of immune cells to infiltrate tumors, leading to a decrease in tumor expansion. Exosomes derived from photothermal methods display heightened damage-associated molecular patterns compared to hyperthermia, resulting in a more potent immunostimulatory effect and tumor growth inhibition. ²¹

Although EXO^{cold−plasma} and EXO^x−ray were able to generate some immune responses, the results were not satisfactory, failed to show favorable outcomes, and could not modify the TME (Tumor Microenvironment) effectively. However, the use of these methods, which induced the formation of free radicals, is very effective in direct methods and is well known for the immunogenic cell death (ICD) in cancer cells. By contrast, the results indicated that EXO^dox possessed promising therapeutic effects. This study suggests that these exosomes were able to successfully change the TME in favor of the immune system and help strengthen immune responses at the tumor site (Fig. 8). We suggest that other anthracycline compounds be investigated for their potential use in indirect cancer treatment, such as exosomes in immune therapy. These compounds, which are being developed as next-generation cancer drugs, may have superior effects compared to doxorubicin and could potentially show improved results in modifying the tumor microenvironment (TME).

Cell line

The 4T1 cells were incubated at 37°C in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2% penicillin/streptomycin, and 1% glutamine

Doxorubicin, X-ray, and Cold plasma treatments

The 4T1 cells were seeded at a density of 5 × 10^5 cells/well in a 6-well plate and incubated overnight at 37°C for cell adhesion. The following day, the culture medium was replaced with a fresh medium, and the cells were treated with 25 ppm of Dox. After 72 hours, the supernatant was collected for exosome isolation.

X-ray irradiation was performed using an RS225 x-ray cabinet (X-Strahl, Camberley, UK), operated at 200 kV and 10 mA (Thoraeus filter, 20 Gy in 20 s) with a single dose of 20 Gy. After 72 hours, the cultured medium was collected, centrifuged at 10,000 g for 5 minutes, and stored at -80°C until further use.

The plasma jet kINPen was used to perform plasma treatment at atmospheric pressure, generating a non-thermal plasma. (neoplas, Germany). Argon (99.9999% pure; Air Liquide, France) was used as the feed gas at a flow rate of 1.5 standard liters per minute (spm). Plasma was generated with a sinusoidal voltage of 3.97 kVpp and a frequency of 68 KHz. The 4T1 cells were treated directly in the 6-well plates for 8 minutes. After 24 hours, the culture medium was harvested.

Exosome Isolation

Exosomes were isolated using the ultracentrifugation method according to Théry et al. ²². The medium of treated-cells was collected and centrifuged at 8000g for 30 minutes to remove debris, followed by centrifugation at 50,000g for 1 hour at 4°C. The resulting pellet was then subjected to a final centrifugation step at 200,000g for 1 hour at 4°C. The exosome pellets were subsequently resuspended in PBS.

Characterization of exosomes

We used Transmission Electron Microscope (TEM) and Dynamic Light Scattering (DLS) to determine the size, shape, and charge of exosomes. We used Nano drop analysis to measure the concentration of exosomes. We also quantified the total protein content of the isolated exosomes with a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

Western blot analysis

The study evaluated biomarkers and immunostimulatory agents of exosomes (HSPs 90, HMGB1, calnexin, CD36) using the western blot method. To measure the protein concentration of isolated exosomes, they were suspended in lysis buffer and analyzed using Micro-BCA and Varian Cary 50 UV-Vis Spectrophotometer. The protein concentration was also measured using the Bradford assay and standard Western blot analysis. The proteins were then transferred onto a Polyvinylidene Difluoride (PVDF) membrane, blocked with 5% Blotting Grade Blocker Non-Fat Dry Milk, and probed with primary antibodies overnight at 4°C. The membranes were washed to remove unbound secondary antibodies. The protein bands were detected using an enhanced chemiluminescence ³⁸ detection kit and recorded with X-Ray film. The intensity of each band was quantified using Fiji software and compared with the loading control, β-actin.

Immunogenicity of exosomes

In the present study, Balb/c mice's peripheral blood mononuclear cells were isolated from their buffy coat using density Ficoll gradient centrifugation. The cells were separated using a buffy coat-histopaque emulsion centrifuge, and the PBMCs were collected, washed, and resuspended in PBS. The viable monocytes were counted and resuspended in RPMI-1640. The cells were then treated with EXO^dox, EXO ^{cold−plasma}, and EXO ^x−ray, while the control groups were taken from the culture plate's wells. After 24 hours, the treated cells were collected for ELISA analysis. The supernatant was used for indicator determination, and the concentrations of IL-1, IL-6, IL-12, and IFN-β were found using an ELISA kit.

Tumor model: A female Balb/C tumor model was obtained from Pishro Mehravaran Azma Pars in Babul, Iran. The breast cancer cell line (4T1) was injected subcutaneously into the right side of a mouse's abdomen at a seeding density of 106 cells/ml. When the tumor volume reached 50 to 100 mm3 (calculated using the formula: volume = 0.5 x length x width2 with a digital caliper), the mice were randomized into two groups: untreated (PBS) and treated with exosomes (n = 3).

Intratumoral treatment the 4T1 model

Ten days after inducing the tumor (day 1), 100 µg of exosomes in 50 µL of PBS were injected into the tumor with 72-hour intervals. The tumor size was monitored daily, and the mice were sacrificed when the tumor volume exceeded 1000 mm3, in accordance with the approved protocol of the Institutional Animal Care and Use Committee (IACUC) of Tabriz University of Medical Sciences.

Immunohistochemistry assay

The Antibodies were supplied by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) to conduct immunohistochemistry on tumors that were harvested on day 22 of treatment. The tumors were preserved in PBS and transported to the pathology department of Imam Khomeini Hospital Urmia University of Medical Sciences for sectioning and staining. The sections were subjected to various antibodies and substrates to investigate the tumor tissue. The sections were initially treated with hydrogen peroxide, then rinsed twice, and subsequently incubated with a blocking buffer. Following this, the primary antibody was applied and allowed to incubate for 24 hours. Finally, the sections were exposed to a polyvalent goat anti-mouse antibody labeled with biotin and washed four times. The protein was then combined with 3,3'-Diaminobenzidine (DAB) chromogen and incubated for 1–10 minutes. Subsequently, the sections were counterstained with hematoxylin, dehydrated, and covered with a coverslip. The cellular densities were quantified using Fiji image analysis software for immunohistochemistry on tumors harvested on day 22 of treatment. The tumors were fixed in PBS and sent to the pathology department of Imam Khomeini Hospital Urmia University of Medical Sciences for sectioning and staining. The study involved incubating sections with various antibodies and substrates to study tumor tissue. The sections were covered with hydrogen peroxide, rinsed twice, and incubated with a blocking buffer. The primary antibody was added and incubated for 24 hours. Subsequently, the sections were covered with a polyvalent goat anti-mouse antibody labeled with biotin and washed four times. The protein was then combined with 3,3'-Diaminobenzidine (DAB) chromogen and incubated for 1–10 minutes. Subsequently, the sections were counterstained with hematoxylin, dehydrated, and covered with a coverslip. Cellular densities were quantified using Fiji image analysis software.

It has been previously reported that injecting exosomes directly into a tumor can induce an antitumor-immune response. These reports indicate that administering exosome nanoparticles can locally reprogram the innate adaptive immune response, making the immune system more resistant to cancer. In the current study, we tested this theory using exosomes derived from cells under stress conditions (Dox, X-ray irradiation, and cold plasma) and injected them directly into the tumor. The exosomes caused several immune changes in the tumor microenvironment

Data availability

The data supporting the findings the conclusion of this article will be made available upon reasonable request to the corresponding author.

Reporting of in vivo experiments

The study was reported in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines³⁹.

Acknowledgements

The authors would like to express their gratitude to those who provided help and support for this project. The authors thank Nooshin Naeimi for the language editing of the manuscript.

Author contributions

Arman Kalami and Zahra Poursalehi have equal first-author-level contribution to this work. They contributed to the study conception and design, performing experiments, data analysis, and manuscript writing. Forzani Hosseini Gharalari contributed to analysis of immunohistochemistry staining results. Hana Molavi helped in performing experiments. Mohammad Tollabi helped in manuscript writing. Behnam Nasiri-Motlagh contributed to thedesign of X-ray therapy. Mehdi Shahgolzari contributed to study design, experimental design, proofreading, and data analysis. Ahmad Yari Khosroushahi provided contributions to the conception of the study, proofread the manuscript, study design, and experimental design.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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Intratumoral Administration Immunogenic Exosomes can Modify Tumor Immune Microenvironment

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Abstract

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Introduction

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Isolation and characterization of 4T1 cells derived exosomes:

Immunogenicity assay of exosomes:

Intratumoral treatment the 4T1 model:

Discussion

Material and Methods

Conclusion

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