Utilizing Tumor-Derived Extracellular Microvesicles for Kidney Regeneration

doi:10.21203/rs.3.rs-3594528/v1

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Utilizing Tumor-Derived Extracellular Microvesicles for Kidney Regeneration

https://doi.org/10.21203/rs.3.rs-3594528/v1

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Background

A comparative study was conducted to assess the potential of extracellular microvesicles (MVs) derived from mesenchymal stem cells (MSCs) and tumor cells in kidney regeneration using an experimental murine model of chronic kidney injury (CKI).

Methods

MVs were obtained from MSCs, L929 sarcoma, LLC carcinoma, or B16 melanoma cells. These MVs were then administered intravenously to CBA mice with CKI. Renal excretory function was evaluated based on serum concentrations of creatinine and fatty acid binding protein-1. Morphological changes in the kidneys were assessed histologically. Flow cytometry analysis was utilized to characterize T cells in spleens and renal cell infiltrates.

Results

The study revealed that both MSCs and MSC-derived MVs (MSC-MVs), as well as tumor-derived MVs (T-MVs), regardless of their origin, exhibited a comparable ability to improve both excretory function and the histological structure of the damaged kidney. Both MSC-MVs and T-MVs reduced the proportion of pro-inflammatory CD4 + CD44 + T cells in renal cell infiltrates and spleens of CKI mice. Furthermore, the MVs studied increased the numbers of natural CD4 + CD25 + FoxP3 + regulatory T cells in the spleen but not in renal cell infiltrates.

Conclusions

These findings suggest that T-MVs, similar to MSC-MVs, possess a universal capacity to stimulate kidney tissue restoration and provide anti-inflammatory immunomodulation of the kidney milieu.

tumor

mesenchymal stem cell

microvesicle

regeneration

chronic kidney injury

Extracellular vesicles are phospholipid bilayer-enclosed particles released by all cell types. These vesicles can be easily separated from tissue culture supernatants and biological fluids, including blood, saliva, breast milk, cerebrospinal fluid, and malignant ascites. Extracellular vesicles can be categorized into three distinct subtypes based on their biogenesis: exosomes, microvesicles (MVs), and apoptotic bodies.

Exosomes are formed through the inward budding of the endosome limiting membrane, leading to the creation of multivesicular bodies that fuse with the plasma membrane and are subsequently released into the extracellular space. Extracellular MVs arise from plasma membrane invagination, forming neck-like structures that eventually undergo vesicle scission. Exosomes are typically 50–150 nm in size, while MVs range from 100 to 1000 nm in diameter. Distinguishing between exosomes and MVs can be challenging due to overlapping sizes, shared surface proteins, and the absence of specific markers.

Cell activation and apoptosis are well-established stimuli for the formation and secretion of extracellular vesicles, which serve as carriers for various biomolecules, including proteins, lipids, DNA, and different RNA species. The loading of exovesicles with molecular cargo occurs through an organized process that controls biomolecule sorting [1]. The primary function of exovesicles is to transport and protect biomolecules from degradation by extracellular enzymes and other aggressive factors. Published evidence suggests that MVs can also transport and protect cell organelles, such as mitochondria. MVs possess intrinsic stability due to their overall negatively charged surface and expression of the surface immunoglobulin CD47 biomarker, which enables evasion from engulfment by mononuclear phagocytic cells [2]. The migrational direction patterns of MVs in the body are determined by the surface receptors they express[3]. Additionally, a low pH environment has been shown to stimulate MV fusion with cell membranes [4]. Recent reports have also demonstrated the efficient crossing of various biological cell membrane barriers, including the blood-brain barrier, by exovesicles [1].

The current paradigm suggests that exovesicles, including MVs, mediate distant intercellular interactions, playing a pivotal role in coordinating all adaptive cellular reactivity in the body.

Mesenchymal stem cells (MSCs) have been extensively studied and applied in regenerative medicine, with numerous clinical trials conducted for a wide range of diseases [5]. Recently, research focus has shifted towards the investigation of exovesicle preparations derived from MSCs. In particular, MSC-derived exovesicles have shown promising potential for in vivo tissue regeneration and the treatment of various diseases in experimental models, including respiratory, renal, hepatic, neurodegenerative, musculoskeletal, and cardiovascular disorders. These studies have highlighted the robust anti-inflammatory, antiapoptotic, pro-angiogenic, and immunomodulatory effects exhibited by MSC-derived exovesicles, similar to those displayed by MSCs in different disease models. Specifically, MSC-derived exovesicles from different sources have demonstrated renoprotective activity in experimental models of chronic kidney injury (CKI), diabetic nephropathy, and kidney fibrosis [6]. Administration of these exovesicles has been found to significantly reduce serum markers of renal injury, such as blood urea nitrogen, creatinine, and transaminase levels, while improving necrotic lesions and ameliorating pronounced signs of tubular dilatation and cast formation [7].

This report presents experimental data demonstrating, for the first time, that tumor cell-derived microvesicles (T-MVs) of various origins, similar to MSCs and MSC-derived microvesicles (MSC-MVs), effectively restore damaged kidney tissue and function. Furthermore, the observed regenerative effect may be partially attributed to the inhibition of pro-inflammatory immune reactivity.

3 − 1. Mice

All experiments were conducted on male CBA mice aged between 4 and 6 months. The mice were provided with autoclaved food and boiled water.

3 − 2. Generation of bone marrow-derived mesenchymal stromal cells

Bone marrow was extracted from intact mice's femoral and tibial bones using a glass homogenizer. The cells were then resuspended in cold RPMI 1640 medium. After washing, the cells were cultured in plastic flasks using a complete cell culture medium consisting of RPMI 1640 medium supplemented with 10% foetal calf serum, 2 mM L-glutamine, and antibiotics. All reagents were sourced from Sigma-Aldrich (St. Louis, MI). Non-adherent cells were progressively removed starting from day 3. The spindle-shaped, plastic-adherent MSCs displayed a classical fibroblast-like phenotype and formed a complete monolayer by week 4. The MSCs were collected using a 0.25% versene-trypsin solution, followed by two washes in serum-free medium.

3–3. Tumor Cell Lines

The L929 murine fibroblast cell line, Lewis lung epidermoid carcinoma (LLC), and B16 melanoma cell lines were maintained in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, and antibiotics.

3–4. Generation, isolation and characterization of extracellular MVs

Apoptosis of MSCs, L929, LLC, or B16 cells (1 × 10⁶/ml) was induced by cultivating them under conditions of oxygen deprivation in serum-free medium for 24 hours. After cultivation, cell debris was removed by centrifugation at 2000 g for 15 minutes, and the supernatants were subjected to a second centrifugation step at 12000 g for 60 minutes at 4ºC. The cell pellet was resuspended in 100 µl of saline, and the size of Annexin V-positive MVs was determined by flow cytometry using a CytoFlex benchtop flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN) with TruCOUNT nanoparticles (TC) for bead size calibration, following the manufacturer’s instructions.

3–5. Induction of chronic kidney injury

To induce rhabdomyolysis, a 50% glycerol solution was injected i.m. at a dose of 8.6 mg/kg three times at weekly intervals. Rhabdomyolysis exerted a mixed influence (ischemic, toxic, and retentional) on the kidneys, predominantly damaging the epithelium of the proximal convoluted tubules.

3–6. Treatment of CKI mice with MSCs or MVs

MSCs (2 × 10⁶/mouse) or MVs (derived from 2 × 10⁶ MSCs or approximately 30–50 µg/mouse) were injected intravenously into the tail veins of the experimental mice 3 weeks after the last glycerol administration. The experiments were terminated 11 days after the administration of MSC/MV preparations. The study included six groups, each consisting of ten male mice:

Control group (mice with CKI without treatment).

CKI group treated with MSCs.

CKI group treated with MSC-MVs.

CKI group treated with L929 cell-derived MVs (L929-MVs).

CKI group treated with LLC-MVs.

CKI group treated with B16-MVs.

At least two independent experiments were performed.

3–7. Biochemical analysis

Blood creatinine levels were measured using a BioVision Creatinine (Mouse) ELISA Kit (rev 03/18, cat. No. E4369-100, Abcam, Cambridge, UK). Fatty acid binding protein-1 (FABP1) was determined in the blood using a Mouse/Rat FABP1/L-FABP Immunoassay Quantikine® ELISA (cat. No. RFBP10, R&DSystems, Minneapolis, MN).

3–8. Histological examinations

Murine kidneys were fixed in a 4% formalin solution, followed by standard steps of tissue processing for histopathology, including dehydration and paraffin embedding. Paraffin blocks were cut into 4–5 µm thick sections using a Rotary Microtome (Microm HM 340E; Carl Zeiss, Germany) and stained with haematoxylin/eosin, Sirius red, or according to Mallory's trichrome staining protocol. Light microscopy and microphotography were performed using a light microscope Axioskop 40 (Carl Zeiss, Germany). Morphometric analysis of the histological kidney structure was conducted on paraffin sections by measuring the following morphometric parameters: diameters of superficial renal glomeruli, diameters of renal collecting tubules, and the size of cells in the middle third of the medullary region. Morphometric measurements were taken in one field of view (ocular lens, 10×25; objective lens 63×).

3–9. Flow cytometry.

Murine kidneys or spleens were cut into small pieces using scissors, followed by gentle homogenization in cold Versene solution using a glass homogenizer. The cell suspension was allowed to settle to remove large cell aggregates, after which density gradient centrifugation was performed in a Ficoll-diatrizoate solution (density: -1.082). After centrifugation, cells were collected, thoroughly washed, and counted. Flow cytometry analysis was conducted using a BD FACSCanto™ II Flow Cytometry System (BD International, Heidelberg, Germany) or CytoFLEX (Beckman Coulter, IN, USA) Flow cytometers with anti-murine CD4-FITC, CD44-PE, CD4-APC, CD25-FITC, and FoxP3-PE antibodies (BD Biosciences, San Jose, California), following the manufacturer's instructions

3–10. Statistics

Data analysis was performed using Graph Prism 8 and one-way ANOVA. Significant differences between groups were assessed using Student's test or Šidák’s multiple comparison test. A p-value < 0.05 was considered significant.

4 − 1. Size distribution of isolated extracellular vesicles

Flow cytometry analysis revealed that the isolated extracellular vesicles were predominantly larger than 100 nm in size. This suggests that our technological approach primarily yielded extracellular MVs (Fig. 1).

4 − 2. T-MVs improve the excretory function of damaged kidneys similar to MSCs and MSC-MVs

Creatinine is a nonenzymatic breakdown product of creatine and creatine phosphate in the body. It is cleared by the kidneys, and elevated creatinine levels typically indicate reduced filtration in renal glomeruli and decreased excretory kidney function. FABP1 is expressed in renal proximal tubule cells and is released into the urine in response to hypoxia caused by decreased peritubular capillary blood flow, serving as an early marker of kidney damage [8].

Our experiments demonstrated that the induction of CKI resulted in a significant increase in serum creatinine levels (from 2.41 to 4.818 ng/ml) and FABP1 levels (from 0.8 to 1.085 ng/ml). Treatment of mice with MSCs or MSC-MVs led to a reduction in blood creatinine and FABP1 levels, nearly approaching the background levels (Table 1). Importantly, administration of T-MVs derived from L929, LLC, or B16 cells exhibited similar effects on creatinine and FABP1 levels in CKI mice. This data strongly suggests that, similar to MSCs and MSC-MVs, T-MVs have the potential to restore the function of damaged kidneys, regardless of the histological origin of the T-MVs used for their generation.

Table 1

Serum concentrations of creatinine and FABP1 (ng/ml) in CKI mice.
Parameter	CKI (control)	Treatment
Parameter	CKI (control)	МSCs	МSC-EVs	L929-MVs	LLC-MVs	В16-MVs
Creatinine	4.818	3.182*	2.767*	2.264*	2.101*	2.095*
FABP1	1.085	0.639*	0.780*	0.785*	0.791*	0.781*
Notes: p < 0.05 as compared to control.

4 − 3. T-MVs improve the histological structure of the damaged kidney similar to MSCs and MSC-MVs.

In terms of morphology, the initiation of chronic kidney injury led to an increase in the size of renal collecting tubules, cellular hypertrophy in the collecting duct (Bellini duct), and cell dystrophy in the Henle's loop within the middle and lower regions of the medullary area (Fig. 2).

However, no signs of dystrophic changes in renal corpuscles were detected upon administration of MSCs or MSC-MVs into CKI-induced mice. Similar therapeutic effects were observed in mice treated with T-MVs. Specifically, morphological examination revealed well-preserved parietal and visceral layers of the renal corpuscle (Bowman's capsule), with the urinary (Bowman's) space clearly visible (Figs. 3–5).

Taken together, the treatment of CKI-induced mice with T-MVs, as well as MSC-MVs, resulted in the restoration of the normal morphological structure of the damaged kidneys. The morphometric data presented in Fig. 6 indicates that both MSC-MVs and T-MVs exhibited similar regenerative effects on the damaged kidney tissue.

The immune system is known to exert constant control over the regenerative processes in the body via cytodestructive and cytoprotective immune reactivity, with T cells playing a pivotal role in defining the direction and strength of such immune reactions. Therefore, we studied the effect of regenerative MVs on the proportion of CD44 + and CD25 + FOXP3 + T cells within the CD4 + T cell population in the renal cell infiltrates and in the spleen of CKI mice.

CD44 is an adhesion molecule that functions as a receptor to glycosaminoglycan of the extracellular matrix (hyaluronan), facilitating migration and mobilization of immune cells into inflammatory loci. The expression of CD44 on T cells is typically associated with their pro-inflammatory activity, including that of pathological origin, which has been confirmed in most chronic inflammatory models of disease [9]. On the other hand, regulatory CD4 + CD25 + FOXP3 + T cells possess pronounced anti-inflammatory properties, down-regulating immune-mediated cytodestructive reactions [10, 11]. The data shown in Fig. 7 suggests that CKI development resulted in an increased number of pro-inflammatory CD4 + CD44 + T cells in renal cell infiltrates, while administration of MSC-MVs in CKI mice brought the levels of CD4 + CD44 + T cells back to nearly normal values. Similar effects were observed in groups of CKI mice treated with B16-MVs or LLC-MVs. In contrast, CKI development did not significantly affect the proportion of CD4 + CD25 + FOXP3 + T cells in renal cell infiltrates (Fig. 8), and this situation remained unchanged upon administration of MVs derived from different origins.

Moreover, similar to the kidneys, CKI development was accompanied by an increased relative content of CD4 + CD44 + T cells in the spleens (Fig. 9), which though returned to baseline levels upon administration of MSC-MVs or T-MVs. In contrast to the unchanged levels of CD4 + CD25 + FOXP3 + T cells in renal cell infiltrates in CKI mice, we observed a significant reduction in this cell population in the spleens. However, upon administration of the MVs studied here in CKI mice, the levels of CD25 + FOXP3 + T cells practically returned to the normal values characteristic of intact animals (Fig. 9).

The tumor is known to possess pathological regenerative activity, capable of modulating the tumor microenvironment to facilitate its own growth, with tumor-derived exovesicles playing an important role in this process. These exovesicles have been described as containing a range of biologically active biomolecules that stimulate tumor growth upon entry into malignant cells. Tumor-derived exovesicles have been shown to interact with immune cells, effectively inhibiting the development of anti-tumor cytotoxic immune reactivity and reprogramming systemic immunity to sustain tumor invasion [12]. Both tumors and normal tissues use similar mechanisms involving the same biomolecules to regulate regenerative processes [13]. In a pioneering study, C. Waddington implicated innate natural regenerative mechanisms as possible regulators of cancer, suggesting that these mechanisms could reach maximal efficiency in malignant cells [14].

In this study, we conducted a comparative analysis of T-MVs, bone marrow MSCs, and MSC-MVs to evaluate their impact on regenerative processes in a murine chronic kidney injury (CKI) model. MSCs and MVs derived thereof are currently being extensively investigated in experimental settings and controlled clinical studies to treat various diseases. MSC-derived MVs have shown a similar spectrum of regenerative activity to MSCs [6] and have demonstrated efficacy in reducing reactive oxygen species (ROS) production in renal tubule epithelial cells in an experimental model of renal hypoxic injury [15]. Importantly, MSCs and MSC-derived exovesicles have been effective in reducing serum markers of kidney failure (urea, creatinine, and transaminases) and promoting morphological kidney regeneration in experimental models of chronic kidney injury, diabetic/hypertensive nephropathy, and renal fibrosis [7]. The therapeutic effects of MSC-derived exovesicles have been partially attributed to small non-coding microRNAs and transforming growth factor β, which stimulate the functional activity of regulatory T cells [6].

Our studies strongly support the idea that T-MVs possess significant regenerative activity in the kidney, which is comparable or even superior to that observed for MSC or MSC-MV preparations. Importantly, all T-MV preparations used in this study were of non-kidney origin, suggesting that T-MVs may exhibit a universal regenerative activity similar to that of MSCs or MSC-MVs. Although both extracellular MVs and exosomes can have regenerative activity, we believe that MVs, as regenerative stimulators, offer superior advantages in comparison to exosomes. First, MVs, similar to cells, are equipped with receptor molecules on their surface that enable them to move purposefully and interact with target cells. Second, in contrast to exosomes, MVs can transfer not only biomolecules but also cellular organelles, such as mitochondria, which are typically non-viable in the extracellular space. Indeed, experimental studies have shown MV-mediated mitochondrial transport and transfection into target cells [16]. The transfected mitochondria were shown to enhance energy production in cells, which likely contributes to the observed regenerative effects in injured organs [17]. We utilized confocal microscopy to show the presence of viable mitochondria inside tumor-derived MVs. In our experiments, T-MVs effectively improved kidney function not only in a CKI model but also in an acute kidney injury model, where there is limited time for regenerative cell growth (paper in preparation for publication). We speculate that, under such circumstances, MV-mediated mitochondrial transfer could provide additional energy support to the injured organ, playing a crucial role in maintaining its functional activity and facilitating subsequent cellular regeneration.

Published data suggests that MSCs and tumor cells, as well as extracellular MVs derived from them, possess immunosuppressive activity that inhibits cytodestructive immune reactivity, at least partially, via enhancing the functional activity of regulatory CD4 + CD25 + FoxP3 + T cells. Regulatory T cells play an important role in creating an immunological cytoprotective microenvironment beneficial for the development of both normal and pathological (tumor) regenerative processes occurring in the body [18, 10, 11]. Our study showed that both MSC-MVs and T-MVs down-regulated the relative content of pro-inflammatory CD4 + CD44 + T cells not only in renal cell infiltrate but also in the spleens of CKI mice. Additionally, MVs restored (up-regulated) the relative numbers of Tregs in the spleens of CKI mice. Clearly, this phenomenon should be further investigated, but the data obtained here strongly suggested that immune-mediated modulation of inflammation plays a significant role in the mechanisms underlying the regenerative effects of both MSC-MVs and T-MVs.

From a practical and cost-effectiveness standpoint, it is important to note that the generation of MSCs is a laborious and technically demanding process that requires well-characterized donor cells. Unlike MSCs, tumor cells of interest can be easily expanded in culture in vitro with no need for donor material. Additionally, it is worth mentioning that T-MV isolation does not involve expensive and cumbersome steps such as ultracentrifugation or microfiltration, unlike exosome isolation. Previous studies have demonstrated that xenogeneic MSC-derived exovesicles can impact the growth and functional activity of target cells [19], suggesting that therapeutic T-MV-based drugs might be separated from both human and animal cells. MV preparations can be stored in frozen and lyophilized forms for extended periods without significant loss of specific activity [20]. Considering these factors, the laboratory-oriented technological cycle of T-MV-based regenerative drugs can be easily adapted to large-scale manufacturing. Such drugs can be standardized based on their physical-chemical and biological properties.

Despite the pronounced regenerative potential of T-MVs demonstrated in this study, there are legitimate concerns regarding the potential oncogenic activity associated with T-MV preparations. Indeed, T-MVs have been shown to enhance tumor growth [21]. However, we emphasize that T-MVs do not contain any substances that are directly mutagenic. Instead, T-MVs are likely to accelerate the growth of cells that have already become malignant as a result of previous carcinogenesis, rather than directly inducing carcinogenesis in healthy cells. In our experimental system, a six-month follow-up of mice treated with T-MV preparations did not reveal an increased incidence of malignancy (data not shown). Nevertheless, the safety of long-term and multiple administrations of T-MVs for treating chronic disorders, including those of age-related origin, requires further meticulous investigation. Concerns about the clinical application of T-MVs to treat life-threatening conditions may diminish when considering that the potential oncogenic risks are outweighed by the unquestionable therapeutic advantages. This notion applies not only to patients with renal failure but also to those with other severe organ abnormalities. We envision that relatively inexpensive T-MV-based preparations could fill the currently underrepresented market niche of regenerative and immunotherapeutic drugs designed for the treatment of various severe conditions, particularly those characterized by high mortality rates.

The data obtained here strongly suggests that T-MVs, like MSC-MVs, are able to stimulate regenerative processes in a CKI-damaged kidney. The observed therapeutic efficacy of T-MVs was independent of the tumour origin used for T-MV preparation. Immunomodulation may play a significant role in the regenerative action of both T-MVs and MSC-MVs.

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Not applicable

Availability of data and materials

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Competing interests

The authors declare no conflict of interest.

Funding

This work was partially supported by the grants FURG-2023-0074 and FURG-2023-0077 from the Ministry of Education and Science of the Russian Federation.

Authors' contributions

G.V.S and V.I.S made equal contributions to this study: conceptualization, study design, data analysis, writing, supervision, and original draft preparation. A.B.D., I.P.I, T.S.K., E.A.B., and A.D. were responsible for experimentation and statistical examination. A.A.D. reviewed and edited the work.

Ethics statement

All animal experiments and procedures were approved by the Institute for Fundamental and Clinical Immunology.

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No competing interests reported.

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You are reading this latest preprint version

Utilizing Tumor-Derived Extracellular Microvesicles for Kidney Regeneration

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

2. Introduction

3. Materials and Methods

3 − 1. Mice

3 − 2. Generation of bone marrow-derived mesenchymal stromal cells

3–3. Tumor Cell Lines

3–4. Generation, isolation and characterization of extracellular MVs

3–5. Induction of chronic kidney injury

3–6. Treatment of CKI mice with MSCs or MVs

3–7. Biochemical analysis

3–8. Histological examinations

3–9. Flow cytometry.

3–10. Statistics

4. Results

4 − 1. Size distribution of isolated extracellular vesicles

4 − 2. T-MVs improve the excretory function of damaged kidneys similar to MSCs and MSC-MVs

4 − 3. T-MVs improve the histological structure of the damaged kidney similar to MSCs and MSC-MVs.

5. Discussion

6. Conclusions

Declarations

References

Additional Declarations

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