Renal clear cancer cells regulate the differentiation of urine‐derived stem cells to carcinoma‐associated fibroblasts via RUNX3/TGF‐β1 signaling axis

Clear cell renal cell carcinoma (ccRCC), the most common pathological subtype of renal cancer, is one of the significant health concerns due to limited clinically effective treatments. Nevertheless, targeting carcinoma‐associated fibroblasts in the tumor microenvironment has emerged as a promising innovative strategy for renal cancer therapy. Thus, this study is aimed to explore the role and molecular mechanism of urine‐derived stem cells (USCs) in the progression and metastasis of ccRCC. Initially, wound‐healing and transwell experiments were used to assess the migration and invasion abilities of the cells. Then, western blot analysis (WB) and quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) analyses were used to demonstrate the relevant protein and messenger RNA expression levels. Finally, hematoxylin–eosin and immunohistochemical stainings were performed to evaluate metastasis and protein expression in lung tumors. The coculture of USCs with the ccRCC cell lines significantly enhanced their migratory and invasive abilities. WB and qRT‐PCR analyses exhibited that ccRCC cell lines significantly increased cell mobility markers transcriptional and protein levels in USCs. Finally, the in vivo investigations in nude mice showed that USCs promoted the proliferation and migration of ccRCC‐based xenograft tumors. In summary, these findings demonstrated that USCs promoted ccRCC tumorigenesis and development in vivo and in vitro by regulating the Runt‐related transcription factor 3/transforming growth factor‐β1 signaling axis.


| INTRODUCTION
Clear cell renal cell carcinoma (ccRCC) is the most common pathological subtype of renal cancer due to limited clinically effective treatments and being highly unresponsive to chemotherapy or radiotherapy (Garje et al., 2018). Nevertheless, tremendous advancements in exploring diverse therapeutic options have resulted in targeted therapy, which has been demonstrated effective even against advanced ccRCC (Armesto et al., 2021;Meng et al., 2021;Wei et al., 2021). The targeted therapy mainly acts by inhibiting the tumor progression by selectively constraining the vascular endothelial growth factor receptor and platelet-derived growth factor receptor (PDGFR) towards preventing tumor angiogenesis and subsequent tumor metastasis Reustle et al., 2020;Wang et al., 2022). Nonetheless, the curative effects of targeted therapy against the notified receptors are effective only for a few months (Gorka et al., 2020;Li et al., 2022;Ribback et al., 2022;Thompson et al., 2018). In recent years, research in exploring the targeted therapy has resulted in several new options for targeting the tumor microenvironment (TME) of ccRCC, such as blocking programmed cell death-1 and programmed cell death ligand 1 of inflammatory cells in the TME (Hu, Chen, et al., 2020;Meng et al., 2021;Yang et al., 2021;Zhang et al., 2021). Therefore, targeting the stromal cells in the TME may provide new therapeutic options for the treatment of ccRCC.
Among various cells in the TME, carcinoma-associated fibroblasts (CAFs) are the most common type of stromal cells in the TME. Notably, the activation of CAFs in the ccRCC tissues remodels the renal extracellular matrix, promoting tumor progression and metastasis, affecting angiogenesis, and improving tumor cell proliferation and chemoresistance by secreting VEGF and PDGF (Guo et al., 2019;Li et al., 2019;Yang et al., 2020). To this end, urine-derived stem cells (USCs), originating from the renal parietal cells, are a kind of pluripotent stem cells extracted from urine (Zhang et al., 2014). It should be noted that every 100 mL of urine contains approximately two to seven USCs, expressing similar surface markers, such as CD44, CD73, CD105, and CD133. Similar to mesenchymal stem cells (MSCs), these USCs possess similar self-renewal and multi-directional differentiation capabilities (Kim et al., 2020;Zhang et al., 2019). Consequently, USCs are also considered a kind of MSCs that could be used in regenerative medicine and tissue engineering. In an instance, Xiong et al. (2020) demonstrated that USCs expressed the myofibroblast markers (α-smooth muscle actin [α-SMA]) when cultured for 14 days in the presence of 2.5 ng/mL of transforming growth factor-β1 (TGF-β1) and 5.0 ng/mL of PDGF-BB. Nevertheless, the role of USCs and their differentiation to CAFs, as well as promoting tumor progression in the TME of ccRCC, remain to be reported. Hence, it is of great significance to explore the regulatory mechanism of ccRCC in promoting the transformation of USCs to CAFs.
Typically, TGF-β1 plays a crucial role in transforming MSCs to CAFs . Previous studies indicated that colon cancer, prostate cancer, and breast cancer cells could promote tumor progression by secreting TGF-β1 and transforming MSCs to CAFs. In contrast, blocking TGF-β1 or CXCR4 could inhibit the transformation process of MSCs to CAFs. The plausible mechanism behind the transformation of MSCs to CAFs might be that TGF-β1 could bind to the TGF-β1 receptor on the surface of MSCs, activating the phosphorylation of Smad2/3 and the SDF-1/CXCR4 pathway. Motivated by these considerations, herein, we demonstrate the role of TGF-β1 in transforming USCs to CAFs, due to its Runt-related transcription factor 3 (RUNX3)dependent regulation to promote the transformation of USCs. In summary, we believe this study will benefit in understanding the interaction mechanism between USCs and ccRCC cells, and providing new ideas for studying renal cancer TME and treatment.

| Cell culture
USCs, 786O, Caki-1, and HEK293 cell lines were purchased from the American type cell culture collection. These cells were cultured in the specific medium and incubated in a humidified atmosphere of 37°C and 5% CO 2 according to the provider's instructions. USCs were cultured in a special medium based on the K-SFM medium (

| Extraction and culture of USCs
The extraction of USCs was performed following the reported procedure.
Initially, 20 mL of urine collection special medium (DMEM containing 100 U/mL of streptomycin, 2.5 µg/mL of amphotericin B, 10% FBS, 50 µg/mL of gentamicin, and 100 U/mL penicillin) was placed into a 250 mL of sterile urine collector tube. Further, the midstream urine of volume ≤200 mL (before was collected and the urethral orifice of men/ women was washed once using potassium permanganate sitz bath). Then, the urine sample was placed in an ice box and sent to the laboratory for isolation and culture of USCs. Further, the urine sample was dispensed and centrifuged at 1500 rpm for 5 min. After separating the supernatant, an appropriate amount of USCs special medium was added and inoculated original urine (500 μL per well) into the 24-well plate and placed in a humidified atmosphere maintained at 37°C and 5% CO 2 .
Notably, the medium in the plate was replenished on the 3rd, 5th, 7th, 9th, and 11th day, and cells were selected to cover 60%-75% of the wells of the plate for passage.

| Coculture assay
The coculture assay was performed using the Corning Transwell coculture chamber (0.4 μm, 6-well plate). Concerning the effect of On the other hand, while studying the influence of USCs on ccRCC cells, ccRCC cells were cultured in the lower chamber, and USCs cells were placed in the upper chamber. Further, 2.6 mL of culture medium was added to the lower chamber and 1.5 mL of culture medium was added to the upper chamber to maintain contact between the upper and lower chambers.

| Generation of engineered cell lines
The engineered cell lines were generated and transfected as follows.

| Western blot (WB) analysis
Briefly, the cell lysate was prepared by lysing the cells in the logarithmic phase using the radioimmunoprecipitation assay buffer and centrifuged at 15,000 rpm for 30 min. Further, the supernatant was subjected to protein determination using the Bicinchoninic acid protein detection kit (ab102536, Abcam). Then, 30-40 μg of protein was loaded and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Afterward, the protein blots were transferred to polyvinylidene fluoride membranes (Millipore) and were blocked with 5% nonfat dry milk for 1 h at room temperature, and then incubated with the corresponding primary antibodies (1:1000) overnight at 4°C.
In this study, the primary antibodies, including anti-TGF-β1

| Statistical analysis
The data were expressed as mean ± SE of three independent measurements. The data were analyzed by comparing the groups using a two-tailed t test, one-way analysis of variance (ANOVA), and two-way ANOVA using GraphPad Prism 6, considering p < .05 as statistically significant. * indicates p < .05, ** defines p < .01, and *** represents p < .001. genes, such as α-SMA, vimentin, FAP, S100A4, and PDGFRα transcription mRNAs ( Figure 1d) and proteins ( Figure 1e). Together, these findings suggested that ccRCC cells significantly promoted the transformation of USCs to CAFs.

| ccRCC cells promote the differentiation of USCs through TGF-β1
Previous studies indicated that TGF-β1 played a crucial role in the transformation of MSCs to CAFs in various cancers . In a case, it was reported that the TGF-β1 signaling pathway was abnormally activated in the ccRCC cells (Hanusek et al., 2022). Similarly, our analysis of KIRC data in the TCGA database indicated that the TGF-β1 expression level was significantly higher in the KIRC tumor tissues than in the paracancerous tissues (Figure 2a).
In addition, qRT-PCR and WB analyses presented that TGF-β1 mRNA Together, these findings indicated that ccRCC cells could substantially promote the transformation of USCs to CAFs through TGF-β1.

| RUNX3 regulates the transformation of USCs to CAFs through TGF-β1
On the other hand, the transformation of USCs to CAFs through TGF-β1 can be regulated by RUNX3. Several reports indicated that RUNX3 could negatively regulate the expression of TGF-β1 (Krishnan & Ito, 2017). In addition, previous reports demonstrated that RUNX3 played an important role in inhibiting the proliferation, invasion, migration, metastasis, and angiogenesis of ccRCC cells (He et al., 2012). Considering these reports, we speculated that RUNX3 could regulate the secretion of TGF-β1 by ccRCC cells toward differentiating USCs into CAFs. Immunohistochemical

| DISCUSSION
Indeed, USCs, originating from the renal parietal cells, are a kind of pluripotent stem cells extracted from urine. The typical count of USCs per 100 mL of urine includes 2-7 in number, expressing surface markers similar to MSCs, such as CD44, CD73, CD105, and CD133 (Kang et al., 2015). Similar to MSCs, these USCs possess self-renewal ability and multi-directional differentiation capability into various cell types, such as osteoblasts, adipocytes, endothelial cells, fibroblasts, and nerve cells, among others (Akiyama et al., 2012;Wu et al., 2021). Previous studies based on tissue engineering research indicated that USCs were found to express the myofibroblast marker, α-SMA when cultured for 14 days in the presence of 2.5 ng/mL of TGF-β1 and 5.0 ng/mL of PDGF-BB (Hu, He, et al., 2020Huang et al., 2021). In this study, consistent with the reported findings, ccRCC cells promoted the transformation of USCs to CAFs through TGF-β1. Moreover, RUNX3 played an important role in inhibiting the proliferation, invasion, migration, metastasis, and angiogenesis of ccRCC cells. In addition, silencing RUNX3 could promote the high expression of TGF-β1 in renal cancer cells, and overexpression of RUNX3 could inhibit the expression of TGF-β1 (Boguslawska et al., 2019;Zheng et al., 2018). Similarly, our findings indicated that RUNX3 regulated the transformation of USCs to CAFs by inhibiting the expression of TGF-β1.

| CONCLUSION
In summary, this study demonstrated that RUNX3 has substantially regulated the USCs differentiation into CAFs by negatively regulating the TGF-β1 secretion in the ccRCC cells. Moreover, the transformed USCs promoted the ccRCC progression. Considering these findings, we believe that this study will substantially help understand the mechanism of interactions between USCs and ccRCC cells, and provide new ideas for studying TME and treating renal cancer.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The figures supporting the results of this study are included in the article and the original data sets are available from the first author or corresponding author upon request.

ETHICS STATEMENT
All the protocols and experiments related to in vivo investigations involving animals have been approved by the Institutional Committee